ABCMdb

ABCG2 p.Arg482Gly

Predicted by SNAP2:	A: D (91%), C: D (85%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (85%), K: D (85%), L: D (91%), M: D (85%), N: D (95%), P: D (95%), Q: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Acquired mutations in the MXR/BCRP/ABCP gene alter... Cancer Res. 2001 Sep 15;61(18):6635-9. Honjo Y, Hrycyna CA, Yan QW, Medina-Perez WY, Robey RW, van de Laar A, Litman T, Dean M, Bates SE
Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells.
Cancer Res. 2001 Sep 15;61(18):6635-9., 2001-09-15 [PMID:11559526]

Abstract [show]
A disparity was noted in the transport of rhodamine 123 among nine MXR/BCRP/ABCP-overexpressing cells studied; all demonstrated mitoxantrone transport, whereas only two effluxed rhodamine 123. When the MXR/BCRP/ABCP gene was sequenced in the cell lines studied, differences were noted at amino acid 482, predicted to be at the start of the third transmembrane domain. Sequencing genomic DNA revealed wild-type MXR/BCRP/ABCP to have an arginine at position 482. Cells having a threonine or glycine at position 482 were able to efflux rhodamine 123, whereas cells having an arginine were not. A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/BCRP/ABCP forms transported mitoxantrone. Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. These results suggest that amino acid 482 has a crucial role in MXR/BCRP/ABCP function and that mutation of a single amino acid residue significantly changes substrate specificity, thus altering the drug resistance phenotype.

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No. Sentence Comment
7 A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/ BCRP/ABCP forms transported mitoxantrone. Login to comment
8 Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. Login to comment
78 C, electropherograms for MCF-7 MX100 (R482, top), S1-M1-80 (R482G, middle), and MCF-7 AdVp3000 (R482T, bottom) MXR/BCRP/ABCP forms. Login to comment
103 However, the MCF-7 AdVp3000 cells with the R482T mutation were more resistant to adriamycin than the R482 wild type (P ϭ 0.001), whereas S1-M180 cells with the R482G mutation were also more resistant to adriamycin (P ϭ 0.004) but appeared to be less resistant to topotecan and SN-38. Login to comment
112 The absence of rhodamine and doxorubicin efflux is a sharp contrast from the phenotype observed in S1-M1-80 cells, with an R482G mutation, and in MCF-7 AdVp cells with an R482T mutation. Login to comment
116 When transmembrane domains for MXR/BCRP/ABCP were identified using the TMpred program, the third transmembrane domain was predicted to span amino acids 483-499 for wild-type MXR/ BCRP/ABCP (R482) but was predicted to span amino acids 478-497 for the R482G mutation and 478-499 for the R482T mutation. Login to comment
132 Four-day cytotoxicity assays were performed on the MCF-7 MX100 (R482), MCF-7 AdVp3000 (R482T), and S1-M1-80 (R482G) cells with topotecan, mitoxantrone, adriamycin, and SN-38. Login to comment
6 A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/ BCRP/ABCP forms transported mitoxantrone. Login to comment
77 C, electropherograms for MCF-7 MX100 (R482, top), S1-M1-80 (R482G, middle), and MCF-7 AdVp3000 (R482T, bottom) MXR/BCRP/ABCP forms. Login to comment
102 However, the MCF-7 AdVp3000 cells with the R482T mutation were more resistant to adriamycin than the R482 wild type (P ϭ 0.001), whereas S1-M180 cells with the R482G mutation were also more resistant to adriamycin (P ϭ 0.004) but appeared to be less resistant to topotecan and SN-38. Login to comment
111 The absence of rhodamine and doxorubicin efflux is a sharp contrast from the phenotype observed in S1-M1-80 cells, with an R482G mutation, and in MCF-7 AdVp cells with an R482T mutation. Login to comment
115 When transmembrane domains for MXR/BCRP/ABCP were identified using the TMpred program, the third transmembrane domain was predicted to span amino acids 483-499 for wild-type MXR/ BCRP/ABCP (R482) but was predicted to span amino acids 478-497 for the R482G mutation and 478-499 for the R482T mutation. Login to comment
131 Four-day cytotoxicity assays were performed on the MCF-7 MX100 (R482), MCF-7 AdVp3000 (R482T), and S1-M1-80 (R482G) cells with topotecan, mitoxantrone, adriamycin, and SN-38. Login to comment

[hide] The role of half-transporters in multidrug resista... J Bioenerg Biomembr. 2001 Dec;33(6):503-11. Bates SE, Robey R, Miyake K, Rao K, Ross DD, Litman T
The role of half-transporters in multidrug resistance.
J Bioenerg Biomembr. 2001 Dec;33(6):503-11., [PMID:11804192]

Abstract [show]
ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.

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No. Sentence Comment
163 NOTE ADDED IN PROOF Recent studies have shown that Mutations Resulting in an altered amino acid at position 482: R482G and R482T in S1-M1-80 and MCF-7AdVp3000, respectively, result in altered substrate specificity. Login to comment

[hide] A mutation hot spot in the Bcrp1 (Abcg2) multidrug... Cancer Res. 2002 Apr 15;62(8):2294-9. Allen JD, Jackson SC, Schinkel AH
A mutation hot spot in the Bcrp1 (Abcg2) multidrug transporter in mouse cell lines selected for Doxorubicin resistance.
Cancer Res. 2002 Apr 15;62(8):2294-9., 2002-04-15 [PMID:11956086]

Abstract [show]
The recent identification of mutations at arginine 482 (R482) in human Breast Cancer Resistance Protein (BCRP) in two drug-selected cell lines largely explains some discrepancies observed in the cross-resistance profiles of human cell lines overexpressing this multidrug transporter. We find that each of three mouse cell lines independently selected for resistance to the anthracycline doxorubicin also acquired mutations in the cognate mouse transporter Bcrp1 exclusively at R482. Although the mouse Bcrp1 amino acid substitutions (M or S) are distinct from those seen in the human cell lines (G or T), they all have similar consequences: (a) greater resistance to anthracyclines (and bisantrene); (b) relatively lower resistance to topotecan; (c) greatly enhanced efflux of the dye rhodamine 123. The ready selection of R482X mutations seen in vitro might also occur in tumors treated with anthracyclines. Thus, it is noteworthy that the efficacy of Bcrp1 inhibitors applicable in vivo was not markedly affected by the presence of the mutations. We found that the Bcrp1 mutations all occurred after previous amplification and overexpression of the wild-type gene under doxorubicin selection; wild-type Bcrp1 is evidently able to mediate substantial resistance to anthracyclines, and this was confirmed in Bcrp1-transduced cell lines. These observations emphasize the general importance of the arginine at amino acid 482 for substrate specificity of the transporter, while reminding us that unmutated Bcrp1 remains a potential source of resistance to anthracyclines and a potential factor in anthracycline pharmacokinetics. The same is most likely true of human BCRP, given its profound similarities to mouse Bcrp1.

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104 The identification of R482T and R482G mutations in human BCRP in highly drug-selected human cell lines (12, 13), which increase resistance to anthracyclines, suggested that the doxorubicin-selected mouse lines might harbor similar mutations. Login to comment
149 Resistance to topotecan was at least 10-fold lower, relative to the anthracyclines and bisantrene, in the 88.6-derived R482M and R482S mutant lines, as was found for the human R482G mutant (13). Login to comment
150 In this context it is also noteworthy that cell lines carrying either the R482M or R482S Bcrp1 mutants showed greatly reduced (and Ko143-reversible) accumulation of the dye rhodamine 123 (Fig. 3C), as was observed previously for the R482G and R482T mutants of human BCRP (13). Login to comment
161 Likewise, these mutations resulted in higher resistance to doxorubicin, possibly lower resistance to topotecan in the R482G mutant, and the capacity to efflux rhodamine 123. Login to comment
102 The identification of R482T and R482G mutations in human BCRP in highly drug-selected human cell lines (12, 13), which increase resistance to anthracyclines, suggested that the doxorubicin-selected mouse lines might harbor similar mutations. Login to comment
147 Resistance to topotecan was at least 10-fold lower, relative to the anthracyclines and bisantrene, in the 88.6-derived R482M and R482S mutant lines, as was found for the human R482G mutant (13). Login to comment
148 In this context it is also noteworthy that cell lines carrying either the R482M or R482S Bcrp1 mutants showed greatly reduced (and Ko143-reversible) accumulation of the dye rhodamine 123 (Fig. 3C), as was observed previously for the R482G and R482T mutants of human BCRP (13). Login to comment
159 Likewise, these mutations resulted in higher resistance to doxorubicin, possibly lower resistance to topotecan in the R482G mutant, and the capacity to efflux rhodamine 123. Login to comment

[hide] Overexpression of wild-type breast cancer resistan... Cancer Res. 2002 Sep 1;62(17):5035-40. Volk EL, Farley KM, Wu Y, Li F, Robey RW, Schneider E
Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance.
Cancer Res. 2002 Sep 1;62(17):5035-40., 2002-09-01 [PMID:12208758]

Abstract [show]
Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.

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No. Sentence Comment
7 In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Login to comment
183 Additionally, we found that the R482T and R482G mutations in BCRP abolish MTX resistance. Login to comment
6 In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Login to comment
182 Additionally, we found that the R482T and R482G mutations in BCRP abolish MTX resistance. Login to comment

[hide] Characterization of drug transport, ATP hydrolysis... J Biol Chem. 2002 Dec 13;277(50):47980-90. Epub 2002 Oct 8. Ozvegy C, Varadi A, Sarkadi B
Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation.
J Biol Chem. 2002 Dec 13;277(50):47980-90. Epub 2002 Oct 8., 2002-12-13 [PMID:12374800]

Abstract [show]
The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.

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No. Sentence Comment
1 An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. Login to comment
5 The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. Login to comment
7 A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. Login to comment
9 Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. Login to comment
38 Previously, we demonstrated that the ABCG2-R482G multidrug transporter could also be expressed in insect cells, allowing the investigation of its membrane ATPase activity (18). Login to comment
43 We generated and expressed the human wild-type ABCG2 and its variants R482G and R482T in Sf9 cells, and we characterized their transport and ATP hydrolytic activity. Login to comment
44 As a control, we mutated a crucial amino acid in the catalytic center of ABCG2-R482G (Lys-86 was changed to Met) in hope of producing a non-functional transporter. Login to comment
59 Generation of Transfer Vector Possessing Different Human ABCG2 cDNAs-pAcUW21-L/ABCG2 (R482G) was constructed as described (18). Login to comment
60 Wild-type ABCG2 (Arg-482) and its variants (R482T and K86M) were created using ABCG2-R482G cDNA as a template (4) by overlap extension PCR (30). Login to comment
64 The PCR products containing the Arg-482 or R482T coding sequence were digested with PstI and MscI enzymes and ligated between the corresponding sites of the pAcUW21-L/ABCG2 vector. The PCR product coding for the K86M variant was digested with NotI and SpeI enzymes and ligated to the NotI and SpeI sites of the pAcUW21-L/ABCG2 (R482G) vector. The mutations were confirmed by sequencing the PstI-MscI or the NotI-SpeI fragments of the constructs, respectively. Login to comment
108 In the present study we utilized this expression system to produce the wild-type human ABCG2 protein and its variants, R482G and R482T, as well as a catalytic center mutant, K86M. Login to comment
125 We found equal expression levels for the wild-type, R482G, R482T, and K86M/ R482G mutant variants (data not shown). Login to comment
132 Lane 1, R482T/Sf9; lane 2, wild-type ABCG2/Sf9; lane 3, R482G/Sf9; lane 4, K86M-R482G/Sf9; lane 5, wild-type ABCG2/ HL60; lane 6, wild-type ABCG2/HL60 grown in the presence of 2.5 ␮g/ml tunicamycin; and lane 7, beta-galactosidase/Sf9. Login to comment
140 These experiments indicate that wtABCG2 and the R482G or R482T variants actively extrude MX in the intact Sf9 cells, whereas the K86M mutant is inactive in this respect. Login to comment
144 As documented, at increasing MX concentrations, the wild-type (R482) ABCG2 was found to be somewhat less effective in protecting Sf9 cells against MX accumulation than the other two amino acid 482 variants; the accumulation of MX was about 15 Ϯ 4% more in wtABCG2- expressing cells than in cells containing the R482G or R482T variants. Login to comment
145 In addition, the MX transport capacities of the amino acid 482 variants R482G and R482T were found to be similar in these experiments. Login to comment
146 Next we performed similar transport studies for the fluorescent dye rhodamine 123, which was indicated to be a transported substrate of the ABCG2 variants R482G and R482T (20). Login to comment
147 As shown in Fig. 3, we found that insect cells expressing the wild-type ABCG2 or the K86M mutant accumulated significantly more rhodamine 123 than cells expressing the R482G or R482T variants. Login to comment
148 In contrast to that found for the wild-type ABCG2, in the R482G or R482T variants the addition of FTC greatly increased rhodamine 123 accumulation indicating an ABCG2-dependent extrusion of this compound. Login to comment
149 These data support the fact that MX is a substrate for wtABCG2 and its amino acid 482 variants, although MX transport may be less efficient by the wild-type protein than by the R482G or R482T variants. Login to comment
150 In contrast, the wild-type ABCG2 is practically inactive for rhodamine 123 transport, whereas the R482G and R482T mutants actively extrude this fluorescent dye. Login to comment
158 Fig. 4A shows a typical Hst dye uptake experiment using intact Sf9 cells harvested post-40 h of recombinant baculovirus infection, expressing either ABCG2-R482G or its K86M mutant variant. Login to comment
161 As documented, Hst dye uptake is significantly slower in the Sf9 cells that express the ABCG2-R482G protein (Fig. 4A, line A) than in cells expressing the K86M/R482G mutant (line B). Login to comment
163 The rate of Hst dye uptake in cells expressing ABCG2-R482G (Fig. 4A, line A) greatly increases upon the addition of the specific inhibitor FTC (or Ko 143, not shown). Login to comment
164 However, there is no change in the rate of Hst accumulation in cells expressing the K86M/R482G mutant (Fig. 4A, line B) or beta-galactosidase (not shown). Login to comment
167 Fig. 4, B and C, shows that the rate of Hst influx was low and was greatly increased by the inhibitor FTC in the wild-type ABCG2 (Fig. 4B) and also in the R482G and R482T variants (Fig. 4, B and C). Login to comment
172 We have shown earlier (18) that ABCG2-R482G expressed in Sf9 cells had a high level of vanadate-sensitive membrane ATPase activity, and this activity could be significantly increased by substrates and decreased by the inhibitors of this protein. Login to comment
173 In the present study we have compared the ATPase activity of the wild-type human ABCG2 and its variants (R482G, R482T, and K86M) in the presence and absence of a variety of potential ABCG2 substrates or inhibitors. Login to comment
174 As shown in Fig. 5A, the basal, vanadate-sensitive ATPase activity was significantly higher in membranes containing any of the wtABCG2 or its amino acid 482 variants than in those containing ABCG2-K86M/R482G or beta-galactosidase (not shown here). Login to comment
175 Despite the similar ABCG2 expression levels, this basal ATPase activity was ϳ1.5-fold higher in case of the R482G variant (71 Ϯ 10.8 nmol of Pi/mg of membrane protein/ min) than in case of the wild-type ABCG2 or the R482T variant (45 Ϯ 8.85 and 46 Ϯ 9.04 nmol of Pi/mg of membrane protein/ min, respectively) and negligible in the ABCG2-K86M mutant (5 Ϯ 0.5 nmol of Pi/mg of membrane protein/min). Login to comment
177 In wtABCG2 and in R482G and R482T proteins FTC (or Ko 143) powerfully inhibited the vanadate-sensitive ATPase activity. Login to comment
179 In case of the R482G and R482T mutants, vanadate-sensitive ATPase activity could be greatly stimulated by several potential ABCG2 substrates (e.g. prazosin, mitoxantrone, adriamycin, rhodamine 123, benzamil, and camptothecin), corresponding to the transport activity of these proteins. Login to comment
183 We found that the effect of different ABCG2 substrates on the ATPase activity of variants R482G and R482T was almost similar. Login to comment
186 Rhodamine 123, a specific substrate of the amino acid 482 mutants, gave a maximum of 30% stimulation, and the Kact values were 4.5 and 4 ␮M, respectively, for the R482G and R482T mutants. Login to comment
187 The maximum extent of stimulation of the R482G and R482T-ATPases by prazosin was 100 and 70%, and the Kact values were 7 and 3 ␮M. Login to comment
188 Mitoxantrone showed a maximum of 50% stimulation of the ATPase activity of the R482G and R482T variants, with Kact values of 1 and 0.8 ␮M, respectively. Login to comment
189 FTC was found to be an effective inhibitor for the vanadate-sensitive ABCG2-ATPase activity both for the wild-type enzyme (76% inhibition at 10 ␮M) and the R482G variant (74% inhibition at 10 ␮M); however, its inhibitory effect was smaller and required higher FTC concentrations in the case of the R482T variant (37% inhibition at 10 ␮M) (see Fig. 5). Login to comment
190 The concentration of FTC causing half-maximal inhibition of the ABCG2-ATPases was 0.4 ␮M for the wild-type enzyme, 0.5 ␮M for the R482G variant, and 0.7 ␮M for the R482T mutant. Login to comment
192 A, MX accumulation in Sf9 cells expressing beta-galactosidase (beta-gal), wild-type ABCG2 (Arg-482), and the R482G, R482T, or K86M mutants. Login to comment
202 We found no significant effect of any of these conditions on the ATPase activity of the wild-type ABCG2 or the R482G variant; neither their basal activity nor the effects of drugs were changed. Login to comment
207 In the presence of 3.1 mM Mg-8-azido-ATP, the vanadate-sensitive ATPase activity of ABCG2-R482G was 77% compared with the activity measured in the presence of a similar concentration of MgATP (data not shown). Login to comment
214 We found that 8-azido-ATP binding was similar in the wild-type and in the R482G, R482T, and K86M mutant variants under these conditions (Fig. 6B). Login to comment
218 As shown in Fig. 7B, in the presence of 1 mM vanadate and 5 ␮M Co-8-azido-[␣-32 P]ATP, ABCG2 (R482G) showed a high level of nucleotide trapping (lane 3). Login to comment
221 As documented in Fig. 7B, the functionally inactive ABCG2-K86M/R482G did not show any nucleotide trapping activity under these conditions (lane 5). Login to comment
224 Rhodamine 123 accumulation in Sf9 cells expressing the wild-type (Arg-482), R482G, R482T, or K86M/R482G variants of ABCG2. Login to comment
228 As shown in Fig. 8, we found significant differences in the adenine nucleotide trapping of the wild-type, R482G, and R482T ABCG2 transporters. Login to comment
229 When we analyzed these data with a quantitative PhosphorImager, in the absence of added drugs the wild-type ABCG2 (Fig. 8, lane 4) showed a more pronounced nucleotide trapping activity, 2.6 Ϯ 0.03- and 2.05 Ϯ 0.5-fold of the R482G and R482T variants (lanes 7 and 10), respectively. Login to comment
230 The addition of prazosin, an activator of the ABCG2-ATPase of the R482G and R482T variants, significantly stimulated nucleotide trapping in the same variants (see Fig. 8, lanes 6 and 9, the stimulations were 2.0 Ϯ 0.22- and 1.7 Ϯ 0.04-fold, respectively). Login to comment
237 DISCUSSION In this paper we describe the expression and detailed functional analysis of the wild-type human ABCG2 multidrug transporter and its mutant variants R482G, R482T, and K86M. Login to comment
241 According to the established sequence in the human genome data base (GenBankTM accession number AF103796), the wild-type ABCG2 contains arginine at this position, whereas the other two variants R482G and R482T most probably were generated during drug selection in tumor cell lines (20, 22). Login to comment
245 The aim of the present study was to provide a quantitative characterization both for the transport and the ATP hydrolytic activity of the wtABCG2 and its variants R482G and R482T. Login to comment
248 We introduced the K86M mutation into the R482G variant of ABCG2, because we expected that the mutation of the key Lys will abolish the function of ABCG2 regardless the amino acid found in position 482. Login to comment
254 A shows the increase in fluorescence due to the uptake of 2.5 ␮M Hst in ABCG2-R482G- (line A) or ABCG2-K86M/R482G (line B) -expressing Sf9 cells. Login to comment
255 B and C, the rate of Hst influx (⌬ fluorescence/⌬ time) into Sf9 cells expressing the wtABCG2 or the R482T mutant (B) and R482G or K86M/R482G (C) was determined at different Hst concentrations with or without the ABCG2 inhibitor FTC. Login to comment
258 ATPase activity measured in membranes of Sf9 cells expressing the wild-type, R482G, R482T, or K86M/R482G variants of human ABCG2. Login to comment
263 ATPase activity determined in membranes of wtABCG2- (B), R482G- (C), or R482T (D)-expressing Sf9 cells. Login to comment
268 On the other hand the R482G variant could be easily investigated in all four assays as described under "Results." Login to comment
273 Comparing the transport activity of the wtABCG2 and its mutant variants, we found that the wild-type and the two amino acid 482 variants actively exported mitoxantrone, whereas rhodamine 123 extrusion could only be observed in cells expressing the R482G and R482T proteins. Login to comment
279 We have already described a high level, vanadate-sensitive ATPase activity, stimulated by several transported compounds, for the human ABCG2-R482G variant in isolated Sf9 cell membranes (18). Login to comment
281 However, stimulation of the ABCG2-ATPase activity was observed only in Sf9 membranes containing the R482G or R482T variants and not the wild-type ABCG2. Login to comment
283 [␣-32 P]8-azido-ATP binding of the wild-type and R482G, R482T, or K86M/R482G mutant ABCG2 proteins. Login to comment
290 [␣-32 P]8-azido-ATP trapping of the R482G and K86M/ R482G mutant ABCG2 proteins. Login to comment
291 Sf9 membranes containing ABCG2R482G (see A, lane 2, and B, lanes 1-3), ABCG2-K86M (B, lane 5), or beta-galactosidase (beta-gal) (see A, lane 1 and B, lane 4) were incubated for 5 min at 37 °C with 5 ␮M 8-azido-[␣-32 P]ATP, 1 mM sodium orthovanadate (except for lane 1) and 2 mM Mg2ϩ (A) or 2 mM Co2ϩ (B) as described under "Experimental Procedures." Login to comment
296 Comparison of the effect of different compounds on the [␣-32 P]8-azido-ATP trapping of the wild-type, and R482G or R482T variants of the ABCG2 protein. Login to comment
297 Sf9 membranes (150 ␮g) containing wtABCG2 (lanes 2-4), ABCG2-R482G (lanes 5-7), ABCG2-R482T (lanes 8-10), or beta-galactosidase (lane 1) were incubated for 2 min at 37 °C with 2 ␮M 8-azido-[␣-32 P]ATP, 1 mM sodium orthovanadate, and 2 mM Co2ϩ as described under "Experimental Procedures." Login to comment
309 In case of the R482G and R482T variants, this endogenous stimulation (caused by endogenous activators, producing what we observe as a high base-line ATPase) is only partial, and further stimulation by the transported compounds is observed. Login to comment
325 We found that the nucleotide trapping characteristics of the R482G or R482T variants were different from the wild-type ABCG2. Login to comment
326 Whereas this transition state formation in variants R482G and R482T could be significantly stimulated by various ABCG2 substrates (prazosin and mitoxantrone), the transition state formation of the wild-type ABCG2 could not be stimulated but rather was inhibited by these compounds. Login to comment

[hide] Breast cancer resistance protein (BCRP/ABCG2) indu... Mol Pharmacol. 2003 Jan;63(1):65-72. Wang X, Furukawa T, Nitanda T, Okamoto M, Sugimoto Y, Akiyama S, Baba M
Breast cancer resistance protein (BCRP/ABCG2) induces cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors.
Mol Pharmacol. 2003 Jan;63(1):65-72., [PMID:12488537]

Abstract [show]
Breast cancer resistance protein (BCRP/ABCG2) is a novel member of ATP- binding cassette transporters, which induce multidrug resistance in cancer cells. We found that a high level of BCRP expression in CD4+ T cells conferred cellular resistance to human immunodeficiency virus type-1 (HIV-1) nucleoside reverse transcriptase inhibitors. The cell line MT-4/DOX 500 was established through the long-term culture of MT-4 cells in the presence of doxorubicin (DOX) and had reduced sensitivity to not only DOX but also zidovudine (AZT). MT-4/DOX 500 cells showed reduced intracellular accumulation and retention of DOX and increased ATP-dependent rhodamine 123 efflux. The cells were also resistant to several anticancer agents such as mitoxantrone, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin, and 7-ethyl-10-hydroxycamptothecin. AZT was 7.5-fold less inhibitory to HIV-1 replication in MT-4/DOX 500 cells than in MT-4 cells. Furthermore, the anti-HIV-1 activity of lamivudine was severely impaired in MT-4/DOX 500 cells. In contrast, the antiviral activity of non-nucleoside reverse transcriptase inhibitors and protease inhibitors was not affected in the cells. MT-4/DOX 500 cells expressed glycosylated BCRP but not P-glycoprotein (ABCB1), multidrug resistance protein 1, 2, or 4 (ABCC1, -2, or -4), or lung resistance-related protein. In addition, the BCRP-specific inhibitor fumitremorgin C completely abolished the resistance of MT-4/DOX 500 cells to AZT as well as to DOX. An analysis for intracellular metabolism of AZT suggests that the resistance is attributed to the increase of ATP-dependent efflux of its metabolites, presumably AZT 5'-monophosphate, in MT-4/DOX 500 cells.

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193 Increased efflux of doxorubicin and rhodamine 123 was observed with the R482T or R482G mutant but not with the wild type of BCRP (Honjo et al., 2001). Login to comment
205 Although this mutation differed from those observed previously in doxorubicin-resistant human cell lines (R482T or R482G), it could not be excluded that the NRTI resistance of MT-4/ DOX500 cells was caused by the mutation but not by the increased expression of BCRP. Login to comment

[hide] Mammalian drug efflux transporters of the ATP bind... Adv Drug Deliv Rev. 2003 Jan 21;55(1):3-29. Schinkel AH, Jonker JW
Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview.
Adv Drug Deliv Rev. 2003 Jan 21;55(1):3-29., 2003-01-21 [PMID:12535572]

Abstract [show]
Active drug efflux transporters of the ATP binding cassette (ABC)-containing family of proteins have a major impact on the pharmacological behavior of most of the drugs in use today. Pharmacological properties affected by ABC transporters include the oral bioavailability, hepatobiliary, direct intestinal, and urinary excretion of drugs and drug-metabolites and -conjugates. Moreover, the penetration of drugs into a range of important pharmacological sanctuaries, such as brain, testis, and fetus, and the penetration into specific cell- and tissue compartments can be extensively limited by ABC transporters. These interactions with ABC transporters determine to a large extent the clinical usefulness, side effects and toxicity risks of drugs. Many other xenotoxins, (pre-)carcinogens and endogenous compounds are also influenced by the ABC transporters, with corresponding consequences for the well-being of the individual. We aim to provide an overview of properties of the mammalian ABC transporters known to mediate significant transport of clinically relevant drugs.

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387 We recently found that Ko134 has R482T and R482G mutants efficiently extruded all low cytotoxicity in vitro and that it can be given at three compounds [14]. Login to comment

[hide] Natural allelic variants of breast cancer resistan... Pharmacogenetics. 2003 Jan;13(1):19-28. Zamber CP, Lamba JK, Yasuda K, Farnum J, Thummel K, Schuetz JD, Schuetz EG
Natural allelic variants of breast cancer resistance protein (BCRP) and their relationship to BCRP expression in human intestine.
Pharmacogenetics. 2003 Jan;13(1):19-28., [PMID:12544509]

Abstract [show]
The aim of this study was to identify the extent of genetic variability in breast cancer resistance protein (BCRP) in humans. We first analysed the sequence of BCRP cDNA from human livers and from human intestines phenotyped for expression of intestinal BCRP. We then determined the frequency of all known coding single nucleotide polymorphisms (cSNPs) using DNA from individuals representing 11 different ethnic populations. Nine SNPs including four non-synonymous and three synonymous cSNPs and two intronic SNPs were identified. Of the missense mutations, exon 2 SNP (G34A) resulted in a V12M change; exon 5 SNP (C421A) resulted in a Q141K substitution; exon 6 SNP (A616C) resulted in an I206L amino acid substitution; and exon 15 SNP (A1768T) resulted in a N590Y change in the BCRP protein. The two most frequent polymorphisms identified in the human population studied were the G34A and C421A transitions. There was marked variation in BCRP genotypes and allele frequencies in the different populations. BCRP mRNA was phenotyped in human small bowel intestinal samples by real-time polymerase chain reaction and BCRP protein was analysed on immunoblots of tissue from the same individuals. There was a 78-fold variation in expression of BCRP mRNA and significant variation in BCRP protein expression in human intestine. Expression of intestinal BCRP mRNA and protein was not different between persons expressing the common Gln141 allele compared to the Lys141 allele. Thus, common natural allelic variants of BCRP have been identified, and did not influence interindividual variation in expression of BCRP mRNA in human intestine, but remain to be tested for their effect on BCRP function.

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125 Unauthorized reproduction of this article is prohibited. G34A V12M Exon 2 C71T1 A24V Exon 2 623C1 F208S Exon 6 A616C I206L Exon 6 C496G1 Q166E Exon 5 C421A Q141K Exon 5 A1444G2 R482G Exon 12 G1445C3 R482T Exon 12 A1768T N590Y Exon 15 Walker A motif: amino acids 80-89 Walker B motif: amino acids 206-210 SNPs found in human samples in this study Reported in ABCP1 Drug selected variants, MXR2 and BCRP3 MXR BCRP Fig. 1 BCRP protein topology and the positions of the identified SNPs resulting in missense mutations. Login to comment

[hide] Application of a human multidrug transporter (ABCG... Hum Gene Ther. 2003 Mar 1;14(4):403-12. Ujhelly O, Ozvegy C, Varady G, Cervenak J, Homolya L, Grez M, Scheffer G, Roos D, Bates SE, Varadi A, Sarkadi B, Nemet K
Application of a human multidrug transporter (ABCG2) variant as selectable marker in gene transfer to progenitor cells.
Hum Gene Ther. 2003 Mar 1;14(4):403-12., 2003-03-01 [PMID:12659681]

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4 In the present study a mutant form of ABCG2 (R482G), showing drug-pumping activity with an altered substrate specificity, was coexpressed with a therapeutic gene by using a bicistronic vector and an efficient retroviraltransduction protocol. Login to comment
17 ABCG2 is normally expressed in the placenta and in stem cells (Zhou et al., 2001; Bunting,2002; Kim et al., 2002).It has been shown that the R482G variant of ABCG2 has a different substrate specificity than the wild-type protein (Honjo et al., 2001; Öz- 1National Medical Center, Institute of Hematology and Immunology, 1113 Budapest, Hungary. Login to comment
37 The NotI-BamHI fragment from the coding sequence of the R482G mutant MXR cDNA was used to replace the previously used LNGFR cDNA. Login to comment
39 In a monocistronicABCG2 vector, LNGFR cDNA was replaced by the cDNAs encoding the R482 (SsARS) and R482G (SsAGS) variants of ABCG2, and the gp91phox cDNA was also removed. Login to comment
82 RESULTS AND DISCUSSION Coexpression of gp91phox and ABCG2 in progenitor cells: effect of drug selection To produce recombinant retroviruses,a bicistronic retroviral vector was constructed, carrying the coding sequences for gp91phox and those for the normal ABCG2, or the R482G variant (denoted as ABCG2-G). Login to comment
145 Selective protection of the transduced cells against cytotoxic drugs by the mutant ABCG2 protein In the above-describedexperiments we used an R482G variant of the ABCG2 protein as a selectable marker. Login to comment
147 It has been reported that the R482G and R482T mutant variants of ABCG2 have altered substrate specificity. Login to comment
149 To investigate the function and expression of these variants, we generated cDNAs encoding both the wild-type and R482G ABCG2 proteins, and inserted each of these cDNAs into the retroviral vector presented above (see Fig. 1). Login to comment
153 As documented in Fig. 5, after retroviral transduction and selection for 4 days in increasing concentrations of MX in the medium, PLB985 cells expressed both the wild-type ABCG2 and the R482G variant of ABCG2 in a functionallyactive form. Login to comment
176 of Western blot lanes (using actin as an internal protein control), PLB985 cells transduced with wild-type ABCG2 and selected in 10 nM MX (Fig. 5A, lane 4) expressed a similar (according to PhosphorImager evaluation, 1.6 times higher) level of ABCG2 protein than did corresponding cells expressing the R482G variant and selected in 100 nM MX (Fig. 5A, lane 5). Login to comment
188 In summary, we have demonstrated that overexpression of R482G ABCG2 protein efficiently protected genetically modified progenitor cells against cytotoxic selection, allowing increased expression of the therapeutic gene. Login to comment

[hide] Transport of methotrexate, methotrexate polyglutam... Cancer Res. 2003 Jul 15;63(14):4048-54. Chen ZS, Robey RW, Belinsky MG, Shchaveleva I, Ren XQ, Sugimoto Y, Ross DD, Bates SE, Kruh GD
Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport.
Cancer Res. 2003 Jul 15;63(14):4048-54., 2003-07-15 [PMID:12874005]

Abstract [show]
ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.

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12 By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Login to comment
29 The results of these experiments show that wild-type ABCG2 is indeed capable of mediating the transport of MTX, but that neither the R482G nor the R482T mutants are able to transport this agent to any extent. Login to comment
49 HEK293 cells transfected with ABCG2-R482, ABCG2-R482G, and ABCG2-R482T were generated using the respective cDNAs cloned into pcDNA3.4 LLC/PK1 cells transfected with MRP2 expression vector, and HEK293 cells transfected with MRP3 expression vector were described previously (19, 20). Login to comment
61 RESULTS Analysis of MTX Transport by Wild-Type ABCG2, ABCG2R482G, and ABCG2-R482T. Login to comment
68 Membrane vesicles were prepared from HEK293 cells transfected with ABCG2-R482G and ABCG2-R482T, so as to analyze both types of R482 mutations that have been identified in drug-resistant cell lines (4). Login to comment
70 However, by contrast with ABCG2-R482, neither of the mutants were able to transport [3 H]MTX to any extent, in that MgATP-dependent uptake of this agent by ABCG2-R482G and ABCG2-R482T-enriched vesicles was indistinguishable from uptake by the same vesicles in the presence of MgAMP, or uptake by negative control vesicles in the presence of either MgATP or MgAMP (Fig. 2, B and C). Login to comment
82 Membrane vesicles were prepared from HEK293 cells transfected with parental vector (Lane 1), ABCG2-R482 (Lane 2), ABCG2-R482G (Lane 3), and ABCG2-R482T (Lane 4). Login to comment
99 We reported previously that several members of the MRP family that are capable of transporting MTX are also competent in mediating the Fig. 2. Time course of ATP-dependent uptake of [3 H]MTX by ABCG2-R482, ABCG2-R482G, and ABCG2-R482T. Login to comment
100 Membrane vesicles (10 ␮g) prepared from HEK293 cells transfected with ABCG2-R482 (A), ABCG2-R482G (B), and ABCG2-R482T (C), and from parental plasmid-transfected control cells, were incubated at 37°C in uptake medium containing 100 ␮M [3 H]MTX and 4 mM MgATP (solid symbols) or 4 mM MgAMP (open symbols). Login to comment
107 The results of experiments in which uptake of FA was examined were in complete accord with the properties of ABCG2 as determined from the MTX transport experiments, in that whereas membrane vesicles prepared from wild-type ABCG2-transfected cells were able to catalyze the uptake of 100 ␮M [3 H]FA, uptake was not detected for R482G or R482T (Fig. 6, A-C). Login to comment
143 Membrane vesicles (10 ␮g) prepared from HEK293 cells transfected with ABCG2-R482 (A and D), ABCG2-R482G (B), ABCG2-R482T (C), and MRP3 (E), from LLC/PK1 cells transfected with MRP2 (F), and from the respective parental plasmid-transfected counterparts, were incubated at 37°C in uptake medium containing 100 ␮M [3 H]FA (A-C) or 100 ␮M [3 H]leucovorin (D-F), and 4 mM MgATP (solid symbols) or 4 mM MgAMP (open symbols). Login to comment

[hide] Quantitative analysis of breast cancer resistance ... Clin Cancer Res. 2003 Aug 15;9(9):3320-8. Nakanishi T, Karp JE, Tan M, Doyle LA, Peters T, Yang W, Wei D, Ross DD
Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients.
Clin Cancer Res. 2003 Aug 15;9(9):3320-8., 2003-08-15 [PMID:12960118]

Abstract [show]
PURPOSE: Flavopiridol is a cyclin-dependent kinase inhibitor currently undergoing human clinical trials. As clinical development is pursued, it becomes important to evaluate resistance mechanisms to flavopiridol. To elucidate the contribution of breast cancer resistance protein (BCRP) to cellular resistance to flavopiridol in acute myeloid leukemia, we studied the relationship between cellular resistance to flavopiridol and mRNA expression of BCRP or P-glycoprotein (P-gp, product of MDR1gene) in blast cells from adult patients with acute leukemia. Experimental Design: Twenty-one blast cell samples from 20 patients were studied. The expression of BCRP, P-gp, or beta-actin mRNA was determined by real-time reverse transcription-PCR, using fluorescent hybridization probes to evaluate codon 482, a known site of mutations in BCRP mRNA. In vitro cell viability and apoptosis were examined after 24 h exposure to flavopiridol. RESULTS: BCRP mRNA expression varied over a 200-fold range. In the blast cell samples with BCRP mRNA expression > 10000 copies/pg beta-actin (n = 9), BCRP mRNA correlated proportionally with cell viability in the presence of 250 nM flavopiridol (r = 0.86, P = 0.003) and with apoptosis induced by flavopiridol (r = 0.71, P = 0.031). In contrast, MDR1mRNA expression did not correlate with either flavopiridol cytotoxicity or induction of apoptosis. Melting point analysis of the hybridization probes determined that all 21 patient samples had arginine at codon 482 of BCRP mRNA, the wild-type form. CONCLUSIONS: These results suggest that unlike P-gp, BCRP may play a role in leukemia cellular resistance to flavopiridol. No mutations at codon 482 were observed in BCRP mRNA in this group of patients.

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50 The codon 482 mutations observed have substitutions of threonine (R482T) or glycine (R482G), which results in the ability of the mutant protein to transport anthracyclines and rhodamine 123 but loss of ability to transport methotrexate (29-31). Login to comment
218 Recently, the R482T and R482G mutations of BCRP have been described. Login to comment
220 Additionally, a recent study (31) indicates that the R482T and R482G mutations result in loss of the ability to transport methotrexate. Login to comment

[hide] Wild-type breast cancer resistance protein (BCRP/A... Cancer Res. 2003 Sep 1;63(17):5538-43. Volk EL, Schneider E
Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter.
Cancer Res. 2003 Sep 1;63(17):5538-43., 2003-09-01 [PMID:14500392]

Abstract [show]
The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown. We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002). Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918. Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system. Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP. In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C. In contrast, no transport was observed in vesicles containing the mutant form of BCRP. Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min. MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport. Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX. Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP. Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.

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29 Together, these data suggested that BCRP can function as a MTX transporter. However, resistance to MTX occurred only in the presence of the wild-type transporter, which contains an arginine at position 482, whereas cells that overexpress a mutated BCRP (R482T and R482G) remained sensitive to MTX. Login to comment
171 In contrast, no uptake of MTX was observed into vesicles containing BCRP with an R482G mutation. Login to comment
174 Thus, it appears that the R482G mutation resulted in a dramatic change in substrate specificity for BCRP. Login to comment

[hide] Single amino acid substitutions in the transmembra... Int J Cancer. 2003 Dec 10;107(5):757-63. Miwa M, Tsukahara S, Ishikawa E, Asada S, Imai Y, Sugimoto Y
Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants.
Int J Cancer. 2003 Dec 10;107(5):757-63., 2003-12-10 [PMID:14566825]

Abstract [show]
Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette transporters that has an N-terminal ATP binding domain and a C-terminal transmembrane domain (TM). Expression of wild-type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN-38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446-mutants in TM2, 15 R482-mutants in TM3, 4 N557-mutants in TM5 and 6 H630-mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446-mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2-BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild-type BCRP-transfected cells. Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482-, N557- or H630-mutant BCRP cDNA. Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild-type BCRP-transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.

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8 Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3 H]mitoxantrone than the wild-type BCRP-transfected cells. Login to comment
13 A doxorubicin-resistant human breast cancer cell line MCF-7 AdVp3000 and a mitoxantrone-resistant human colon carcinoma cell line S1-M1-80 expressed R482T- and R482G-BCRP, respectively and showed high resistance to mitoxantrone and doxorubicin.5,6,13 Doxorubicin-resistant murine fibroblast cell lines also expressed R482M- or R482S-BCRP and showed high resistance to mitoxantrone and doxorubicin.14 We recently identified the substitution R482M in a doxorubicin-resistant human T cell line MT-4/DOX500.23 We made 32 mutant BCRP cDNAs with an amino acid substitution in the TMs and examined the effect of the substitutions on cellular drug resistance. Login to comment
64 PA/R482N, PA/R482C, PA/R482M, PA/R482S, PA/R482T, PA/R482V, PA/R482A, PA/R482G, PA/R482E PA/R482W and PA/R482D (Group 2) showed higher degrees of resistance to mitoxantrone than to SN-38. Login to comment
76 As shown in Figure 3, E446G-, R482G-, N557D- and H630L-BCRP were expressed on the cell surface. Login to comment
77 Expression levels of BCRP in PA/ WT2, PA/E446G and PA/R482G were similar. This suggests that trafficking problem is not a main reason for the loss of BCRP function in E446-mutants. Login to comment
78 Anthracycline resistance of R482-mutant BCRP transfectants Resistance to anthracycline antibiotics of PA/R482G and PA/ R482S, typical members of Group 2 transfectants, was examined (Fig. 4). Login to comment
80 PA/R482G and PA/R482S showed resistance to doxorubicin, epirubicin and daunorubicin, but not to aclarubicin (Fig. 4). Login to comment
84 Estrone is the first physiological substrate that was shown to circumvents BCRP-mediated drug resistance.17 As shown in Table I, estrone effectively reversed mitoxantrone resistance and SN-38 resistance of PA/R482G, PA/R482S, PA/N557H, PA/N557D, PA/H630E and PA/H630L. Login to comment
85 Estrone also reversed doxorubicin resistance of PA/ R482G and PA/R482S (Table II). Login to comment
86 Similarly, fumitremorgin C, a well-known BCRP inhibitor, strongly reversed the mitoxantrone resistance and SN-38 resistance of PA/R482G, PA/R482S, PA/ N557H, PA/N557D, PA/H630E and PA/H630L (Table III). Login to comment
88 Uptake of [3 H]mitoxantrone To confirm that the high mitoxantrone resistance of PA/R482G and PA/R482S is attributable to decreased drug accumulation, the uptake of [3 H]mitoxantrone in PA317, PA/WT2, PA/R482G and PA/R482S cells was examined. As shown in Figure 6, intracellular accumulation of [3 H]mitoxantrone was significantly lower in PA/ R482G and PA/R482S than in PA/WT2. Login to comment
89 The [3 H]mitoxantrone uptake reached plateau levels in 4 hr. The results clearly showed that steady-state levels of mitoxantrone accumulation in PA/ R482S and R482G were lower than that in PA/WT2 (Fig. 6). Login to comment
117 Mitoxantrone uptake in PA317 cells was 10-fold higher than that in PA/R482S or PA/R482G cells. Login to comment
119 PA/R482S and PA/R482G cells show approximately 100-200-fold more resistance to mitoxantrone than the parental cells. Login to comment
122 This concentration was much higher than the IC50 values of FIGURE 4 - Resistance to mitoxantrone, SN-38 and anthracyclines of R482-mutant BCRP transfectants. PA/WT2, PA/R482G and PA/ R482S were mixed populations of the transfected cells established after the 2-step selection with 120 ng/ml methotrexate and 1 ng/ml mitoxantrone. Login to comment
123 PA317 (closed circle), PA/WT2 (open triangle), PA/ R482G (open square) or PA/R482S (open lozenge) cells were cultured for 5 days with increasing concentrations of anthracyclines. Login to comment
131 PA/R482S or PA/R482G and transporter function might not be so efficient in the presence of such a high concentration of mitoxantrone. Login to comment
135 MCF-7 AdVp3000 established by treating MCF-7 cells with 3 ␮g/ml of doxorubicin and 5 ␮g/ml of verapamil overexpressed R482T-BCRP.5,13 S1-M1-80 established by treating human colon carcinoma S1 cells with 80 ␮M mitoxantrone overexpressed R482G-BCRP.6,13 These resistant cells exhibited high resistance to doxorubicin and mitoxantrone.5,6,13 We found recently that MT-4/DOX500 cells established by treating human T cell MT-4 cells with 500 ng/ml of doxorubicin overexpressed R482M-BCRP.23 Two doxorubicin-resistant murine fibroblast lines 88.6/D800-A and 88.6/D800-B overexpressed R482M-BCRP and R482S-BCRP, respectively.14 Another doxorubicin-resistant cell line KOT52/D800 from mouse fibroblasts co-expressed wild-type BCRP and R482M-BCRP.14 In addition to anthracyclines and mitoxantrone, cells transfected with R482-mutant BCRP cDNAs also showed high resistance to methotrexate.28 Theoretically, 9 FIGURE 6 - Accumulation of [3 H]mitoxantrone in R482-mutant BCRP transfectants. PA/WT2, PA/R482G and PA/R482S were mixed populations of the transfected cells established after the 2-step selection with 120 ng/ml methotrexate and 1 ng/ml mitoxantrone. Login to comment
136 PA317 (closed circle), PA/WT2 (open triangle), PA/R482S (open lozenge) or PA/R482G (open square) cells were incubated at 37°C with 50 nM [3 H]mitoxantrone. Login to comment
140 TABLE I - REVERSAL OF MITOXANTRONE RESISTANCE AND SN-38 RESISTANCE BY ESTRONE1 Cell line Degree of Mitoxantrone resistance Reversal index Degree of SN-38 resistance Reversal index Control 10 ␮M estrone Control 10 ␮M estrone PA/WT1 7.1 Ϯ 0.6 2.2 Ϯ 0.4 3.2 28 Ϯ 1 4.7 Ϯ 0.3 6.0 PA/R482S 120 Ϯ 20 6.8 Ϯ 0.4 18 37 Ϯ 2 4.4 Ϯ 1.1 8.4 PA/R482G 84 Ϯ 17 3.7 Ϯ 0.3 23 22 Ϯ 1 3.3 Ϯ 0.1 6.7 PA/N557H 3.3 Ϯ 0.1 1.0 Ϯ 0.1 3.3 4.8 Ϯ 0.5 3.4 Ϯ 0.1 1.4 PA/N557D 7.4 Ϯ 0.2 1.4 Ϯ 0.1 5.3 3.8 Ϯ 0.6 2.9 Ϯ 0.2 1.3 PA/H630E 5.8 Ϯ 0.2 1.1 Ϯ 0.1 5.3 20 Ϯ 2.8 6.0 Ϯ 0.1 3.3 PA/H630L 3.3 Ϯ 0.1 0.9 Ϯ 0.1 3.7 8.6 Ϯ 0.4 2.7 Ϯ 0.1 3.2 1 Cells were cultured in the absence or presence of 10 ␮M estrone with increasing concentrations of mitoxantrone or SN-38. Degree of resistance is the ratio of the IC50 value for BCRP-expressing cells divided by that for parental PA317. Login to comment
141 The reversal index is calculated by dividing degree of resistance without estrone by that with estrone. TABLE II - REVERSAL OF DOXORUBICIN RESISTANCE BY ESTRONE Cell line Degree of Doxorubicin resistance Reversal index Control 10 ␮M Estrone PA/WT1 1.2 Ϯ 0.1 1.0 Ϯ 0.0 1.2 PA/R482S 10 Ϯ 1 1.3 Ϯ 0.1 7.7 PA/R482G 8.0 Ϯ 0.6 1.2 Ϯ 0.1 6.7 These cells were cultured in the absence or presence of 10 ␮M estrone with increasing concentrations of doxorubicin. Login to comment
143 The reversal index is calculated by dividing degree of resistance without estrone by that with estrone. TABLE III - REVERSAL OF MITOXANTRONE RESISTANCE AND SN-38 RESISTANCE BY FUMITREMORGIN C1 Cell line Degree of Mitoxantrone resistance Reversal index Degree of SN-38 resistance Reversal index Control 3 ␮M Fumitremorgin C Control 3 ␮M Fumitremorgin C PA/WT1 11 Ϯ 1 1.0 Ϯ 0.1 11 23 Ϯ 1 1.1 Ϯ 0.1 21 PA/R482S 140 Ϯ 10 1.2 Ϯ 0.1 120 41 Ϯ 1 0.9 Ϯ 0.1 46 PA/R482G 89 Ϯ 21 0.9 Ϯ 0.1 99 17 Ϯ 2 0.9 Ϯ 0.1 19 PA/N557H 3.3 Ϯ 0.1 1.0 Ϯ 0.1 3.3 4.8 Ϯ 0.5 1.1 Ϯ 0.1 4.4 PA/N557D 7.4 Ϯ 0.2 0.8 Ϯ 0.0 9.3 3.8 Ϯ 0.6 1.2 Ϯ 0.1 3.2 PA/H630E 5.8 Ϯ 0.2 1.1 Ϯ 0.1 5.3 20 Ϯ 2.8 1.4 Ϯ 0.1 14 PA/H630L 3.3 Ϯ 0.1 0.9 Ϯ 0.1 3.7 8.6 Ϯ 0.4 1.4 Ϯ 0.1 6.1 1 These cells were cultured in the absence or presence of 3 ␮M fumitremorgin C with increasing concentrations of mitoxantrone or SN-38. Degree of resistance is the ratio of IC50 value for BCRP-expressing cells divided by that for parental PA317. Login to comment
147 Cells transfected with R482G- (GGG), R482M- (ATG), R482T- (ACG), or R482S- (AGT and AGC) BCRP cDNA showed greater resistance to mitoxantrone and doxorubicin than PA/WT2 (Fig. 2). Login to comment
148 As described above, human drug-resistant cell lines MCF-7 AdVp3000, S1-M1-80, MT-4/DOX500 and a mouse drug-resistant line 88.6/D800-B overexpressed R482T- (ACG), R482G- (GGG), R482M- (ATG) and R482S- (AGT) BCRP, respectively.5,6,13,14,23 The other possible mutations are R482W (TGG) and R482K (AAG). Login to comment
149 PA317 cells expressing R482W- BCRP (PA/R482W) showed somewhat higher levels of resistance to mitoxantrone and doxorubicin than PA/WT2, but the resistance levels were lower than those in PA/ R482G, PA/R482M, PA/R482T and PA/R482S. Login to comment
163 Group 2 members (PA/R482N, PA/R482C, PA/R482M, PA/R482S, PA/R482T, PA/R482V, PA/ R482A, PA/R482G, PA/R482E PA/R482W and PA/R482D) showed higher degrees of resistance to mitoxantrone than to SN-38. Login to comment

[hide] Multidrug resistance mediated by the breast cancer... Oncogene. 2003 Oct 20;22(47):7340-58. Doyle LA, Ross DD
Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2).
Oncogene. 2003 Oct 20;22(47):7340-58., 2003-10-20 [PMID:14576842]

Abstract [show]
Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance. In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found. The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells. BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2. Like all members of the ABC G (white) subfamily, BCRP is a half transporter. Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer. BCRP maps to chromosome 4q22, downstream from a TATA-less promoter. The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors. Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482. Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition. Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics. BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype. Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers. More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.

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154 Sequencing of BCRP derived from normal tissues such as placenta reveal arginine at amino acid position 482, indicating that R482 is the 'wild-type` configuration, and that the threonine or glycine substitutions are mutations (R482T or R482G). Login to comment
161 This conclusion was refuted by another study demonstrating high levels of mitoxantrone resistance in cell lines expressing wild-type R482 as well as mutated R482G and R482T (Volk et al., 2002a). Login to comment
167 This realization led to further studies of methotrexate transport, which revealed that methotrexate resistance, reversible with GF120918, correlated with BCRP expression in cell lines that overexpressed wild-type BCRP, but not the mutant forms (R482T or R482G) (Volk et al., 2002b). Login to comment

[hide] Mutations at amino-acid 482 in the ABCG2 gene affe... Br J Cancer. 2003 Nov 17;89(10):1971-8. Robey RW, Honjo Y, Morisaki K, Nadjem TA, Runge S, Risbood M, Poruchynsky MS, Bates SE
Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.
Br J Cancer. 2003 Nov 17;89(10):1971-8., 2003-11-17 [PMID:14612912]

Abstract [show]
Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

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10 Cells overexpressing wild-type ABCG2 with an arginine at amino-acid 482 have been shown by flow cytometric analysis to transport mitoxantrone, while those overexpressing ABCG2 with a threonine or glycine at position 482 (R482G, R482T) transported mitoxantrone and also exhibited a gain in function with the transport of rhodamine 123 and daunorubicin (Honjo et al, 2001). Login to comment
133 Mutations at amino-acid 482 have included R482G and R482T in human cancer cells; R482S and R482M in mouse fibroblast lines (Honjo et al, 2001; Allen et al, 2002); and a recently reported R482M mutation in a doxorubicin-selected human T-cell line (Wang et al, 2003). Login to comment
159 This would suggest that, if the described R482T or R482G mutations in ABCG2 were to occur in patients, they could render currently known ABCG2 inhibitors less effective. Login to comment

[hide] ABC of oral bioavailability: transporters as gatek... Gut. 2003 Dec;52(12):1788-95. Dietrich CG, Geier A, Oude Elferink RP
ABC of oral bioavailability: transporters as gatekeepers in the gut.
Gut. 2003 Dec;52(12):1788-95., [PMID:14633964]

Abstract [show]
MDR1 (ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) are members of the family of ATP binding cassette (ABC) transporters. These are plasma membrane transporters that are expressed in various organs. The role of MDR1 and MRP2 in the hepatobiliary system is well defined; both contribute to bile formation by transport of drugs, toxins, and waste products across the canalicular membrane. As they transport exogenous and endogenous substances, they reduce the body load of potentially harmful compounds. The role of ABCG2, which is also expressed in the canalicular membrane of hepatocytes, has not yet been fully characterised. All three proteins are also expressed in the apical membrane of enterocytes where they probably control oral availability of many substances. This important "gatekeeper" function of ABC transporters has been recognised recently and is currently under further investigation. Expression and activity of these transporters in the gut may differ between individuals, due to genetic polymorphisms or pathological conditions. This will lead to individual differences in bioavailability of different drugs, toxins, and (food derived) carcinogens. Recent information on substrates, transport mechanisms, function, and regulation of expression of MDR1, MRP2, and BCRP in different species is summarised in this review.

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108 substrate specificity, can be the result of single amino acid mutations in the protein.88 While the wild-type protein with an arginine on position 482 confers resistance to mitoxantrone and irinotecan, R482T or R482G mutations (arginine replaced by threonine or glycine, respectively) result in additional outward transport of rhodamine and doxorubicin by BCRP. Login to comment

[hide] Functional characterization of human breast cancer... Mol Pharmacol. 2003 Dec;64(6):1452-62. Nakanishi T, Doyle LA, Hassel B, Wei Y, Bauer KS, Wu S, Pumplin DW, Fang HB, Ross DD
Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis.
Mol Pharmacol. 2003 Dec;64(6):1452-62., [PMID:14645676]

Abstract [show]
To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. High expression of BCRP was observed on the oocyte surface. Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Transport activity was completely inhibited by fumitremorgin C, a known inhibitor of BCRP. Injection of oocytes with cRNA containing mutations of serine 187 in the ATP-binding cassette signature motif (S187T or S187A) resulted in strong expression of the mutant forms; however, these oocytes were devoid of transporter activity. When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Substrate interaction studies found that no two substrates reciprocally inhibited the efflux of the other. Although FLV proved to be an effective inhibitor of both MX and DNR transport, and MX inhibited DNR transport, the other substrates tested had only weak or no inhibitory activity, indicating a complex nature of substrate interaction with the BCRP homodimer. We conclude that the X. laevis oocyte heterologous expression system is a valid and effective means of studying BCRP function and substrate specificity.

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28 Mutant forms of BCRP with threonine or glycine in place of arginine at codon 482 (R482T or R482G) have been described in drug-selected cell lines (Honjo et al., 2001; Komatani et al., 2001), including the original isolate of BCRP from MCF-7/ AdrVp cells (Doyle et al., 1998), which express the R482T mutation. Login to comment
29 Compared with the wild-type form, the R482T or R482G mutations are able to transport anthracyclines and Rho123 (Honjo et al., 2001; Ozvegy et al., 2002) but not methotrexate (Volk et al., 2002). Login to comment

[hide] Single nucleotide polymorphisms result in impaired... Int J Cancer. 2004 Mar 20;109(2):238-46. Mizuarai S, Aozasa N, Kotani H
Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2.
Int J Cancer. 2004 Mar 20;109(2):238-46., 2004-03-20 [PMID:14750175]

Abstract [show]
ABCG2/MXR/ABCP1/BCRP is a member of the ATP-binding cassette membrane transporter, which consists of six transmembrane regions and one ATP-binding cassette. The transporter is known to be involved in the efflux of various anticancer compounds such as mitoxantrone, doxorubicin and topoisomerase I inhibitor. In this study, we analyzed the effects of polymorphisms in ABCG2, V12M and Q141K on transporter function. When polarized LLC-PK1 cells were transfected with variant ABCG2, drug-resistance to topoisomerase I inhibitor of cells expressing V12M or Q141K was less than 1/10 that of wild-type ABCG2 transfected cells, and was accompanied by increased drug accumulation and decreased drug efflux in the variant ABCG2-expressing cells. We further elucidated the molecular mechanisms of the transport dysfunction by investigating membrane localization and ATPase activity. Confocal microscopic analysis revealed that apical plasma membrane localization of V12M was disturbed, while the localization of wild-type transporters occurred specifically in the apical plasma membrane of polarized LLC-PK1 cells. Also, ATPase activities measured in the membrane of SF9 cells infected with variant ABCG2 showed that Q141K decreased activity by 1.3 below that of wild-type ABCG2. In addition, kinetic analysis of ATPase activity showed that the K(m) value in Q141K was 1.4-fold higher than that of wild-type ABCG2. These results indicated that naturally occurring SNPs alter transport functions of ABCG2 transporter and analysis of SNPs in ABCG2 may hold great importance in understanding the response/metabolism of chemotherapy compounds that act as substrates for ABCG2.

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61 Drug efflux assay LLC-PK1 cells were incubated with the indicated concentration of indolocarbazole compound for 120 min under energy-depleted TABLE I - SINGLE NUCLEOTIDE POLYMORPHISMS IN ABCG21 SNP Effect Region Domain Frequency in 30 cell lines Frequency in 150 clinical samples Hetero Home Hetero Homo Allele (%) G34A V12M Exon2 N-terminal 5 (16.7%) 0 27 (18.0%) 2 (1.3%) 10.3 Aϩ10G Intron3 ND ND 21 (14.0%) 4 (2.7%) 9.7 C369T Wobble Exon4 ABC 0 0 1 (0.67%) 0 0.3 C376T Q126Term Exon4 ABC 0 1 (3.3%) 0 0 0.0 C421A Q141K Exon5 ABC 5 (16.7%) 1 (3.3%) 22 (15.3%) 2 (1.3%) 9.0 C458T T153M Exon5 ABC 1 (3.3%) 0 0 0 0.0 C474T Wobble Exon5 ABC 0 0 1 (0.67%) 0 0.3 Aϩ20G Intron11 ND ND 34 (22.7%) 10 (6.7%) 18.0 A1444G R482G Exon12 TM3 0 0 0 0 0.0 G1445C R482T Exon12 TM3 0 0 0 0 0.0 C-21T Intron13 ND ND 32 (21.3%) 4 (2.7%) 13.3 A1768T N590Y Exon15 EC3 0 0 1 (0.67%) 0 0.3 G2237T Exon16 3ЈUTR 1 (3.3%) 0 0 0 0.0 G2393T Exon16 3ЈUTR 1 (3.3%) 0 0 0 0.0 The positions of the polymorphisms correspond to that of the ABCG2 cDNA (GenBank accession number AB051855) with the first base of the ATG start codon set to 1. Login to comment

[hide] Multidrug resistance in cancer chemotherapy and xe... Curr Med Chem Anticancer Agents. 2004 Jan;4(1):31-42. Han B, Zhang JT
Multidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the half ATP-binding cassette transporter ABCG2.
Curr Med Chem Anticancer Agents. 2004 Jan;4(1):31-42., [PMID:14754410]

Abstract [show]
ABCG2, also termed BCRP/MXR/ABCP, is a half ATP-binding cassette (ABC) transporter expressed on plasma membranes. ABCG2 was independently cloned from placenta as well as cell lines selected for resistance to mitoxantrone or anthracyclines. ABCG2 consists of a nucleotide-binding domain (NBD) at the amino terminus and a transmembrane domain (TMD) at the carboxyl terminus and it is postulated to form a homodimer to perform its biological functions. Over-expression of ABCG2 in cell lines confers resistance on a wide variety of anticancer drugs including mitoxantrone, daunorubicin, doxorubicin, topotecan and epirubicin. The expression of ABCG2 has been implicated in multidrug resistance (MDR) of acute myeloid leukemia and some solid tumors. In addition, ABCG2 can transport several fluorescent dyes or toxins. ABCG2 is found to be expressed in epithelial cells of intestine and colon, liver canaliculi, and renal tubules, where it serves to eliminate the plasma level of orally administered anticancer drugs as well as ingested toxins. ABCG2 is found to be highly expressed in placenta and the luminal surface of microvessel endothelium blood-brain barrier where it may play a role in limiting the penetration of drugs, such as topotecan from the maternal plasma into the fetus and from blood to brain. A variety of inhibitors for ABCG2 including GF120918 may prove useful for sensitizing cancer cells to chemotherapy or altering the distribution of orally administered drug substrates of ABCG2. Interestingly, ABCG2 is also expressed highly in hematopoietic stem cells. However, the function of ABCG2 in stem cells is currently unknown, although it may provide protection to stem cells from a variety of xenobiotics.

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106 However, no polymorphism at amino acid 482 was identified to correspond to the Arg482 Gly, Arg482 Met, or Arg482 Thr mutation that has been identified in drug selected cell lines. Login to comment

[hide] Pheophorbide a is a specific probe for ABCG2 funct... Cancer Res. 2004 Feb 15;64(4):1242-6. Robey RW, Steadman K, Polgar O, Morisaki K, Blayney M, Mistry P, Bates SE
Pheophorbide a is a specific probe for ABCG2 function and inhibition.
Cancer Res. 2004 Feb 15;64(4):1242-6., 2004-02-15 [PMID:14973080]

Abstract [show]
Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2(-/-) knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649-15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 micro M of the cyclin-dependent kinase inhibitor UCN-01 or 1 micro M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 micro M tariquidar, and ABCG2-transfected cells were 6-7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.

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94 The ability of the cyclin-dependent kinase inhibitor UCN-01 to inhibit PhA transport was Table 1 Selected cell lines examined in this study Cell line Selecting drug Transporter MCF-7a - - MCF-7 MX100a 100 nM mitoxantrone ABCG2 (482R) MCF-7 FLV100a 100 nM flavopiridol ABCG2 (482R) MCF-7 FLV250a 250 nM flavopiridol ABCG2 (482R) MCF-7 FLV500a 500 nM flavopiridol ABCG2 (482R) MCF-7 FLV1000a 1000 nM flavopiridol ABCG2 (482R) MCF-7 AdVp10a 5 ␮g/ml verapamil ABCG2 (R482T) 10 ng/ml Adriamycin MCF-7 AdVp3000a 5 ␮g/ml verapamil ABCG2 (R482T) 3000 ng/ml Adriamycin MCF-7/VP 4 ␮M etoposide MRP1b SF295a - - SF295 MX20a 20 nM mitoxantrone ABCG2 (482R) SF295 MX50a 50 nM mitoxantrone ABCG2 (482R) SF295 MX100a 100 nM mitoxantrone ABCG2 (482R) SF295 MX250a 250 nM mitoxantrone ABCG2 (482R) SF295 MX500a 500 nM mitoxantrone ABCG2 (482R) NCI-H460a - - NCI-H460 MX10a 10 nM mitoxantrone ABCG2 (482R) NCI-H460 MX20a 20 nM mitoxantrone ABCG2 (482R) S1 - - S1-M1-80a 80 ␮M mitoxantrone ABCG2 (R482G) SW620 - - SW620 Ad300 300 ng/ml Adriamycin Pgpc M14a - - IGROVa - - a Cells used to correlate fumitremorgin C-inhibitable efflux with surface ABCG2 expression. Login to comment

[hide] Identification of a novel estrogen response elemen... Cancer Res. 2004 Feb 15;64(4):1247-51. Ee PL, Kamalakaran S, Tonetti D, He X, Ross DD, Beck WT
Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene.
Cancer Res. 2004 Feb 15;64(4):1247-51., 2004-02-15 [PMID:14973083]

Abstract [show]
The breast cancer resistance protein (BCRP) is an ATP-binding cassette half transporter that confers resistance to anticancer drugs such as mitoxantrone, anthracyclines, topotecan, and SN-38. Initial characterization of the BCRP promoter revealed that it is TATA-less with 5 putative Sp1 sites downstream from a putative CpG island and several AP1 sites (K. J. Bailey-Dell et al., Biochim. Biophys. Acta, 1520: 234-241, 2001). Here, we examined the sequence of the 5'-flanking region of the BCRP gene and found a putative estrogen response element (ERE). We showed that estrogen enhanced the expression of BCRP mRNA in the estrogen receptor (ER)-positive T47D:A18 cells and PA-1 cells stably expressing ERalpha. In BCRP promoter-luciferase assays, sequential deletions of the BCRP promoter showed that the region between -243 and -115 is essential for the ER effect. Mutation of the ERE found within this region attenuated the estrogen response, whereas deletion of the site completely abrogated the estrogen effect. Furthermore, electrophoretic mobility shift assays revealed specific binding of ERalpha to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.

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15 Cells that overexpress a mutated BCRP (R482T and R482G) remained sensitive to methotrexate (10-12). Login to comment

[hide] ABCG2 mediates differential resistance to SN-38 (7... J Pharmacol Exp Ther. 2004 Aug;310(2):836-42. Epub 2004 Apr 9. Bates SE, Medina-Perez WY, Kohlhagen G, Antony S, Nadjem T, Robey RW, Pommier Y
ABCG2 mediates differential resistance to SN-38 (7-ethyl-10-hydroxycamptothecin) and homocamptothecins.
J Pharmacol Exp Ther. 2004 Aug;310(2):836-42. Epub 2004 Apr 9., [PMID:15075385]

Abstract [show]
One activity potentially limiting the efficacy of camptothecin anticancer agents is their cellular efflux by the ATP-binding cassette half-transporter, ABCG2. Homocamptothecins are novel anticancer drugs that inhibit topoisomerase 1 with a greater potency than camptothecins. Homocamptothecins differ from camptothecins by their E-ring, which is seven-membered instead of the six-membered ring of camptothecins. We report herein that, like camptothecins, homocamptothecin and its difluoro derivative BN80915 are substrates for ABCG2. However, the resistance of three selected cell lines overexpressing wild-type or mutant ABCG2 to homocamptothecin or BN80915 was less than resistance to SN-38 (7-ethyl-10-hydroxycamptothecin), indicating that both the seven-membered E-ring present in homocamptothecin and the A- and B-ring modifications present in SN-38 are involved in substrate recognition by ABCG2. HEK-293 cells transfected with vectors encoding wild-type or mutant ABCG2 were found to be less resistant to both homocamptothecins than to SN-38. However, transfectants overexpressing mutant ABCG2 had relative resistance values for homocamptothecin and BN80915 4- to 14-fold higher than cells expressing wild-type ABCG2, suggesting that the gain of function resulting from mutation at amino acid 482, although not affecting SN-38, extends to the homocamptothecins. Resistance was reversed by the ABCG2 inhibitor fumitremorgin C. BN80915 was 17-fold more potent than SN-38 in wild-type ABCG2-transfected cells, suggesting that BN80915 has the potential to overcome ABCG2-related resistance to SN-38, the active metabolite of CPT-11 (irinotecan).

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31 To determine whether homocamptothecins are ABCG2 substrates, we compared the cytotoxicity of homocamptothecin and its clinical difluoro derivative, BN80915, to CPT and to SN-38 in three ABCG2-overexpressing selected cell lines expressing either wild type (R482) or mutant (R482T, R482G) ABCG2. Login to comment
79 In agreement with previous results (Brangi et al., 1999), we found that resistance to SN-38 was high in all three drug-selected cell lines overexpressing either wild-type 482R (MCF-7 MX100), mutant R482T (MCF-7 AdVp3000), or R482G (S1M1-80) ABCG2. Login to comment
98 Interestingly, cells transfected with a mutant R482G or R482T ABCG2 gene were 4-to 7-fold more resistant to homocamptothecin and 7-to 14-fold more resistant to BN80915 than cells transfected with wild-type R482 ABCG2, suggesting that amino acid 482 affects the ability of ABCG2 to confer resistance to these compounds. Login to comment
99 Previous studies had demonstrated that in addition to the absence of anthracycline resistance and rhodamine transport, the wild-type R482 protein was less effective in transporting mitoxantrone compared with mutant (R482G, R482T) ABCG2 (Robey et al., 2003). Login to comment
129 Three drug-selected cell lines overexpressing wild-type (R482) or mutant (R482G, R482T) ABCG2 as well as HEK-293 cells expressing wild-type or mutant ABCG2 were found to be resistant to homocamptothecin and BN80915. Login to comment
144 Resistance to homocamptothecin and BN80915 was highest in cells transfected with mutant (R482G, R482T) compared with wild-type Fig. 4. Login to comment
147 B, 4-day cytotoxicity assays were performed on HEK-293 cells transfected with empty vector (filled squares), wild-type (R482, triangles), or mutant (R482G, circles; R482T, hatched squares) ABCG2 with SN-38, camptothecin (CPT), homocamptothecin (hCPT), or BN80915. Login to comment

[hide] ABCG2 -- a transporter for all seasons. FEBS Lett. 2004 Jun 1;567(1):116-20. Sarkadi B, Ozvegy-Laczka C, Nemet K, Varadi A
ABCG2 -- a transporter for all seasons.
FEBS Lett. 2004 Jun 1;567(1):116-20., 2004-06-01 [PMID:15165903]

Abstract [show]
The human ABCG2 (ABCP/MXR/BCRP) protein is a recently recognized ABC half-transporter, which forms homodimers in the plasma membrane and actively extrudes a wide variety of chemically unrelated compounds from the cells. This protein protects our cells and tissues against various xenobiotics, with a crucial role in the intestine, liver, placenta, and the blood-brain barrier. Moreover, ABCG2 seems to have a key function in stem cell protection/regulation, and also in hypoxic defense mechanisms. Widely occurring single nucleotide polymorphisms in ABCG2 may affect absorption and distribution, altering the effectiveness and toxicity of drugs in large populations. At the clinics, overexpression of ABCG2 in tumor cells confers cancer multidrug resistance to a variety of newly developed anticancer agents. On the other hand, specific substrate mutants of ABCG2 are advocated for use as selectable markers in stem-cell based gene therapy.

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109 Transported substrates of the wild-type and R482G ABCG2 protein. Login to comment
119 The mutants having R482G or R482T (R482M or R482S in the mouse abcg2) showed altered substrate specificity as compared to the wild-type protein, i.e., they conferred increased mitoxantrone or doxorubicin (DOX) resistance and rhodamine 123 transport capacity (see Fig. 2, [42,43]). Login to comment
121 However, the R482G and R482T mutants were not able to transport methotrexate, which is a transported substrate of the wild-type ABCG2 [15,44]. Login to comment
135 Since the R482G variant of ABCG2 has different substrate specificity than the wild-type protein (Fig. 2), this mutant has a special advantage in gene therapy applications. Login to comment

[hide] Imatinib mesylate (STI571) is a substrate for the ... Blood. 2004 Nov 1;104(9):2940-2. Epub 2004 Jul 13. Burger H, van Tol H, Boersma AW, Brok M, Wiemer EA, Stoter G, Nooter K
Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump.
Blood. 2004 Nov 1;104(9):2940-2. Epub 2004 Jul 13., 2004-11-01 [PMID:15251980]

Abstract [show]
Imatinib mesylate (STI571), a potent tyrosine kinase inhibitor, is successfully used in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. However, the intended chronic oral administration of imatinib may lead to development of cellular resistance and subsequent treatment failure. Indeed, several molecular mechanisms leading to imatinib resistance have already been reported, including overexpression of the MDR1/ABCB1 drug pump. We examined whether imatinib is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump that is frequently overexpressed in human tumors. Using a panel of well-defined BCRP-overexpressing cell lines, we provide the first evidence that imatinib is a substrate for BCRP, that it competes with mitoxantrone for drug export, and that BCRP-mediated efflux can be reversed by the fumitremorgin C analog Ko-143. Since BCRP is highly expressed in the gastrointestinal tract, BCRP might not only play a role in cellular resistance of tumor cells but also influence the gastrointestinal absorption of imatinib.

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13 MCF7 (ATCC, HTB-22); MCF7/MR, a mitoxantrone-resistant and BCRP-overexpressing subline; MCF7/AdVp3000 overexpressing the R482T BCRP variant; HEK293 cells transfected with pcDNA3 (HEK293/Neo); wild-type BCRP/ABCG2-R482R (HEK293/R); BCRP/ ABCG2-R482G (HEK293/G); and BCRP/ABCG2-R482T (HEK293/T) were used. Login to comment

[hide] Mutational analysis of ABCG2: role of the GXXXG mo... Biochemistry. 2004 Jul 27;43(29):9448-56. Polgar O, Robey RW, Morisaki K, Dean M, Michejda C, Sauna ZE, Ambudkar SV, Tarasova N, Bates SE
Mutational analysis of ABCG2: role of the GXXXG motif.
Biochemistry. 2004 Jul 27;43(29):9448-56., 2004-07-27 [PMID:15260487]

Abstract [show]
ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.

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3 All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. Login to comment
54 This vector was selected for ease of study with rhodamine 123, a substrate for ABCG2 only when the R482G mutation is present. Login to comment
124 In the present study we used this R482G "gain-of-function" mutation in all of the mutants in order to be able to study them with flow cytometry using rhodamine 123 as a substrate. Login to comment
130 When compared to the R482G transfectant clone, lower levels in most of the leucine mutants were consistently observed by immunoblot analysis. Login to comment
138 Rhodamine 123 is transported by ABCG2 only when the R482G amino acid substitution is present. Login to comment
140 Assessment of mitoxantrone accumulation revealed some transport in the same mutants, but activity was impaired when compared to the R482G transfected clones. Login to comment
146 Changes in fluorescence when FTC was added were almost identical to the R482G mutant, indicating that alanine is able to replace glycine without interfering with protein function. Flow cytometry also proved that the G406A and G410A mutants had intact transport of these substrates (data not shown). Login to comment
151 Using DSS, which cross-links proteins 11.4 Å apart, a dimer or higher order multimer was observed by immunoblot analysis in the G406L and G406L/G410L mutants and in the R482G control cells, as shown in Figure 4A. Next, cross-linking studies were performed with DSP, an agent that can be cleaved with the addition of DTT or -mercaptoethanol. Login to comment
153 (A) Protein expression levels on immunoblot with the BXP21 monoclonal anti-ABCG2 antibody for two clones of each of the leucine mutants (25 µg of protein per lane), in most cases revealing significantly lower levels than in the R482G clone, from which they were derived. Login to comment
160 Levels are compared to the R482G transfected cells. Plots: ABCG2 (---); negative control (s). Login to comment
161 Transport capacity for BODIPY-prazosin, rhodamine 123, mitoxantrone, and pheohorbide A in the R482G, G406A/G410A, G406L/G410L, G406L, and G410L transfected cells. Plots: Accumulation without FTC (s) and with FTC (---). Login to comment
163 The single leucine mutants (G406L, G410L) show impaired transport for BODIPY-prazosin, mitoxantrone, and pheophorbide a when compared to the fully functional R482G control cell line. Login to comment
166 (A) Cross-linking of ABCG2 is observed with DSS in HEK293 cells transfected with R482G or with the functionally impaired leucine mutants. Login to comment
167 (B) Cross-linking is observed with the cleavable cross-linker DSP in the R482G mutant; note that the same higher molecular weight band can be seen under nonreducing conditions and that it can be reduced to the monomeric 72 kDa band with the addition of -ME or DTT. Login to comment
172 Cross-linking of the R482G transfected cells with DSP resulted in a high molecular weight protein, representing a dimer or multimer, which could be reduced to the monomeric 72 kDa form by exposure to reducing agents (Figure 4B). Login to comment
179 We determined the vanadate-sensitive component of the ATPase activity of the G406L/G410L mutant in the presence of 0, 1, 10, and 100 µM prazosin and compared it to the R482G and empty pcDNA3.1 vector transfected cells. Login to comment
180 Figure 5 shows that 100 µM prazosin increased the ATP hydrolysis activity of the R482G transfected clone approximately 3-fold over basal level, while the empty vector transfected control cell line (pcDNA) had a significantly lower basal level with a negligible increase in activity in the presence of the drug. Login to comment
185 Cells were thus incubated in 2-10 µM mitoxantrone overnight, membranes harvested, and proteins separated by electrophoresis. Figure 6A shows a significant increase in protein levels on imunoblot for the GXXXG motif mutants, with a less pronounced increase in the cells expressing protein without this mutation (R482G), reaching a plateau at 5 µM mitoxantrone. Login to comment
201 The vanadate-sensitive ATP hydrolysis in the presence of the indicated concentrations of prazosin for crude membranes of HEK293 cells expressing ABCG2 R482G (0), G406L/G410L mutants (]), and empty vector transfected cells (pcDNA; O) is shown. Login to comment
225 Although the mutant proteins are located on the cell surface, they are expressed at a lower level than the R482G mutant, to which they should be compared. Login to comment
231 (A) Overnight treatment of intact cells with 2-10 µM mitoxantrone substantially increases the level of ABCG2 in the GXXXG single and double leucine mutants (two clones for each shown) on immunoblot, while the change is much less pronounced in the control R482G transfected cells. Login to comment

[hide] Arginine-482 is not essential for transport of ant... Biochem J. 2005 Jan 15;385(Pt 2):419-26. Janvilisri T, Shahi S, Venter H, Balakrishnan L, van Veen HW
Arginine-482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2).
Biochem J. 2005 Jan 15;385(Pt 2):419-26., 2005-01-15 [PMID:15362954]

Abstract [show]
The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance. To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482-->Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G. In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L. lactis. Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Since Arg482 is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate. Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg482 and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg482.

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1 To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482 → Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Login to comment
2 Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. Login to comment
5 Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Login to comment
19 In a previous work [4], the R482G mutant BCRP (BCRP-G) was functionally expressed in L. lactis and evidence for the interaction of BCRP-G with sterols was obtained. Login to comment
31 Genetic manipulations The BCRP R482G (BCRP-G) cDNA was subcloned as an NcoI- XbaI fragment from the lactococcal expression vector pNZ-BCRP-G [4] into the Escherichia coli plasmid pGEM, yielding pGEM-BCRP-G. Login to comment

[hide] Breast cancer resistance protein (BCRP/ABCG2) does... Leukemia. 2004 Nov;18(11):1914-7. Walter RB, Raden BW, Thompson J, Flowers DA, Kiem HP, Bernstein ID, Linenberger ML
Breast cancer resistance protein (BCRP/ABCG2) does not confer resistance to gemtuzumab ozogamicin and calicheamicin-gamma1 in acute myeloid leukemia cells.
Leukemia. 2004 Nov;18(11):1914-7., [PMID:15385942]

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33 Evidence to date suggests that BCRP mainly serves to protect cells from xenobiotic toxins, although additional functions in stem cell populations are discussed.4,5 BCRP confers resistance to certain chemotherapeutic agents; however, the substrate specificity in cells containing a mutation at codon 482 (R482T or R482G) differs somewhat from cells expressing wild-type protein.4,5 BCRP mRNA and protein are variably expressed in AML.5,6 So far, only wild-type BCRP (ie arginine at codon 482) has been Received 27 April 2004; accepted 29 June 2004; Published online 16 September 2004 Correspondence: Dr RB Walter, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D2-373, Seattle, WA 98109-1024, USA; Fax: þ 1 206 667 6084; E-mail: rwalter@fhcrc.org This work has been presented in part at the 45th annual meeting of the American Society of Hematology (December 6-9, 2003; San Diego, CA, USA; Blood 2003; 102: 208b-209b; abstract #4553) Correspondence Leukemia found in primary AML samples7 (and A Suvannasankha et al. Blood 2002; 100: 67a; abstract), whereas R482T and R482G variants have been identified in drug-selected cell lines.4,5 It remains unknown whether mutations at codon 482 might occur in patients after treatment with BCRP substrate drugs. Login to comment

[hide] Functional assessment of ABCG2 (BCRP) gene polymor... Drug Metab Dispos. 2005 Jan;33(1):94-101. Epub 2004 Oct 8. Kobayashi D, Ieiri I, Hirota T, Takane H, Maegawa S, Kigawa J, Suzuki H, Nanba E, Oshimura M, Terakawa N, Otsubo K, Mine K, Sugiyama Y
Functional assessment of ABCG2 (BCRP) gene polymorphisms to protein expression in human placenta.
Drug Metab Dispos. 2005 Jan;33(1):94-101. Epub 2004 Oct 8., [PMID:15475413]

Abstract [show]
The aim of the present study was to assess the contribution of polymorphisms in the breast cancer resistance protein/ATP-binding cassette transporter G2 (BCRP/ABCG2) gene to the placental expression from a new perspective, allelic imbalance. Polymorphisms were screened by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis followed by sequencing with DNA extracted from 100 placentas. To examine whether polymorphisms of the BCRP gene correlate with the placental BCRP expression, we determined mRNA and protein levels by quantitative real-time PCR and Western blotting, respectively. In placentas, G34A (Val(12)Met) and C421A (Gln(141)Lys) were frequently observed (18-36%), but C376T, which creates a stop codon (Gln(126) stop codon), was found with an allelic frequency of 1%. The mean of the BCRP protein level was significantly lower (p < 0.05) in homozygotes for the A421 allele than in those for the C421 allele, and heterozygotes had an intermediate value. To evaluate whether the C421A polymorphism acts as a cis-element in BCRP transcription, allelic imbalance was determined using informative lymphoblasts and 56 samples of placental cDNA. In most of the placental samples we tested, the difference in expression levels between the two alleles was small, and only two samples indicated a monoallelic expression (i.e., preferential expression of one allele). These results suggest that 1) the predominant allelic expression pattern of BCRP in placental samples is biallelic, and 2) the mutation C421A is not a genetic variant acting in cis, but is considered to influence the translation efficiency.

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197 Other nonsynonymous variants, Arg482Gly and Arg482Thr, have been reported to have a crucial role in protein function and in altering the multidrug resistance phenotype by changing substrate specificity (Honjo et al., 2001; Allen et al., 2002). Login to comment

[hide] Cells resistant to HTI-286 do not overexpress P-gl... Mol Cancer Ther. 2004 Oct;3(10):1319-27. Loganzo F, Hari M, Annable T, Tan X, Morilla DB, Musto S, Zask A, Kaplan J, Minnick AA Jr, May MK, Ayral-Kaloustian S, Poruchynsky MS, Fojo T, Greenberger LM
Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin.
Mol Cancer Ther. 2004 Oct;3(10):1319-27., [PMID:15486199]

Abstract [show]
HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [(3)H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [(14)C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major beta-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in alpha-tubulin that substitutes Ser for Ala(12) near the nonexchangeable GTP-binding site of alpha-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala(12) mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of alpha-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.

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75 S1-M1-3.2, which overexpress a mutant ABCG2 (Arg482 Gly),5 and parental S1, which express low levels of wild-type ABCG2, were used as positive and negative control cell lines for ABCG2 (18). Login to comment

[hide] Cloning and functional analysis of the rhesus maca... J Biol Chem. 2005 Jan 14;280(2):991-8. Epub 2004 Oct 30. Ueda T, Brenner S, Malech HL, Langemeijer SM, Perl S, Kirby M, Phang OA, Krouse AE, Donahue RE, Kang EM, Tisdale JF
Cloning and functional analysis of the rhesus macaque ABCG2 gene. Forced expression confers an SP phenotype among hematopoietic stem cell progeny in vivo.
J Biol Chem. 2005 Jan 14;280(2):991-8. Epub 2004 Oct 30., 2005-01-14 [PMID:15516692]

Abstract [show]
Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.

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198 Further, coexpression of gp91phox and the R482G variant of ABCG2 corrected the phenotype of gp91phox knockout cells and protected the transduced cells from the toxic effects of mitoxantrone (39). Login to comment

[hide] Flow cytometry-based approach to ABCG2 function su... Cytometry A. 2004 Dec;62(2):129-38. Garcia-Escarp M, Martinez-Munoz V, Sales-Pardo I, Barquinero J, Domingo JC, Marin P, Petriz J
Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs.
Cytometry A. 2004 Dec;62(2):129-38., [PMID:15517563]

Abstract [show]
BACKGROUND: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively. METHODS: ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter. RESULTS: ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (Rho123), SYTO13, doxorubicin, and daunorubicin. After incubation with MXR, intracellular accumulations were 9- and 22-fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux showed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 min vs. 27.8 and 44.5 min, respectively). MXRG cells also extruded Rho123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A, and PSC-833, but not by verapamil or probenecid. MXRG cells displayed the highest level of resistance to MXR, doxorubicin, and daunorubicin in the cytotoxicity assays. CONCLUSIONS: Glycine mutations at position 482 have a significant impact on ABCG2 function by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters.

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0 Flow Cytometry-Based Approach to ABCG2 Function Suggests That the Transporter Differentially Handles the Influx and Efflux of Drugs Marta Garcı´a-Escarp,1 Vanessa Martı´nez-Mun˜oz,3 Irene Sales-Pardo,3 Jordi Barquinero,1 Joan Carles Domingo,2 Pedro Marin,3 and Jordi Petriz3 * 1 Unitat de Diagno`stic i Tera`pia Molecular, Centre de Transfusio´ i Banc de Teixits, Barcelona, Spain 2 Departament de Bioquı´mica i Biologia Molecular, Universitat de Barcelona, Barcelona, Spain 3 Area de Criopreservacio´, Centre de Diagno`stic Biome`dic, Hospital Clı´nic, Institut d`Investigacions Biome`diques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain Received 3 February 2004; Revision Received 25 February 2004; Accepted 23 April 2004 Background: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively. Login to comment
117 KB cells transfected with ABCG2-R482A, R482G, and R482T were seeded in the presence of 0.8 ␮M MXR for 1 h at 37°C. Login to comment
156 Moreover, MXR is pumped out of the cells more efficiently by R482T and R482G mutants than by wt ABCG2. Login to comment
174 In this study, we found that the mutation R482G, which changes the amino acid arginine to a glycine, results in a faster efflux of MXR when compared with MXRT cells, suggesting that the enhanced ability to transport MXR increases resistance to this drug. Login to comment
178 Functional assay for transport kinetics in R482G transfected cell lines. Efflux assays were performed with 0.8 ␮M MXR, 100 ng/ml Rho123, and 50 nM SYTO-13 in the selected cell line. Login to comment
182 This supports the hypothesis that the R482G mutation is crucial for enhanced MXR resistance, because it allows an increased efflux of the drug, but it may also significantly decrease the intracellular levels of MXR. Login to comment
200 The R482G mutation seems to be essential to increase the velocity of this ABCG2 form and to decrease the intracellular accumulation of MXR. Login to comment

[hide] Function-dependent conformational changes of the A... J Biol Chem. 2005 Feb 11;280(6):4219-27. Epub 2004 Nov 22. Ozvegy-Laczka C, Varady G, Koblos G, Ujhelly O, Cervenak J, Schuetz JD, Sorrentino BP, Koomen GJ, Varadi A, Nemet K, Sarkadi B
Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface.
J Biol Chem. 2005 Feb 11;280(6):4219-27. Epub 2004 Nov 22., 2005-02-11 [PMID:15557326]

Abstract [show]
The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.

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54 In the present study we used the K86M variant introduced into the wild-type (R482) ABCG2 by cloning the NotI-SpeI fragment of pAcUW21-L/K86M-R482G (31) into the corresponding site of the pAcUW21-L/R482 vector. Login to comment
161 We obtained essentially similar data in the MCF-7/MX cells and the PLBs expressing the gain-of-function R482G mutant of ABCG2 (not shown). Login to comment

[hide] Lack of an effect of breast cancer resistance prot... Biochem Pharmacol. 2005 Jan 1;69(1):123-32. Rhee MS, Schneider E
Lack of an effect of breast cancer resistance protein (BCRP/ABCG2) overexpression on methotrexate polyglutamate export and folate accumulation in a human breast cancer cell line.
Biochem Pharmacol. 2005 Jan 1;69(1):123-32., 2005-01-01 [PMID:15588721]

Abstract [show]
Accumulation of methotrexate (MTX) and its polyglutamates (PGs) has been recognized as an important factor in MTX efficacy. We have previously described a multidrug-resistant human breast cancer cell line, MCF7/MX, that exhibits reduced accumulation of total MTX as well as MTX-PGs, and that is resistant to continuous MTX exposure [Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, et al. Methotrexate cross-resistance in a mitoxantrone selected multidrug-resistant MCF7 breast cancer cell line is due to enhanced energy-dependent drug efflux. Cancer Res 2000;60:3514-21]. These cells express high levels of the breast cancer resistance protein (BCRP/ABCG2) that has been shown to actively transport MTX and short-chain MTX-PGs in vitro. However, the effect of BCRP on MTX-PG accumulation in intact cells was unclear. Here, we show that MTX transport by BCRP is required for the observed lower levels of MTX-PGs in the resistant cells. When BCRP was inhibited with fumitremorgin C, or in cells expressing a mutated form of BCRP that is unable to transport MTX, MTX-PG accumulation was similar or even higher than that in the parental cells that do not express BCRP. Concomitantly, there was increased inhibition of thymidylate synthase. It had previously been suggested that BCRP-mediated efflux of MTX-PGs contributed to the reduced MTX-PG accumulation. However, we found no evidence of BCRP-mediated efflux of MTX-PGs from intact cells, suggesting that direct efflux of MTX-PGs does not play a major role in MTX resistance. Together, these data show that BCRP overexpression can cause a reduction in total MTX accumulation as well as a reduction in the proportion of long-chain MTX-PGs. In contrast, BCRP overexpression did not affect natural folate accumulation or the relative distribution of folylpolyglutamates in the resistant, as compared to the parental, cells. Thus, it appears that BCRP overexpression affects the metabolism of the antifolate MTX, but not that of natural folates, although indirect effects cannot be excluded.

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46 MCF7/MX cells express the wild-type form of BCRP with an arginine at amino acid position 482 (482R), whereas the M1-80 cells have acquired a mutation at this position from arginine to glycine (R482G) [30]. Login to comment
79 However, in contrast to the MCF7/MX cells, M1-80 cells are not cross-resistant to MTX [24], because their BCRP contains the R482G mutation [30] that has been shown to abolish the transport of MTX [6,22,23]. Login to comment

[hide] Differential sensitivities of the human ATP-bindin... Mol Pharmacol. 2005 Mar;67(3):902-11. Epub 2004 Dec 14. Ejendal KF, Hrycyna CA
Differential sensitivities of the human ATP-binding cassette transporters ABCG2 and P-glycoprotein to cyclosporin A.
Mol Pharmacol. 2005 Mar;67(3):902-11. Epub 2004 Dec 14., [PMID:15598974]

Abstract [show]
Several ATP-binding cassette (ABC) transporters can confer multidrug resistance to cancer cells by functioning as energy-dependent efflux pumps. The half-transporter ABCG2 and the widely studied P-glycoprotein (P-gp) are two ABC transporters that, when overexpressed, are capable of extruding a variety of structurally unrelated chemotherapy agents from cells. In this study, we demonstrate that human ABCG2 and P-glycoprotein, despite overlapping substrate specificities, differ in sensitivity to the immunomodulator cyclosporin A. In this study, we used human ABCG2 and human P-gp, each expressed separately in drug-selected MCF-7 sublines and transiently transfected HeLa cells. By flow cytometric analysis using the fluorescent substrates rhodamine 123 and mitoxantrone, we showed that cyclosporin A inhibits P-gp function at low micromolar concentrations, whereas ABCG2 function was unaffected. Furthermore, P-gp, but not ABCG2, was able to transport [3H]cyclosporin A directly in intact cells. We also demonstrated, for the first time, that [125I]iodoarylazidoprazosin, a photoaffinity analog of the substrate prazosin, labels multiple variants of ABCG2 specifically and that this labeling, although competed by some ABCG2 substrates, is unaffected by cyclosporin A. These labeling data also suggest the presence of multiple drug binding sites in ABCG2. In addition, cyclosporin A had no effect on the basal or prazosin-stimulated ATPase activity of ABCG2, whereas both the basal and verapamil-stimulated ATPase activities of P-gp were inhibited markedly. Together, our results suggest that cyclosporin A is neither a substrate nor an inhibitor of the human ABCG2 transporter, under the conditions and concentrations examined.

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31 A selection of substrates transported by wild-type ABCG2 or the R482G or R482T variants, include the fluorescent dye rhodamine 123, the anthracenes bisantrene and mitoxantrone, the anthracyclines doxorubicin and daunorubicin, and the camptothecins topotecan and SN-38 (Litman et al., 2001; Gottesman et al., 2002). Login to comment
97 Our data show that ABCG2 is localized at the surface in both HeLa cells transfected with any of the three ABCG2 variants (R482G, R482, or R482T) and in MCF-7/AdVp3000 cells (Fig. 2, A and B). Login to comment
98 The R482 (wild type), R482G, and R482T ABCG2 variants are expressed equivalently in HeLa cells (Fig. 2A). Login to comment
113 Rhodamine 123 is a well established substrate of the R482G and R482T variants of ABCG2 (Honjo et al., 2001) and of P-gp (Hrycyna et al., 1998), but it is not a substrate of wild-type R482 variant of ABCG2 (Honjo et al., 2001). Login to comment
117 As can be seen in Fig. 3A, cells expressing ABCG2 (R482G or R482T) or P-gp exclude rhodamine 123 equivalently in the absence of inhibitor. Login to comment
123 Transport of mitoxantrone by the R482T ABCG2 variant seems to be the least sensitive to addition of 5 ␮M cyclosporin A, followed by the R482G and the wild-type R482 ABCG2 proteins. Login to comment
130 A, transiently transfected HeLa cells expressing ABCG2 labeled with 5D3; R482G (-), R482 (⅐ ⅐ ⅐), R482T (-), R482G cells labeled with IgG2b (- ⅐ - ⅐ -). Login to comment
143 In contrast, it has been reported that 5 ␮M cyclosporin A increased the accumulation of 99m Tc-tetrofosmin 1.8-fold and 99m Tc-sestamibi 1.3-fold in S1-M1/S1-M1-80 cells that express the R482G variant of ABCG2 (Chen et al., 2000b). Login to comment
156 A to F, histograms show HeLa cells transfected with the empty pTM1 vector (shaded peak), R482G (-), R482 (- ⅐ - ⅐ -), R482T (- - -), or P-gp (-). Login to comment
168 These experiments show that cells expressing ABCG2 (R482T or R482G) or P-gp extrude daunomycin in the absence of the inhibitor and that this transport is inhibited in the presence of GF120918. Login to comment
170 Together, our data demonstrate that cyclosporin A is only transported by P-gp, whereas daunomycin is transported by P-gp and the R482G and R482T variants of ABCG2 but not the wild-type ABCG2 (R482). Login to comment
175 To assess whether different variants of ABCG2 can be labeled with [125 I]IAAP, we performed the experiment with membrane isolates from S1-M1-80, MCF-7/FLV1000, and MCF-7/AdVp3000 cells that express the R482G, R482, and R482T variants of ABCG2, respectively. Login to comment
197 The ATPase activity of the R482G and R482T variants of ABCG2 are stimulated 2.2- and 1.4-fold, respectively, by the addition of 40 ␮M prazosin (Fig. 6, A and C). Login to comment
203 C, 60 ␮g of membrane protein from S1 or S1-M1-80 expressing the R482G variant of ABCG2. Login to comment
211 A previous study reported that cyclosporin A inhibits both basal and prazosin-stimulated ATPase activity of ABCG2 (R482G variant) expressed in Sf9 insect cells (O¨ zvegy et al., 2001). Login to comment
224 The assays were performed both in the absence (᭜) and presence (E) of 40 ␮M prazosin (for ABCG2) or 30 ␮M verapamil (for P-gp), at increasing concentrations of cyclosporin A. ATPase activity of ABCG2 proteins transiently expressed in HeLa cells R482G (A), R482 (B), and R482T (C), and in MCF-7/AdVp3000 cells (D). Login to comment
228 In this study, we used both the drug-resistant cell lines MCF-7/AdVp3000 that overexpress ABCG2 with the R482T gain of function mutation (Miyake et al., 1999) and MCF-7/ DX1 that overexpress P-gp, as well as HeLa cells transiently expressing ABCG2 (R482G, R482, or R482T variants) or P-gp to explore, in detail, the molecular effects that the immunomodulator cyclosporin A has on these transporters. Login to comment
233 In addition, we observe subtle differences in the transport of mitoxantrone in the absence and presence of cyclosporin A between the three ABCG2 variants (R482G, Arg482, and R482T) (Fig. 3, D-F), corroborating previous data suggesting amino acid 482 is an important determinant of substrate specificity. Login to comment
254 In conclusion, our findings demonstrate that the immunomodulator cyclosporin A is a substrate and an inhibitor of human P-glycoprotein, but that under the conditions examined, it is neither a substrate nor an inhibitor of any of the variants (R482G, R482, and R482T) of the related ABC transporter ABCG2. Login to comment

[hide] Eight novel single nucleotide polymorphisms in ABC... Drug Metab Pharmacokinet. 2003;18(3):212-7. Itoda M, Saito Y, Shirao K, Minami H, Ohtsu A, Yoshida T, Saijo N, Suzuki H, Sugiyama Y, Ozawa S, Sawada J
Eight novel single nucleotide polymorphisms in ABCG2/BCRP in Japanese cancer patients administered irinotacan.
Drug Metab Pharmacokinet. 2003;18(3):212-7., [PMID:15618737]

Abstract [show]
Eight novel single nucleotide polymorphisms (SNPs) were found in the gene encoding the ATP-binding cassette transporter, ABCG2/BCRP, from 60 Japanese individuals administered the anti-cancer drug irinotecan. The detected SNPs were as follows: 1) SNP, MPJ6_AG2005 (IVS2-93T>C); Gene Name, ABCG2; Accession Number, NT_006204; 2) SNP, MPJ6_AG2007 (IVS3+71_72 insT); Gene Name, ABCG2; Accession Number, NT_006204; 3) SNP, MPJ6_AG2012 (IVS6-204C>T); Gene Name, ABCG2; Accession Number, NT_006204; 4) SNP, MPJ6_AG2015 (at nucleotide 1098G>A (exon 9) from the A of the translation initiation codon); Gene Name, ABCG2; Accession Number, NT_006204; 5) SNP, MPJ6_AG2017 (1291T>C (exon 11)); Gene Name, ABCG2; Accession Number, NT_006204; 6) SNP, MPJ6_AG2019 (IVS11-135G>A); Gene Name, ABCG2; Accession Number, NT_006204; 7) SNP, MPJ6_AG2020 (1465T>C (exon 12)); Gene Name, ABCG2; Accession Number, NT_006204; 8) SNP, MPJ6_AG2023 (IVS13+65T>G); Gene Name, ABCG2; Accession Number, NT_006204.MPJ6_AG2015 was a synonymous SNP (E366E). MPJ6_AG2017 and MPJ6_AG2020 resulted in amino acid alterations, F431L and F489L, respectively.

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108 Honjo et al. recently reported that R482T and R482G variants were found in cells after drug selection.11) ABCG2-ATPase activity was in‰uenced by amino acid residue 482,12) which might be located within the transmembrane domain.10) Currently, the functional signiˆcance of these SNPs is unknown; however, the clinical outcome of those who have these non-synonymous SNPs should carefully be evaluated. Login to comment

[hide] Folate pathway gene expression differs in subtypes... J Clin Invest. 2005 Jan;115(1):110-7. Kager L, Cheok M, Yang W, Zaza G, Cheng Q, Panetta JC, Pui CH, Downing JR, Relling MV, Evans WE
Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics.
J Clin Invest. 2005 Jan;115(1):110-7., [PMID:15630450]

Abstract [show]
The ability of leukemia cells to accumulate methotrexate polyglutamate (MTXPG) is an important determinant of the antileukemic effects of methotrexate (MTX). We measured in vivo MTXPG accumulation in leukemia cells from 101 children with acute lymphoblastic leukemia (ALL) and established that B-lineage ALL with either TEL-AML1 or E2A-PBX1 gene fusion, or T-lineage ALL, accumulates significantly lower MTXPG compared with B-lineage ALL without these genetic abnormalities or compared with hyperdiploid (fewer than 50 chromosomes) ALL. To elucidate mechanisms underlying these differences in MTXPG accumulation, we used oligonucleotide microarrays to analyze expression of 32 folate pathway genes in diagnostic leukemia cells from 197 children. This revealed ALL subtype-specific patterns of folate pathway gene expression that were significantly related to MTXPG accumulation. We found significantly lower expression of the reduced folate carrier (SLC19A1, an MTX uptake transporter) in E2A-PBX1 ALL, significantly higher expression of breast cancer resistance protein (ABCG2, an MTX efflux transporter) in TEL-AML1 ALL, and lower expression of FPGS (which catalyzes formation of MTXPG) in T-lineage ALL, consistent with lower MTXPG accumulation in these ALL subtypes. These findings reveal distinct mechanisms of subtype-specific differences in MTXPG accumulation and point to new strategies to overcome these potential causes of treatment failure in childhood ALL.

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130 It is noteworthy that only wild-type BCRP, but not the mutant forms (R482T or R482G), transports MTX and MTXPGs (13, 39). Login to comment

[hide] Single amino acid (482) variants of the ABCG2 mult... Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63. Ozvegy-Laczka C, Koblos G, Sarkadi B, Varadi A
Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition.
Biochim Biophys Acta. 2005 Feb 1;1668(1):53-63., 2005-02-01 [PMID:15670731]

Abstract [show]
The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

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29 The mutants, containing R482G, T or M (R482M or S in the mouse abcg2), showed altered substrate specificity [22-24]. Login to comment
30 Previously, we have shown that the R482G and T mutants have increased ATP hydrolytic activity, therefore they are bgain of functionQ mutants in this regard [25]. Login to comment
31 However, the R482G and T mutants were unable to transport methotrexate, which is a substrate of the wtABCG2 [6,26]. Login to comment
44 2.2. Generation of transfer vectors possessing different human ABCG2 cDNAs pAcUW21-L/ABCG2 (wild-type, R482G, T or K86M/ R482G) was constructed as described earlier [25]. Login to comment
45 In this study, we used the K86M-R482 single mutant, which was generated by cloning the NotI-SpeI fragment of pAcUW21-L/K86M-R482G [25] into the corresponding site of pAcUW21-L/R482. Login to comment
46 The seven additional Arg-482 variants were created using ABCG2-R482G cDNA as a template by overlap extension PCR [25,28]. Login to comment
116 Prazosin is a substrate of the wtABCG2, R482G and R482T [34], and it stimulates the ATPase activity of R482G and T mutants, but it has no major effect on the ATPase activity of wtABCG2 [25]. Login to comment
131 Characterization of [3 H]methotrexate transport by ABCG2 mutants It has been shown earlier that methotrexate is a transported substrate of the wtABCG2 but not of R482G or T [6,26]. Login to comment
149 Flow cytometry assay of mitoxantrone and rhodamine 123 extrusion from intact Sf9 cells expressing wtABCG2 and its Arg-482 mutants It has been documented earlier that mitoxantrone (MX) is a transported substrate both of the wtABCG2 and its R482G or T mutants, while rhodamine 123 (R123) is transported only by the R482G and R482T variants [22,25]. Login to comment
155 We found that cells containing wtABCG2 or R482G, I, M, S, T, D, N, and Y mutants accumulate less mitoxantrone than cells expressing the inactive K86M mutant. Login to comment
161 3.5. Measurement of Hoechst 33342 transport by the ABCG2-R482 mutants It has been shown that Hoechst 33342 is a transported substrate of the wtABCG2 protein, as well as of its R482G and T mutant variants. Login to comment
167 We found that the R482G, M, S, T, D and N mutants were active in Hoechst transport, and there was no significant difference between the Hst-transport kinetics of these Arg-482 mutants and the wtABCG2. Login to comment
183 All Arg-482 variants (including R482G, T, wild-type, and K86M, as a negative control) were expressed in insect cells, which produce high levels of ABCG2 and allow the detailed characterization of the function of the protein. Login to comment
192 We have shown previously that the transported substrates could not stimulate the ATPase activity of the wtABCG2, while they stimulated the ATPase activity of the R482G and T mutants (26). Login to comment
204 As shown in Fig. 3, none of the Arg-482 mutants showed any MTX transport, similarly to the entirely inactive K86M mutant and the R482G or T mutants [6,26]. Login to comment
208 4 and 5), we found that most of the Arg-482 mutants (R482G, I, M, S, T, D and N) were able to actively extrude these compounds. Login to comment
224 Mutants R482G, M, S, T, D and N were shown to confer higher resistance against mitoxantrone (MX) and doxorubicin than the wtABCG2 [22]. Login to comment
231 As shown recently, ABCG2-R482G has been successfully applied as a selectable marker protein in ex vivo gene transfer experiments. Login to comment

[hide] ABCG2-mediated transport of photosensitizers: pote... Cancer Biol Ther. 2005 Feb;4(2):187-94. Epub 2005 Feb 8. Robey RW, Steadman K, Polgar O, Bates SE
ABCG2-mediated transport of photosensitizers: potential impact on photodynamic therapy.
Cancer Biol Ther. 2005 Feb;4(2):187-94. Epub 2005 Feb 8., [PMID:15684613]

Abstract [show]
In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered followed by activation of the photosensitizer by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and 5-aminolevulinic acid, respectively. ABCG2-transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither multidrug resistance-associated protein 1 expression nor P-glycoprotein expression appreciably decreased the intracellular fluorescence of any of the photosensitizers examined as determined by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.

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90 Mutations in the ABCG2 gene which cause amino acid changes at position 482 are known to alter the substrate specificity of the protein.26,27,34 Thus, accumulation studies with the photosensitizers were repeated in HEK-293 cells stably transfected with wild-type (R482) or mutant (R482G, R482T) ABCG2. Login to comment
94 ABCG2-transfected HEK-293 cells expressing comparable levels of wild-type (482R) or mutant (R482G, R482T) ABCG2 were incubated in the desired photosensitizer (50 µM Ce6, 10 µM MPPa, 25 µM m-THPP, 25 µM m-THPC or 25 µM HpIX) for 30 min with or without 10 µM FTC, washed, and then incubated for 60 min continuing with (dashed line) or without (solid line) FTC. Login to comment

[hide] Breast cancer resistance protein (BCRP/ABCG2). Int J Biochem Cell Biol. 2005 Apr;37(4):720-5. Staud F, Pavek P
Breast cancer resistance protein (BCRP/ABCG2).
Int J Biochem Cell Biol. 2005 Apr;37(4):720-5., [PMID:15694832]

Abstract [show]
Breast cancer resistance protein (BCRP) was identified 7 years ago as the most recent member of ABC drug efflux membrane transporters. It is a 655 amino acid peptide with an ability to extrude a wide variety of chemical compounds from the cells. Today, it is considered as one of three major transporters causing drug resistance in mammalian cells. It is also distributed in epithelia involved in drug disposition with major role in the placenta, liver and intestine. In addition, BCRP is responsible for the "side population" phenotype of stem cells and seems to play a significant role in protection against hypoxia. BCRP inhibitors are currently searched for to overcome drug resistance and to improve the pharmacokinetics, mainly intestinal absorption, of substrate drugs. Mutant BCRP has also been used as a selectable marker in stem cell gene therapy applications.

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57 The fact that R482G variant of BCRP has substrate specificity different from the wild-type protein holds a special potential for gene therapy applications, which has been exploited in a recent study by Ujhelly et al. (2003). Login to comment

[hide] Expression, localization, and functional character... Drug Metab Dispos. 2005 May;33(5):637-43. Epub 2005 Feb 16. Xia CQ, Liu N, Yang D, Miwa G, Gan LS
Expression, localization, and functional characteristics of breast cancer resistance protein in Caco-2 cells.
Drug Metab Dispos. 2005 May;33(5):637-43. Epub 2005 Feb 16., [PMID:15716365]

Abstract [show]
The function of breast cancer resistance protein (BCRP) and its role in drug absorption, distribution, and elimination has recently been evaluated. The objective of the present study was to examine the expression, localization, and functional characteristics of BCRP in Caco-2 cells, a widely used human intestinal epithelial cell model for investigating intestinal drug absorption. The expression of BCRP in Caco-2 cells was measured by Western blotting using the antibody BXP-21. Localization of BCRP was determined by an immunofluorescence technique using both antibodies BXP-21 and BXP-34. The drug efflux function of BCRP was evaluated via the epithelial transport of methotrexate (MTX) and estrone-3-sulfate (E3S) across Caco-2 cell monolayers in the presence or absence of the BCRP inhibitors Ko143 or GF120918 (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)- 9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide). Results from Western blot assay indicated that Caco-2 cells in the late passage (p56) expressed a higher level of BCRP as compared with the level in the early passages (p33). The total amount of BCRP protein did not change after the cells were confluent. Immunofluorescence studies revealed the positive staining of BCRP on the apical membrane of Caco-2 cells but not on the basolateral membrane after cell confluence. MTX and E3S showed a preferential basolateral-toapical (B-to-A) transport across Caco-2 cell monolayers. Both BCRP inhibitors Ko143 and GF120918 increased the apical-to-basolateral (A-to-B) transport but decreased the B-to-A transport of MTX and E3S. Caco-2 cells may therefore be used as an in vitro model to study the transport characteristics of BCRP.

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192 The BCRP-mediated efflux of MTX in our Caco-2 cells indicated that the BCRP expressed on Caco-2 cells might be the wild type because R482T and R482G mutation were unable to transport MTX to any extent (Chen et al., 2003). Login to comment
193 The conclusion is further demonstrated by the efflux of rhodamine 123, to which BCRP-conferred resistance is observed in the R482T or R482G mutation but not the wild-type expressed cell lines (Allen et al., 2002a). Login to comment

[hide] Effect of Walker A mutation (K86M) on oligomerizat... J Cell Sci. 2005 Apr 1;118(Pt 7):1417-26. Epub 2005 Mar 15. Henriksen U, Gether U, Litman T
Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2.
J Cell Sci. 2005 Apr 1;118(Pt 7):1417-26. Epub 2005 Mar 15., 2005-04-01 [PMID:15769853]

Abstract [show]
The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement of prazosin-stimulated ATPase activity revealed a dominant-negative effect of ABCG2-K86M on ABCG2-wt function in co-transfected HEK293 cells. This is consistent with the requirement for at least two active NBDs for transporter activity and suggests that the transporter is a functional dimer. Finally, we analyzed targeting of ABCG2-wt and ABCG2-K86M and observed that they localize to two distinct subcellular compartments: ABCG2-wt targets the cell surface whereas ABCG2-K86M is targeted to the Golgi apparatus followed by retrieval to the endoplasmic reticulum. This suggests an as yet unknown role of the NBDs in assisting proper surface targeting of ABC transporters.

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6 It should be noted that an amino acid substitution at position 482 distinguishes MXR (R482G), BCRP (R482T) and ABCP (R482, wt) (Allikmets et al., 1998; Doyle et al., 1998; Miyake et al., 1999), which are synonymous designations for ABCG2. Login to comment

[hide] N-Linked glycosylation of the human ABC transporte... Biochemistry. 2005 Apr 12;44(14):5420-9. Diop NK, Hrycyna CA
N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane.
Biochemistry. 2005 Apr 12;44(14):5420-9., 2005-04-12 [PMID:15807535]

Abstract [show]
The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa plasma membrane protein that can confer multidrug resistance to cells in culture when overexpressed. Both transiently and stably expressed ABCG2 are glycosylated, and treatment with peptide N-glycosidase F reduces the apparent molecular mass on SDS-PAGE gels to approximately 60 kDa. Sequence analysis revealed three potential N-linked glycosylation sites in human ABCG2 at amino acids 418, 557, and 596. Site-directed mutagenesis experiments, in which each Asn was changed to Gln independently, revealed that only asparagine 596 is N-linked glycosylated. These data provide the first direct identification of the modified residue in ABCG2 and evidence for the localization of loop 5 to the extracellular space, previously only predicted from hydropathy analysis. Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 (N596Q) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives. Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein. The ABCG2 (N596Q) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2. Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated ( approximately 2-fold) ATPase activities of ABCG2 (N596Q) were affected compared to glycosylated ABCG2. Although subtle defects in transporter trafficking and function may exist, these data taken together suggest that N-glycosylation at arginine 596 is not essential for the expression, trafficking to the plasma membrane, or the overall function of ABCG2.

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19 Arginine 482 (R482) has been reported in the literature as the wild-type form of ABCG2, while glycine 482 (R482G) and threonine 482 (R482T) arose as a result of drug selection (6-8). Login to comment
51 Single point mutations were introduced by sequence overlap extension (SOE) PCR using pTM1-ABCG2 (R482G) DNA as the expression vector template. Login to comment
113 The ABCG2 protein variant R482G was chosen for this study because it has broader substrate specificity than the wild-type ABCG2 (R482) and can transport the dye rhodamine 123, our model substrate (28). Login to comment
114 All N-glycosylation variants constructed and described here have ABCG2 (R482G) as the protein backbone, and in this study, the ABCG2 designation will refer to the ABCG2 (R482G) protein unless otherwise specified. Login to comment
180 The drug prazosin (20 µM), a substrate for ABCG2 (R482G) (30), was used to stimulate the ATPase activity. Login to comment

[hide] Single nucleotide polymorphisms modify the transpo... Cancer Chemother Pharmacol. 2005 Aug;56(2):161-72. Epub 2005 Apr 19. Morisaki K, Robey RW, Ozvegy-Laczka C, Honjo Y, Polgar O, Steadman K, Sarkadi B, Bates SE
Single nucleotide polymorphisms modify the transporter activity of ABCG2.
Cancer Chemother Pharmacol. 2005 Aug;56(2):161-72. Epub 2005 Apr 19., [PMID:15838659]

Abstract [show]
Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P = 0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.

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37 containing full-length ABCG2 encoding wild-type (R482), mutant (R482T, R482G), or SNP variants (V12M, Q141K, or D620N) of ABCG2. Login to comment
76 Cells were also infected with mutant R482G ABCG2 encoding a K86M mutation that has been shown to abolish the function of the resulting protein [34]. Login to comment
95 Results Establishment of stable transfectants We had previously generated stable transfectants containing empty vector, wild-type (R482) or mutant (R482G, R482T) ABCG2 [38]. Login to comment
122 Representative results are shown clones each of ABCG2-transfected cells expressing R482, R482T, R482G, V12M, Q141K or D620N ABCG2. Login to comment
132 Nearly all the values for the efflux and expression for cells transfected with mutant R482G and R482T ABCG2 fell above the regression line for wild-type ABCG2, confirming a gain-of-function in the transport of mitoxantrone in these cells. Login to comment
138 Efflux per unit expression values in cells transfected with mutant 482G and 482T ABCG2 were significantly higher than for wild-type ABCG2 (P=0.0003 R482G; P=0.0013 R482T). Login to comment
163 To examine whether the nonsynonymous SNPs in ABCG2 affect the transport of this compound, Hoechst 33342 dye transport was measured in intact Sf9 cells expressing wild-type, V12M, Q141K, or D620N ABCG2, as well as the nonfunctional mutant, R482G/K86M. Login to comment
167 In Sf9 cells expressing the R482G/K86M ATP binding-site mutant, Hoechst 33342 uptake was comparable to the level of Hoechst 33342 accumulation found in the Ko143-inhibited cells. Login to comment
189 Values from the experiment in a were obtained for ABCG2-transfected HEK-293 clones expressing varying levels of 482R, R482G, R482T, V12M, Q141K, and D620N ABCG2 and a box plot was generated. Login to comment

[hide] Mechanisms of resistance to anticancer drugs: the ... Pharmacogenomics. 2005 Mar;6(2):115-38. Lepper ER, Nooter K, Verweij J, Acharya MR, Figg WD, Sparreboom A
Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2.
Pharmacogenomics. 2005 Mar;6(2):115-38., [PMID:15882131]

Abstract [show]
ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein) and ABCG2 (breast cancer-resistance protein [BCRP], mitoxantrone-resistance protein [MXR], or ABC transporter in placenta [ABCP]), are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenetics of the ABC transporters ABCB1 and ABCG2, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.

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140 However, cells expressing either the Arg482Gly or Arg482Thr mutant both demonstrated greater resistance to mitoxantrone than the wild type, suggesting that the mutant is a better transporter for this agent [97]. Login to comment
157 Position in gene* Nucleotide‡ Region Wild-type allele Variant allele Amino acid Change -19572 to -19569 5`-Flanking region CTCA - CTCA deletion -19202 5` UTR G C -18845 5` UTR T C -18604 5` UTR A - Deletion -18482 -113 Exon 1 C T Non-coding -18398 -29 Exon 1 A G Non-coding 34 34 Exon 2 G A 12 Val to Met 71 71 Exon 2 C T 24 Ala to Val 114 114 Exon 2 T C 38 Synonymous 239 Intron 2 A G 7268 Intron 2 T C 7420 Intron 3 - T Insertion 8007 Intron 3 G A 8184 369 Exon 4 C T 123 Synonymous 8191 376 Exon 4 C T 126 Gln to Term 8825 421 Exon 5 C A 141 Gln to Lys 8862 458 Exon 5 C T 153 Thr to Met 8878 474 Exon 5 C T 158 Synonymous 8900 496 Exon 5 C G 166 Gln to Glu 18186 Intron 5 A G 18286 616 Exon 6 A C 206 Ile to Leu 18293 623 Exon 6 T C 208 Phe to Ser 21530 Intron 6 C T 21718 Intron 6 A G 21903 Intron 7 A G 24618 Intron 7 T A 26297 1098 Exon 9 G A 366 Synonymous 38389 1291 Exon 11 T C 431 Phe to Leu 38485 Intron 11 A G 40111 Intron 11 G A 40303 1425 Exon 12 A G 475 Synonymous 40322 1444 Exon 12 A G 482 Arg to Gly 40323 1445 Exon 12 G C 482 Arg to Thr 40343 1465 Exon 12 T C 489 Phe to Leu 40419 Intron 12 G T 42314 Intron 13 T G 44997 Intron 14 A G 45022 Intron 14 C T 45073 1768 Exon 15 A T 590 Asn to Tyr 47355 1858 Exon 16 G A 620 Asp to Asn 47734 2237 Exon 16 G T Non-coding 47890 2393 Exon 16 G T Non-coding 47891 2394 Exon 16 C A Non-coding ABC: ATP-binding cassette; UTR: Untranslated region. Login to comment

[hide] Flavonoid structure-activity studies identify 6-pr... Cancer Res. 2005 Jun 1;65(11):4852-60. Ahmed-Belkacem A, Pozza A, Munoz-Martinez F, Bates SE, Castanys S, Gamarro F, Di Pietro A, Perez-Victoria JM
Flavonoid structure-activity studies identify 6-prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein ABCG2.
Cancer Res. 2005 Jun 1;65(11):4852-60., 2005-06-01 [PMID:15930306]

Abstract [show]
Overexpression of breast cancer resistance protein ABCG2 confers multidrug resistance in cancer cells. The GF120918-sensitive drug efflux activity of human wild-type (R482) ABCG2-transfected cells was used for rational screening of inhibitory flavonoids and establishment of structure-activity relationships. Flavones were found more efficient than flavonols, isoflavones, and flavanones. Differentially substituted flavone derivatives indicated positive OH effects at position 5, in contrast to positions 3 and 7. A methoxy at position 7 was slightly positive in tectochrysin, whereas a strong positive effect was produced by prenylation at position 6. The potency of 6-prenylchrysin was comparable with that of GF120918 (IC50 = 0.3 micromol/L). Both 6-prenylchrysin and tectochrysin seemed specific for ABCG2 because no interaction was detected with either P-glycoprotein or MRP1. The ABCG2 resistance profile in vitro is altered by mutation at amino acid 482. The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Whereas GF120918 strongly inhibited the ATPase activity of wild-type ABCG2, neither 6-prenylchrysin nor tectochrysin altered the activity. In contrast, all three inhibitors stimulated the ATPase activity of mutant ABCG2. 6-Prenylchrysin at 0.5 micromol/L efficiently sensitized the growth of wild-type ABCG2-transfected cells to mitoxantrone, whereas higher concentrations were required for the mutant ones. In contrast, 1 micromol/L tectochrysin was sufficient to fully sensitize mutant ABCG2-transfected cells, whereas higher concentrations were required for the wild-type ones. Both flavones exhibited a lower intrinsic cytotoxicity than GF120918 and were apparently not transported by ABCG2. 6-Prenylchrysin and tectochrysin therefore constitute new and promising inhibitors for the reversal of ABCG2-mediated drug transport.

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24 The precise substrate profile depends on a hotspot mutation at position 482 such that methotrexate is only transported by wild-type ABCG2 (R482) and anthracyclines and rhodamine 123 only by mutant ABCG2 (R482T or R482G), whereas Hoechst33342 and mitoxantrone are transported by all variants (9). Login to comment

[hide] Oligomerization of the human ABC transporter ABCG2... Biochemistry. 2005 Aug 16;44(32):10893-904. Bhatia A, Schafer HJ, Hrycyna CA
Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers.
Biochemistry. 2005 Aug 16;44(32):10893-904., 2005-08-16 [PMID:16086592]

Abstract [show]
Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.

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47 All experiments detailed in this paper make use of these gain-of-function mutations, either R482T in the MCF-7/AdVp3000 cell line or R482G in the transiently transfected HeLa cells. Login to comment
49 HeLa cells (cervical epitheliod carcinoma) were cultured in DMEM complete media at 37 °C. ABCG2 (R482G) was overexpressed by transient transfection using the T7 vaccinia virus system as described previously (43). Login to comment
59 ABCG2-ABCG2 encodes two identical ABCG2 (R482G) proteins connected by a SacII restriction enzyme site which adds a proline-arginine linker (PR). Login to comment
60 ABCG2-M-ABCG2 encodes two ABCG2 (R482G) proteins linked by the sequence (PR-NEVELENAADE- SKSEIDALEMSSNDSRSSLIRKRSTRRSVRGSQAQDR- KLSTKEALD-PR) derived from the flexible linker region of human P-glycoprotein. Login to comment
61 ABCG2-P-ABCG2 encodes two ABCG2 (R482G) proteins linked by the sequence (PR- KRMKKRGVLTEKNANDPENVGERSDLSSDRKMLQ- ESSEEESDTYGEIGLSKSEAIFHWRNLCYEVQIKAE- PR) derived from the flexible linker region of yeast ABC transporter PDR5. Login to comment
64 ABCG2 (D210N) was constructed by site-directed mutagenesis of aspartate 210 of the parent plasmid pTM1-ABCG2 (R482G). Login to comment
67 ABCG2∆EC-C, which contains all three endogenous extracellular cysteines replaced with alanine (C592A, C603A, C608A), was constructed by site-directed mutagenesis of the parent plasmid pTM1-ABCG2 (R482G). Login to comment
109 RESULTS ABCG2 (R482G) Chimeric Dimers Are Expressed at the Cell Surface and Transport ABCG2 Substrates. Login to comment
111 In all of the experiments presented here involving transient expression of ABCG2, we used the ABCG2 (R482G) variant because it has a broader substrate specificity than the wild-type protein (39-41, 46). Login to comment
112 Throughout, the designation ABCG2 refers to the R482G variant unless otherwise noted. Login to comment
133 HeLa cells were co-infected/transfected with plasmid DNA constructs of the empty vector pTM1 (gray shaded peak), ABCG2 (R482G) (black thick line), ABCG2-ABCG2 (gray thick line), ABCG2-M-ABCG2 (black thin line), or ABCG2-P-ABCG2 (dashed line). Login to comment
136 HeLa cells were co-infected/transfected in six-well plates with plasmid DNA constructs of the empty vector pTM1 (lane 1), ABCG2 (R482G) (lane 2), ABCG2-ABCG2 (lane 3), ABCG2-M-ABCG2 (lane 4), and ABCG2-P-ABCG2 (lane 5). Login to comment
153 After 24 h at 32 °C, cells were harvested and incubated with the fluorescent ABCG2 (R482G) substrates rhodamine 123 or mitoxantrone for 30 min at 37 °C, followed by a second incubation for 30 min in drug-free media to allow for transporter-mediated efflux. Cellular drug accumulation was analyzed using flow cytometry. Login to comment
159 A Single Mutation in the Walker B Motif of ABCG2 (R482G) Monomers and Chimeric Dimers Abrogates Function but Not Cell Surface Expression. Login to comment
160 To examine the effects of mutations to the ATP binding site on ABCG2 function, we constructed a variant of the monomeric ABCG2 (R482G) in the Walker B motif (D210N), thought to be involved in magnesium binding. Login to comment
164 The ABCG2 (D210N) monomer is recognized better by the conformation-sensitive 5D3 antibody than the ABCG2 (R482G) protein, as demonstrated by the greater shift in fluorescence intensity, suggesting that the presence of the mutation may have caused a conformational change in the protein (47). Login to comment
186 HeLa cells were co-infected/transfected with plasmid DNA constructs of the empty vector pTM1 (gray shaded peak), ABCG2 (R482G) (black thick line), ABCG2-ABCG2 (gray thick line), ABCG2 (D210N) (black thin line), or ABCG2 (D210N)-ABCG2 (dashed line). Login to comment
189 HeLa cells were co-infected/transfected in six-well plates with plasmid DNA constructs containing ABCG2 (R482G) (lane 1), ABCG2 (D210N) (lane 2), ABCG2-ABCG2 (lane 3), and ABCG2 (D210N)-ABCG2 (lane 4). Login to comment
219 Crude HeLa cell membrane preparations of cells transiently overexpressing the ABCG2 and ABCG2∆EC-C were incubated in the presence (stimulated) and absence (basal) of 20 µM ABCG2 (R482G) substrate prazosin. Login to comment
220 The ABCG2∆EC-C variant demonstrated drug-stimulated ATPase activity comparable to the parent protein, ABCG2 (R482G), with perhaps a slight increase in the basal activity (Figure 5D). Login to comment
235 Basal (gray bars) and drug-stimulated ATPase activity (black bars) of pTM1 (negative control empty vector), ABCG2 (R482), and ABCG2∆EC-C in the ABCG2 (R482G) background. Login to comment
244 By cell surface biotinylation experiments, we showed that ABCG2 (D210N) is expressed at the surface similarly to ABCG2 (R482G) (Figure 2B) whereas ABCG2 (K86M) is found predominantly in the endoplasmic reticulum with a small percentage localized to the plasma membrane (26). Login to comment

[hide] Identification of intra- and intermolecular disulf... J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17. Henriksen U, Fog JU, Litman T, Gether U
Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2.
J Biol Chem. 2005 Nov 4;280(44):36926-34. Epub 2005 Aug 17., 2005-11-04 [PMID:16107343]

Abstract [show]
ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.

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32 MATERIALS AND METHODS Plasmids, Drugs, and Antibodies-pcDNA3.1(-)MXR (R482G) and fumitremorgin C (FTC) was kindly provided by Dr. Susan Bates, NCI, National Institutes of Health. Login to comment

[hide] Breast cancer resistance protein: molecular target... Cancer Sci. 2005 Aug;96(8):457-65. Sugimoto Y, Tsukahara S, Ishikawa E, Mitsuhashi J
Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/pharmacodynamics.
Cancer Sci. 2005 Aug;96(8):457-65., [PMID:16108826]

Abstract [show]
Breast cancer resistance protein (BCRP) is a half-molecule ATP-binding cassette transporter that forms a functional homodimer and pumps out various anticancer agents, such as 7-ethyl-10-hydroxycamptothecin, topotecan, mitoxantrone and flavopiridol, from cells. Estrogens, such as estrone and 17beta-estradiol, have been found to restore drug sensitivity levels in BCRP-transduced cells by increasing the cellular accumulation of such agents. Furthermore, synthetic estrogens, tamoxifen derivatives and phytoestrogens/flavonoids have now been identified that can effectively circumvent BCRP-mediated drug resistance. Transcellular transport experiments have shown that BCRP transports sulfated estrogens and various sulfated steroidal compounds, but not free estrogens. The kinase inhibitor gefitinib inhibited the transporter function of BCRP and reversed BCRP-mediated drug resistance both in vitro and in vivo. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) and BCRP-transduced human non-small cell lung cancer PC-9 (PC-9/BCRP) cells showed gefitinib resistance. Physiological concentrations of estrogens (10-100 pM) reduced BCRP protein expression without affecting its mRNA levels. Two functional polymorphisms of the BCRP gene have been identified. The C376T (Q126Stop) polymorphism has a dramatic phenotype as active BCRP protein cannot be expressed from a C376T allele. The C421A (Q141K) polymorphism is also significant as Q141K-BCRP-transfected cells show markedly low protein expression levels and low-level drug resistance. Hence, individuals with C376T or C421A polymorphisms may express low levels of BCRP or none at all, resulting in hypersensitivity of normal cells to BCRP-substrate anticancer agents. In summary, both modulators of BCRP and functional single nucleotide polymorphisms within the BCRP gene affect the transporter function of the protein and thus can modulate drug sensitivity and substrate pharmacokinetics and pharmacodynamics in affected cells and individuals.

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64 Cells transfected with R482G-BCRP or R482S-BCRP cDNA showed less intracellular accumulation of 3 H-mitoxantrone than wild-type BCRP-transfected cells. Login to comment

[hide] Role of the breast cancer resistance protein (ABCG... AAPS J. 2005 May 11;7(1):E118-33. Mao Q, Unadkat JD
Role of the breast cancer resistance protein (ABCG2) in drug transport.
AAPS J. 2005 May 11;7(1):E118-33., [PMID:16146333]

Abstract [show]
The 72-kDa breast cancer resistance protein (BCRP) is the second member of the subfamily G of the human ATP binding cassette (ABC) transporter superfamily and thus also designated as ABCG2. Unlike P-glycoprotein and MRP1, which are arranged in 2 repeated halves, BCRP is a half-transporter consisting of only 1 nucleotide binding domain followed by 1 membrane-spanning domain. Current experimental evidence suggests that BCRP may function as a homodimer or homotetramer. Overexpression of BCRP is associated with high levels of resistance to a variety of anticancer agents, including anthracyclines, mitoxantrone, and the camptothecins, by enhancing drug efflux. BCRP expression has been detected in a large number of hematological malignancies and solid tumors, indicating that this transporter may play an important role in clinical drug resistance of cancers. In addition to its role to confer resistance against chemotherapeutic agents, BCRP actively transports structurally diverse organic molecules, conjugated or unconjugated, such as estrone-3-sulfate, 17beta-estradiol 17-(beta-D-glucuronide), and methotrexate. BCRP is highly expressed in the placental syncytiotrophoblasts, in the apical membrane of the epithelium in the small intestine, in the liver canalicular membrane, and at the luminal surface of the endothelial cells of human brain microvessels. This strategic and substantial tissue localization indicates that BCRP also plays an important role in absorption, distribution, and elimination of drugs that are BCRP substrates. This review summarizes current knowledge of BCRP and its relevance to multidrug resistance and drug disposition.

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91 Hence, anthracyclines do not appear to be transported by wild-type BCRP but are substrates of the BCRP mutants R482G and R482T. Login to comment
96 Robey et al36 showed that rhodamine 123 and Lyso-Tracker Green are substrates of BCRP mutants R482G and R482T but not substrates of the wild-type protein. Login to comment

[hide] The ABC transporter Abcg2/Bcrp: role in hypoxia me... Biometals. 2005 Aug;18(4):349-58. Krishnamurthy P, Schuetz JD
The ABC transporter Abcg2/Bcrp: role in hypoxia mediated survival.
Biometals. 2005 Aug;18(4):349-58., [PMID:16158227]

Abstract [show]
ABC (ATP-binding cassette) transporters have diverse roles in many cellular processes. These diverse roles require the presence of conserved membrane spanning domains and nucleotide binding domains. Bcrp (Abcg2) is a member of the ATP binding cassette family of plasma membrane transporters that was originally discovered for its ability to confer drug resistance in tumor cells. Subsequent studies showed Bcrp expression in normal tissues and high expression in primitive stem cells. Bcrp expression is induced under low oxygen conditions consistent with its high expression in tissues exposed to low oxygen environments. Moreover, Bcrp interacts with heme and other porphyrins. This finding and its regulation by hypoxia suggests it may play a role in protecting cells/tissue from protoporphyrin accumulation under hypoxia. These observations are strengthened by the fact that porphyrins accumulate in tissues of the Bcrp knockout mouse. It is possible that humans with loss of function Bcrp alleles may be more susceptible to porphyrin-induced phototoxicity. We propose that Bcrp plays a role in porphyrin homoeostasis and regulates survival under low oxygen conditions.

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115 The mutants having R482G or R482T (R482M or R482S in the mouse Bcrp) showed altered transport properties as compared to the wild-type protein (Honjo et al. 2001; Allen et al. 2002). Login to comment
117 However, the R482G and R482T mutants were not able to transport methotrexate, which is a substrate only transported by the wild-type Bcrp (Volk et al. 2002; Chen et al. 2003). Login to comment

[hide] Pharmacogenomics of the human ABC transporter ABCG... Naturwissenschaften. 2005 Oct;92(10):451-63. Ishikawa T, Tamura A, Saito H, Wakabayashi K, Nakagawa H
Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design.
Naturwissenschaften. 2005 Oct;92(10):451-63., [PMID:16160819]

Abstract [show]
In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

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118 For this purpose, we have created variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, E334stop, N590Y, D620N, R482G, and R482T) by site-directed mutagenesis. Login to comment
132 No MTX-transport activity was observed in the Q126stop and E334stop variants, as well as in acquired mutation variants (R482G and R482T). Login to comment
133 Several laboratories have shown that R482G and R482T mutations greatly affect the substrate specificity of ABCG2, suggesting a critical role of the arginine-482 residue in the ABCG2 protein (Mitomo et al. 2003; ¨Ozvegy et al. 2002; Volk et al. 2002; Chen et al. 2003). Login to comment
141 R482G and R482T are acquired mutations. Login to comment

[hide] Membrane transporters and channels in chemoresista... Cancer Lett. 2006 Aug 8;239(2):168-82. Epub 2005 Oct 5. Huang Y, Sadee W
Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells.
Cancer Lett. 2006 Aug 8;239(2):168-82. Epub 2005 Oct 5., 2006-08-08 [PMID:16169662]

Abstract [show]
Membrane transporters play important roles in mediating chemosensitivity and -resistance of tumor cells. ABC transporters, such as ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP, are frequently associated with decreased cellular accumulation of anticancer drugs and multidrug resistance of tumors. SLC transporters, such as folate, nucleoside, and amino acid transporters, commonly increase chemosensitivity by mediating the cellular uptake of hydrophilic drugs. Ion channels and pumps variably affect sensitivity to anticancer therapy by modulating viability of tumor cells. A pharmacogenomic approach, using correlations between drug potency and transporter gene expression in multiple cancer cell lines, has shown promise for identifying potential drug-transporter relationships and predicting anticancer drug response, in an effort to optimize chemotherapy for individual patients.

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76 A mutational hot spot is located in position 482 of ABCG2 where a single amino acid change (R482G or R482T) occurs [25]. Login to comment

[hide] Genetic polymorphisms of ATP-binding cassette tran... Expert Opin Pharmacother. 2005 Nov;6(14):2455-73. Sakurai A, Tamura A, Onishi Y, Ishikawa T
Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications.
Expert Opin Pharmacother. 2005 Nov;6(14):2455-73., [PMID:16259577]

Abstract [show]
Pharmacogenomics, the study of the influence of genetic factors on drug action, is increasingly important for predicting pharmacokinetics profiles and/or adverse reactions to drugs. Drug transporters, as well as drug metabolism play pivotal roles in determining the pharmacokinetic profiles of drugs and their overall pharmacological effects. There is an increasing number of reports addressing genetic polymorphisms of drug transporters. However, information regarding the functional impact of genetic polymorphisms in drug transporter genes is still limited. Detailed functional analysis in vitro may provide clear insight into the biochemical and therapeutic significance of genetic polymorphisms. This review addresses functional aspects of the genetic polymorphisms of human ATP-binding cassette transporters, ABCB1 and ABCG2, which are critically involved in the pharmacokinetics of drugs.

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249 R482G and R482T are acquired mutations. Login to comment
250 COOH H2N N590Y V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L F489L D620N R482G R482T S441N F571I EXTRACELLULAR INTRACELLULAR R160Q R575stop ATP-binding site (transient or stable expression), the copy number of cDNA incorporated in genomic DNA or other cellular determinants may variably affect the cellular processing and sorting of these proteins. Login to comment
255 For this purpose, variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, E334stop, N590Y, D620N, R482G and R482T) were created by site-directed mutagenesis (Figure 3). Login to comment
269 No MTX-transport activity was observed in the Q126stop and E334stop variants, as well as in acquired mutation variants (R482G and R482T). Login to comment
270 Several laboratories have shown that the R482G and R482T mutations greatly affect the substrate specificity of ABCG2, suggesting a critical role for the arginine-482 residue in the ABCG2 protein [140,144,145]. Login to comment

[hide] Use of P-glycoprotein and BCRP inhibitors to impro... Trends Pharmacol Sci. 2006 Jan;27(1):17-24. Epub 2005 Dec 5. Breedveld P, Beijnen JH, Schellens JH
Use of P-glycoprotein and BCRP inhibitors to improve oral bioavailability and CNS penetration of anticancer drugs.
Trends Pharmacol Sci. 2006 Jan;27(1):17-24. Epub 2005 Dec 5., [PMID:16337012]

Abstract [show]
P-glycoprotein (ABCB1) and breast cancer resistance protein [BCRP (also known as ABCG2)] are drug efflux transporters of the ATP binding cassette (ABC) family of proteins. Both P-glycoprotein and BCRP are located in the apical membrane of epithelial cells (e.g. in the intestinal wall and blood-brain barrier), where they can actively extrude a variety of structurally diverse drugs and drug metabolites. Consequently, the oral uptake and CNS penetration of substrate drugs can be low and variable. Inhibition of P-glycoprotein and/or BCRP is therefore a logical strategy to improve oral absorption, CNS penetration and delivery of anticancer agents to brain tumors or CNS metastases. As outlined in this review, this concept of improved oral pharmacokinetics has been demonstrated extensively for the anticancer drugs paclitaxel and topotecan both in preclinical models and in patients, and improved CNS penetration has been shown for paclitaxel, docetaxel and imatinib in preclinical models.

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35 Later studies revealed that the first cloned BCRP cDNA [28] encoded a mutant BCRP that deviated from the 'wild-type` BCRP at Arg482 (R482), which was replaced with either threonine (R482T) or glycine (R482G) [32,33]. Login to comment

[hide] The role of the human ABCG2 multidrug transporter ... Cancer Lett. 2006 Mar 8;234(1):62-72. Epub 2005 Dec 7. Cervenak J, Andrikovics H, Ozvegy-Laczka C, Tordai A, Nemet K, Varadi A, Sarkadi B
The role of the human ABCG2 multidrug transporter and its variants in cancer therapy and toxicology.
Cancer Lett. 2006 Mar 8;234(1):62-72. Epub 2005 Dec 7., 2006-03-08 [PMID:16337740]

Abstract [show]
The human multidrug resistance ABC transporters provide a protective function in our body against a large number of toxic compounds. These proteins, residing in the plasma membrane, perform an active, ATP-dependent extrusion of such xenobiotics. However, the same proteins are also used by the tumor cells to fight various anticancer agents. ABCG2 is an important member of the multidrug resistance proteins, an 'ABC half transporter', which functions as a homodimer in the cell membrane. In this review, we provide a basic overview of ABCG2 function in physiology and drug metabolism, but concentrate on the discussion of mutations and polymorphisms discovered in this protein. Interestingly, a single nucleotide mutation, changing amino acid 482 from arginine to threonine or glycine in ABCG2, results in a major increase in the catalytic activity and a wider drug recognition by this protein. Still, this mutation proved to be an in vitro artifact, produced only in heavily drug-selected cell lines. In contrast, at least two, but possibly more polymorphic variants of ABCG2 were found to be present in large human populations with different ethnic background. However, currently available experimental data regarding the cellular expression, localization and function of these ABCG2 variants are strongly contradictory. Since, the proteins produced by these variant alleles may differently modulate cancer treatment, general drug absorption and toxicity, may represent risk factors in fetal toxicity, or alter the differentiation of stem cells, their exact characterization is a major challenge in this field.

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80 The cDNA sequence analysis of the MCF-7/AdVP3000 human breast cancer cell line and the S1M1-80 human colon cancer cell line revealed that in these cell lines, each showing anthracycline resistance and an ability to extrude rhodamine 123, a single amino acid change occurred at position 482, resulting an R482T (c.1445GOC) mutant in the MCF-7/AdVP3000 cell line, an R482G replacement (c.1444AOG) in S1M1-80 cells, and an R482M substitution (c.1445GOT) in the MT-4/DOX500 human T cell line [27,41,54]. Login to comment
87 In vitro experiments revealed that the expression of the wild-type, R482G and R482T variant forms of ABCG2 in HEK-293 cells conferred 15-fold, 47-fold, and 54-fold resistance against mitoxantrone, respectively [50]. Login to comment
88 It was also demonstrated that the R482G and R482T mutants showed an increased transport activity for various compounds and increased ATP hydrolytic activity in Sf9 cell membranes [51]. Login to comment

[hide] High-speed screening of human ATP-binding cassette... Methods Enzymol. 2005;400:485-510. Ishikawa T, Sakurai A, Kanamori Y, Nagakura M, Hirano H, Takarada Y, Yamada K, Fukushima K, Kitajima M
High-speed screening of human ATP-binding cassette transporter function and genetic polymorphisms: new strategies in pharmacogenomics.
Methods Enzymol. 2005;400:485-510., [PMID:16399366]

Abstract [show]
Drug transporters represent an important mechanism in cellular uptake and efflux of drugs and their metabolites. Hitherto a variety of drug transporter genes have been cloned and classified into either solute carriers or ATP-binding cassette (ABC) transporters. Such drug transporters are expressed in various tissues such as the intestine, brain, liver, kidney, and, importantly, cancer cells, where they play critical roles in the absorption, distribution, and excretion of drugs. We developed high-speed functional screening and quantitative structure-activity relationship analysis methods to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide powerful and practical tools for screening synthetic and natural compounds, and the deduced data can be applied to the molecular design of new drugs. Furthermore, we demonstrate a new "SNP array" method to detect genetic polymorphisms of ABC transporters in human samples.

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115 For this purpose, variant forms (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, E334stop, N590Y, D620N, R482G, and R482T) have been created by site‐ directed mutagenesis with the QuikChange site‐directed mutagensis kit (Stratagene, La Jolla, CA). Login to comment
125 No MTX transport activity was observed in the variants Q126stop, E334stop, R482G, and R482T. Login to comment
136 R482G and R482T are acquired mutations. Login to comment

[hide] Cyclosporin A, tacrolimus and sirolimus are potent... Cancer Chemother Pharmacol. 2006 Sep;58(3):374-83. Epub 2006 Jan 11. Gupta A, Dai Y, Vethanayagam RR, Hebert MF, Thummel KE, Unadkat JD, Ross DD, Mao Q
Cyclosporin A, tacrolimus and sirolimus are potent inhibitors of the human breast cancer resistance protein (ABCG2) and reverse resistance to mitoxantrone and topotecan.
Cancer Chemother Pharmacol. 2006 Sep;58(3):374-83. Epub 2006 Jan 11., [PMID:16404634]

Abstract [show]
PURPOSE: Several studies have demonstrated significant interactions between immunosuppressants (e.g., cyclosporin A) and chemotherapeutic drugs that are BCRP substrates (e.g., irinotecan), resulting in increased bioavailability and reduced clearance of these agents. One possible mechanism underlying this observation is that the immunosuppressants modulate the pharmacokinetics of these drugs by inhibiting BCRP. Therefore, the aim of this study was to determine whether the immunosuppressants cyclosporin A, tacrolimus and sirolimus are inhibitors and/or substrates of BCRP. METHODS: First, the effect of the immunosuppressants on BCRP efflux activity in BCRP-expressing HEK cells was measured by flow cytometry. RESULTS: Cyclosporin A, tacrolimus and sirolimus significantly inhibited BCRP-mediated efflux of pheophorbide A, mitoxantrone and BODIPY-prazosin. The EC(50) values of cyclosporin A, tacrolimus and sirolimus for inhibition of BCRP-mediated pheophorbide A efflux were 4.3 +/- 1.9 microM, 3.6 +/- 1.8 microM and 1.9 +/- 0.4 microM, respectively. Cyclosporin A, tacrolimus and sirolimus also effectively reversed resistance of HEK cells to topotecan and mitoxantrone conferred by BCRP. When direct efflux of cyclosporin A, tacrolimus and sirolimus was measured, these compounds were found not to be transported by BCRP. Consistent with this finding, BCRP did not confer resistance to the immunosuppressants in HEK cells. CONCLUSION: These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs.

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163 CsA has been shown to inhibit ATPase activity of wild-type BCRP and its R482G mutant [27, 30], therefore, it is presumed Table 1 Effect of CsA, tacrolimus and sirolimus on mitoxantrone and topotecan toxicity in HEK/482R and HEK/vector control cells DMSO CsA Tacrolimus Sirolimus 2 lM 5 lM 2 lM 5 lM 2 lM 5 lM IC50 (nM) of topotecan HEK/482R 182.1±30.2 66.7±11.2* 47.3±13.4* 45.7±14.6* 22.5±17.1** 39.8±25.1* 17.8±14.4** HEK/vector 10.9±0.4 11.9±0.9 13.2±4.4 9.1±2.7 12.9±4.0 12.0±4.0 10.4±1.7 RR 16.7 5.6 3.6 5.0 1.7 2.3 1.7 IC50 (nM) of mitoxantrone HEK/482R 2574.9±2134.6 63.6±9.7* 36.1±17.8* 64.8±35.7* 17.1±13.8* 51.4±37.3* 22.6±12.8* HEK/vector 198.8±105.7 14.9±13.2* 21.9±18.1* 24.9±30.4* 16.9±10.5* 20.5±17.9* 24.2±38.2* RR 13.0 4.3 1.7 2.6 1.0 2.5 0.9 Cytotoxicity studies were performed and IC50 values (mean ± S.D., n=3-4 independent experiments) were determined as described in ''Materials and methods``. Login to comment

[hide] Modulation of p-glycoprotein and breast cancer res... Eur J Pharmacol. 2006 Feb 15;531(1-3):25-33. Epub 2006 Jan 25. Cooray HC, Shahi S, Cahn AP, van Veen HW, Hladky SB, Barrand MA
Modulation of p-glycoprotein and breast cancer resistance protein by some prescribed corticosteroids.
Eur J Pharmacol. 2006 Feb 15;531(1-3):25-33. Epub 2006 Jan 25., 2006-02-15 [PMID:16442095]

Abstract [show]
Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood-brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamic-pituitary-adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 microM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 microM the same three increased sensitivity of p-glycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

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46 Bacterial cell culture, preparation of inside-out membrane vesicles and ATPase assay The R482G-BCRP gene (glycine mutant) was amplified from pcDNA3-BCRP by PCR and cloned into the lactococcal pNZ8048 expression vector to give pNZ-BCRP as described (Janvilisri et al., 2003). Login to comment

[hide] A new strategy of high-speed screening and quantit... J Pharmacol Exp Ther. 2006 Jun;317(3):1114-24. Epub 2006 Feb 17. Saito H, Hirano H, Nakagawa H, Fukami T, Oosumi K, Murakami K, Kimura H, Kouchi T, Konomi M, Tao E, Tsujikawa N, Tarui S, Nagakura M, Osumi M, Ishikawa T
A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions.
J Pharmacol Exp Ther. 2006 Jun;317(3):1114-24. Epub 2006 Feb 17., [PMID:16489126]

Abstract [show]
The human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR1/ABCP) plays a critical role in cellular protection against xenobiotics as well as pharmacokinetics of drugs in our body. In the present study, we aimed to analyze the quantitative structure-activity relationship (QSAR) latently residing in ABCG2-drug interactions. We first established standard methods for expression of human ABCG2 in insect cells, quality control of plasma membrane samples by using electron microscopy techniques, and high-speed screening of ABCG2 inhibition with test compounds. Plasma membrane vesicles prepared from ABCG2-expressing Sf9 cells were used as a model system to measure the ATP-dependent transport of [3H]methotrexate (MTX). Forty-nine different therapeutic drugs and natural compounds were tested for their ability to inhibit ABCG2-mediated MTX transport. Based on their inhibition profiles, we performed QSAR analysis using chemical fragmentation codes deduced from the structures of test compounds. Multiple linear regression analysis delineated a relationship between the structural components and the extent of ABCG2 inhibition, allowing us to identify one set of structure-specific chemical fragmentation codes that are closely correlated with the inhibition of ABCG2 transport activity. Based on the QSAR analysis data, we predicted the potency of gefitinib to inhibit ABCG2. The validity of our QSAR-based prediction for gefitinib was examined by actual experiments. Our kinetic analysis experiments suggest that the ABCG2-ATP complex binds gefitinib. The present study provides a new strategy for analyzing ABCG2-drug interactions. This strategy is considered to be practical and useful for the molecular designing of new ABCG2 modulators.

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202 The wild type of ABCG2 transports MTX, whereas acquired mutants, i.e., R482G and R482T, do not (Mitomo et al., 2003). Login to comment

[hide] Inhibitors of cancer cell multidrug resistance med... Anticancer Drugs. 2006 Mar;17(3):239-43. Ahmed-Belkacem A, Pozza A, Macalou S, Perez-Victoria JM, Boumendjel A, Di Pietro A
Inhibitors of cancer cell multidrug resistance mediated by breast cancer resistance protein (BCRP/ABCG2).
Anticancer Drugs. 2006 Mar;17(3):239-43., [PMID:16520651]

Abstract [show]
Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP-binding cassette (ABC) transporter superfamily. It is able to efflux a broad range of anti-cancer drugs through the cellular membrane, thus limiting their anti-proliferative effects. Due to its relatively recent discovery in 1998, and in contrast to the other ABC transporters P-glycoprotein (MDR1/ABCB1) and multidrug resistance-associated protein (MRP1/ABCC1), only a few BCRP inhibitors have been reported. This review summarizes the known classes of inhibitors that are either specific for BCRP or also inhibit the other multidrug resistance ABC transporters. Information is presented on structure-activity relationship aspects and how modulators may interact with BCRP.

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15 In addition, the effects of the hotspot mutation R482G/T, which are well known to change the pattern of transported substrates, have not been systematically characterized for the inhibitors. Login to comment
19 It chemosensitized S1M1-3.2 and MCF-7/BCRP cells to mitoxantrone, doxorubicin and topotecan, promoted the accumulation of radioactive doxorubicin, and also efficiently inhibited (IC50 = 1.3 mmol/l) the ATPase activity of insect cell membranes enriched with recombinant R482G mutant ABCG2 [8]. Login to comment
34 Similar effects were produced by homocamptothecins, containing a seven-membered ring E [15]; interestingly, the best compound, BN-80915, was better transported by mutant (R482G/T) than wild-type (R482) ABCG2. Login to comment

[hide] Functional validation of the genetic polymorphisms... Mol Pharmacol. 2006 Jul;70(1):287-96. Epub 2006 Apr 11. Tamura A, Watanabe M, Saito H, Nakagawa H, Kamachi T, Okura I, Ishikawa T
Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport.
Mol Pharmacol. 2006 Jul;70(1):287-96. Epub 2006 Apr 11., [PMID:16608919]

Abstract [show]
The ATP-binding cassette (ABC) transporter ABCG2 has been implicated to play a significant role in the response of patients to medication and/or the risk of diseases. To clarify the possible physiological or pathological relevance of ABCG2 polymorphisms, we have functionally validated single nucleotide polymorphisms (SNP) of ABCG2. In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Because porphyrins are considered to be endogenous substrates for ABCG2, we have investigated the porphyrin transport activity of those variant forms in vitro. We herein provide evidence that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in porphyrin transport, whereas F489L exhibited impaired transport, approximately 10% of the activity observed for the wild type. Furthermore, Flp-In-293 cells expressing those variants were photosensitive. Thus, among those genetic polymorphisms of ABCG2, at least the hitherto validated alleles of Q126stop, S441N, and F489L are suggested to be of clinical importance related to the potential risk of porphyria.

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2 In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Login to comment
63 The variants R482G and R482T are acquired mutations. Login to comment
144 For this purpose, based on the currently available data on SNPs and acquired mutations, we generated variant forms (i.e., V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis. Login to comment
166 The F431L variant as well as the acquired mutants R482G and R482T transported hematoporphyrin (Fig. 5, top), although they did not transport methotrexate (Fig. 5, bottom). Login to comment
186 None of the SNP variants of F431L, S441N, and F489L conferred Flp-In-293 cells resistance to doxorubicin or daunorubicin (Table 3), being different from the acquired mutants of R482G and R482T (Yoshikawa et al., 2004). Login to comment
196 Acquired mutations (R482G and R482T) at amino acid 482 did not affect the transport of those photosensitizers, being consistent with our findings (Fig. 5). Login to comment
214 In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Login to comment

[hide] Genetic variation and haplotype structure of the A... Drug Metab Pharmacokinet. 2006 Apr;21(2):109-21. Maekawa K, Itoda M, Sai K, Saito Y, Kaniwa N, Shirao K, Hamaguchi T, Kunitoh H, Yamamoto N, Tamura T, Minami H, Kubota K, Ohtsu A, Yoshida T, Saijo N, Kamatani N, Ozawa S, Sawada J
Genetic variation and haplotype structure of the ABC transporter gene ABCG2 in a Japanese population.
Drug Metab Pharmacokinet. 2006 Apr;21(2):109-21., [PMID:16702730]

Abstract [show]
The ATP-binding cassette transporter, ABCG2, which is expressed at high levels in the intestine and liver, functions as an efflux transporter for many drugs, including clinically used anticancer agents such as topotecan and the active metabolite of irinotecan (SN-38). In this study, to elucidate the linkage disequilibrium (LD) profiles and haplotype structures of ABCG2, we have comprehensively searched for genetic variations in the putative promoter region, all the exons, and their flanking introns of ABCG2 from 177 Japanese cancer patients treated with irinotecan. Forty-three genetic variations, including 11 novel ones, were found: 5 in the 5'-flanking region, 13 in the coding exons, and 25 in the introns. In addition to 9 previously reported nonsynonymous single nucleotide polymorphisms (SNPs), 2 novel nonsynonymous SNPs, 38C>T (Ser13Leu) and 1060G>A (Gly354Arg), were found with minor allele frequencies of 0.3%. Based on the LD profiles between the SNPs and the estimated past recombination events, the region analyzed was divided into three blocks (Block -1, 1, and 2), each of which spans at least 0.2 kb, 46 kb, and 13 kb and contains 2, 24, and 17 variations, respectively. The two, eight, and five common haplotypes detected in 10 or more patients accounted for most (>90%) of the haplotypes inferred in Block -1, Block 1, and Block 2, respectively. The SNP and haplotype distributions in Japanese were different from those reported previously in Caucasians. This study provides fundamental information for the pharmacogenetic studies investigating the relationship between the genetic variations in ABCG2 and pharmacokinetic/pharmacodynamic parameters.

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17 In vitro studies have also indicated that a number of anticancer drugs are good substrates for ABCG2: e.g. topotecan, an irinotecan metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), and its glucuronide conjugate, SN-38G.810) Indeed, inhibition of the murine ABCG2 homologue, Bcrp 1, increases the bioavailability of topotecan when orally administered to mdr1aW1b- decient mice.11) In a clinical study, coadministration of topotecan with GF120918, a dual inhibitor for ABCG2 and P-glycoprotein, was shown to markedly increase the bioavailability and systemic exposure of topotecan.12) The cloning of ABCG2 from drug-selected cell lines revealed that acquired amino acid substitutions at residue 482 (Arg482Gly and Arg482Thr) of ABCG2 resulted in marked alterations in substrate recognition and transport ability.13) Thereafter, naturally occurring genetic variations in ABCG2 have been extensively examined in various ethnic populations1421) because they were expected to explain interindividual dierences in oral bioavailability and clearance of ABCG2 substrate drugs.22) Two nonsynonymous polymorphisms, 34GÀA (Val12Met) and 421CÀA (Gln141Lys), were detected at relatively high frequencies in most ethnic groups including Caucasians, Asians, and Africans.1416,1821,23) Both polymorphisms were reported to be associated with reduced protein expression in vitro andWor the increased sensitivity of the expressed cells toward several anticancer drugs although conicting data were also reported.16,2426) The expression of ABCG2 protein in placenta was signicantly lower in homozygotes with the 421A alleles than in those with the 421C alleles, while 34GÀA (Val12Met) did not aect ABCG2 protein expression.23) However, in intestinal samples, no association was found between the ABCG2 protein levels and the 421CÀA (Gln141Lys) genotype.18) A pharmacokinetic study showed that 421A (Gln141Lys) was unlikely to inuence the in vivo disposition of irinotecan in European Caucasian cancer patients.27) On the other hand, diomotecan pharmacokinetics were signicantly aected by the 421A genotype.28) To explain these inconsistencies, the elucidation of the haplotype structure of ABCG2 would be helpful; however, only limited information is available for the linkage disequilibrium (LD) prole and haplotype structure of this gene.20,21) Also, to facilitate future pharmacogenetic studies on ABCG2 genetic variations, haplotype analysis using its high-density SNPs found in a large number of samples is warranted. Login to comment

[hide] ATP-binding cassette transporter G2 mediates the e... Pharm Res. 2006 Jun;23(6):1235-42. Epub 2006 May 25. Asashima T, Hori S, Ohtsuki S, Tachikawa M, Watanabe M, Mukai C, Kitagaki S, Miyakoshi N, Terasaki T
ATP-binding cassette transporter G2 mediates the efflux of phototoxins on the luminal membrane of retinal capillary endothelial cells.
Pharm Res. 2006 Jun;23(6):1235-42. Epub 2006 May 25., [PMID:16715370]

Abstract [show]
PURPOSE: The purpose of this study was to clarify the localization and function of the ATP-binding cassette transporter G2 (ABCG2; BCRP/MXR/ABCP) in retinal capillary endothelial cells, which form the inner blood-retinal barrier, as an efflux transport system. METHODS: The expression was determined by reverse transcriptase polymerase chain reaction and Western blotting. The localization was identified by immunostaining. The transport function of ABCG2 was measured by flow cytometry. RESULTS: Western blotting indicated that ABCG2 was expressed as a glycosylated disulfide-linked complex in the mouse retina and in peripheral tissues, including liver, kidney, and small intestine. Double immunolabeling of ABCG2 and glucose transporter 1 suggested that ABCG2 was localized on the luminal membrane of mouse retinal capillary endothelial cells. ABCG2 mRNA and protein were found to be expressed in a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB, and rat retina. Treatment with Ko143, an ABCG2 inhibitor, restored the accumulation of pheophorbide a and protoporphyrin IX in TR-iBRB cells. CONCLUSION: ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where ABCG2 acts as the efflux transporter for photosensitive toxins such as pheophorbide a and protoporphyrin IX. ABCG2 could play an important role at the inner blood-retinal barrier in restricting the distribution of phototoxins and xenobiotics in retinal tissue.

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164 As far as R482 is concerned, the amino acid corresponding to R482 in the isolated rat ABCG2 is also arginine (14), and human ABCG2 variants of R482T and R482G have been reported to transport PhA (22). Login to comment

[hide] The nature of amino acid 482 of human ABCG2 affect... Protein Sci. 2006 Jul;15(7):1597-607. Ejendal KF, Diop NK, Schweiger LC, Hrycyna CA
The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding.
Protein Sci. 2006 Jul;15(7):1597-607., [PMID:16815914]

Abstract [show]
Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.

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7 Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125 I]iodoarylazidoprazosin. Login to comment
67 ABCG2 variants with amino acids containing side chains with a ring structure have a reduced ability to transport rhodamine 123, compared with those with a small amino acid at this position, such as the R482G variant. Login to comment
71 Analysis of the substrate binding properties of wild-type and six mutant ABCG2 proteins In order to further investigate the effects of the R482X mutations, we studied the drug-binding ability of a selection of ABCG2 mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type ABCG2 (R482wt). Login to comment
74 R482K was chosen because it is completely devoid of any transport, and the R482G and R482T mutants were chosen because these two mutants have been used in many different studies and would therefore be of broader interest. Login to comment
86 We analyzed expression of ABCG2 in the membranes using the monoclonal antibody BXP-21 (Fig. 5A), which shows that the R482G, R482wt, and R482T membranes used here express less ABCG2, compared with the membranes expressing the R482H, R482K, R482P, and R482Y variants. Login to comment
89 The R482G, R482P, and R482T variants are stimulated 1.9-, 2.1-, and 1.8-fold, respectively, by the addition of 20 mM prazosin. Login to comment
96 Specific [125 I]IAAP photoaffinity labeling of crude membranes derived from HeLa cells expressing wild-type ABCG2 (R482wt) and the ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y. Login to comment
106 Basal and drug-stimulated ATPase activity of wild-type ABCG2 (R482wt) and ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y. Login to comment
124 We have previously shown that the prazosin substrate analog [125 I]iodoarylazidoprazosin ([125 I]IAAP) can be photoaffinity cross-linked to R482wt, R482G, and R482T variants of ABCG2 when overexpressed in MCF-7/FLV1000, S1-M1-80, and MCF-7/AdVp3000 cells, respectively (Ejendal and Hrycyna 2005). Login to comment
131 Previously, we have shown that, out of seven agents tested, only prazosin and GF120918 can compete for [125 I]IAAP labeling of R482G, R482wt, and R482T and that known substrates such as rhodamine 123 and mitoxantrone do not (Ejendal and Hrycyna 2005). Login to comment
174 To ensure that this substitution does not affect the function or the surface expression of the ABCG2 protein, we mutated the alanine back to serine in the R482G variant of ABCG2 and performed flow cytometric analysis to test for both function and expression; the two constructs were indistinguishable (data not shown). Login to comment

[hide] Multidrug resistance: retrospect and prospects in ... Curr Med Chem. 2006;13(16):1859-76. Perez-Tomas R
Multidrug resistance: retrospect and prospects in anti-cancer drug treatment.
Curr Med Chem. 2006;13(16):1859-76., [PMID:16842198]

Abstract [show]
Conventional cancer chemotherapy is seriously limited by the multidrug resistance (MDR) commonly exhibited by tumour cells. One mechanism by which a living cell can achieve multiple resistances is via the active efflux of a broad range of anticancer drugs through the cellular membrane by MDR proteins. Such drugs are exported in both ATP-dependent and -independent manners, and can occur despite considerable concentration gradients. To the ATP-dependent group belongs the ATP-binding cassette (ABC) transporter family, which includes P-gp, MRP, BCRP, etc. Another protein related to MDR, though not belonging to the ABC transporter family, is lung resistance-related protein (LRP). All of these proteins are involved in diverse physiological processes, and are responsible for the uptake and efflux of a multitude of substances from cancer cells. Many inhibitors of MDR transporters have been identified over the years. Firstly, MDR drugs were not specifically developed for inhibiting MDR; in fact, they had other pharmacological properties, as well as a relatively low affinity for MDR transporters. They included compounds of diverse structure and function, such as verapamil and cyclosporine, and caused side effects. Secondly, the new drugs were more inhibitor-specific, in terms of MDR transport, and were designed to reduce such side effects (e.g., R-verapamil, dexniguldipine, etc.). Unfortunately, they displayed poor response in clinical studies. Recently, new compounds obtained from drug development programs conducted by the pharmaceutical industry are characterized by a high affinity to MDR transporters and are efficient at nanomolar concentrations. Some of these compounds (e.g., MS-209) are currently under clinical trials for specific forms of advanced cancers. We aim to provide an overview of the properties associated with those mammalian MDR transporters known to mediate significant transport of relevant drugs in cancer treatments. We also summarize recent advances concerning resistance to cancer drug therapies with respect to the function and overexpression of ABC and LRP multidrug transporters.

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216 Indeed, the R482G BCRP variant has been used as a selectable marker and co-transfected with a therapeutic gene in human haematopoietic stem cells. Login to comment

[hide] Breast cancer resistance protein in pharmacokineti... Expert Opin Drug Metab Toxicol. 2005 Dec;1(4):595-611. Xia CQ, Yang JJ, Gan LS
Breast cancer resistance protein in pharmacokinetics and drug-drug interactions.
Expert Opin Drug Metab Toxicol. 2005 Dec;1(4):595-611., [PMID:16863427]

Abstract [show]
Breast cancer resistance protein (BCRP), also known as ABCG2, ABCP and MXR, is a member of the ATP-binding cassette transporter G family. BCRP functions as a biological barrier that extrudes xenobiotics out of cells. The broad substrate specificity and tissue distributions of BCRP in the body make this transporter one of the major efflux transporters in chemotherapy. Recent studies have demonstrated that BCRP exerts a great impact on drug absorption and disposition. This review focuses on the role of BCRP in pharmacokinetics as well as in vitro and in vivo strategies to evaluate hepatic/intestinal BCRP-mediated drug transports and drug-drug interactions. The impacts of polymorphism and gender difference of BCRP are also discussed.

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53 R482T and R482G mutations confer strong resistance to anthracycline and rhodamine 123 [30], and are unable to transport methotrexate [31]. Login to comment
88 Breast cancer resistance protein modulators [8,38,124] Substrates Inhibitors Antitumour drugs Xenobiotics Mitoxantrone Fumitremorgin C and Ko-143 Topotecan GF-120918 Irinotecan BIB-E SN-38 and its glucuronide Flavopiridol 9-Aminocamptothecin CI-1033 Diflomotecan Novobiocin NB-506 Reserpine J-107088 Prazosin UCN-01 VX-710 and VX-853, diethylstillbesterol Doxorubicin* Tamoxifen and derivatives (TAG-111 and TAG-139), toremifene Daunorubicin* Imatinib Epirubicin* Gefitinib Idarubicinol* HIV protease inhibitors (e.g., ritonavir, saquinavir) Etoposide Tryprostatin Prazosin UCN-01 Flavopiridol Cyclosporin A CI-1033 Digoxin BBR-3390 Pantoprazole and omeprazole Methotrexate and polyglutamate methotrexate Statins (e.g., lovastatin, simvastatin, cerivastatin, pitavastatin) Bisantrene Steroids Imatinib Gefitinib Beclomethasone, 6α-methylprednisolone, corticosterone, triamcinolone, dexamethasone, betamethasone Nucleoside reverse transcriptase inhibitors Endogenous steroid and conjugates Zidovudine, lamivudine Oestrone-3-sulfate Dyes 17β-Oestradiol sulfate Rhodamine 123* 17β-Oestradiol dehydroepiandrosterone sulfate LysoTracker Taurolithocholate Hoechst 33342 Taurolithocholate sulfate Prazosin-BODIPY Dietary compounds Toxins Pheophorbide A Flavonoids (e.g. apigenin, biochanin A, chrysin, genistein, kaempferol, hesperetin, naringenin and silymarin) PhIP Isothiocyanates Endogenous substrates Dihydropyridines (e.g. niguldipine, nicardipine and nitrendipine) Oestrone sulfate 17β-Oestradiol sulfate 17β-Oestradiol glucuronide Folic acid Protiporphyrin IX Others Cimetidine *Substrates for R482G mutant breast cancer resistance protein. Login to comment
119 Breast cancer resistance protein modulators [8,38,124] (continued) Substrates Inhibitors *Substrates for R482G mutant breast cancer resistance protein. Login to comment
187 The R482T or R482G mutant of BCRP, in which the wild-type arginine on the amino acid position 482 is replaced by threonine or glycine, alters substrate specificity [30,31]. Login to comment

[hide] Human ABC transporter ABCG2 in xenobiotic protecti... Drug Metab Rev. 2006;38(3):371-91. Wakabayashi K, Tamura A, Saito H, Onishi Y, Ishikawa T
Human ABC transporter ABCG2 in xenobiotic protection and redox biology.
Drug Metab Rev. 2006;38(3):371-91., [PMID:16877258]

Abstract [show]
Human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR/ABCP) is regarded as a member of the phase III system of xenobiotic metabolism. This efflux pump is suggested to be responsible for protecting the body from toxic xenobiotics and for removing toxic metabolites. The aim of this review article is to address new aspects of ABCG2 related to redox biology, namely the posttranslational modification (intra- and intermolecular disulfide bond formation) of ABCG2 protein and the transport of porphyrin and chlorophyll metabolites, as well as the high-speed screening and QSAR analysis method to evaluate ABCG2-drug interactions.

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176 Based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in Sf9 insect cells. Login to comment
185 The wild type of ABCG2 transports MTX, whereas acquired mutants (i.e., R482G and R482T) do not (Chen et al., 2003; Mitomo et al., 2003; Volk and Schneider, 2003). Login to comment

[hide] Multiple drugbinding sites on the R482G isoform of... Br J Pharmacol. 2006 Nov;149(5):506-15. Epub 2006 Sep 18. Clark R, Kerr ID, Callaghan R
Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter.
Br J Pharmacol. 2006 Nov;149(5):506-15. Epub 2006 Sep 18., [PMID:16981002]

Abstract [show]
BACKGROUND & PURPOSE: Drug-resistant cancer cells frequently display efflux pumps such as P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1) or the transporter ABCG2. These transporters are each capable of mediating the active efflux of numerous anticancer drugs and display relatively distinct substrate preferences. The last, most recently discovered member, ABCG2, plays a major role in resistance in several types of cancer and the precise pharmacology of this multidrug transporter remain unresolved as does the nature of substrate binding. EXPERIMENTAL APPROACH: Plasma membranes from insect cells expressing ABCG2 were used to characterise binding of [3H]daunomycin to the multidrug transporter. The kinetics of association and dissociation for this substrate and several other compounds were also determined in this experimental system. KEY RESULTS: The dissociation constant for [3H]daunomycin binding was 564 +/- 57 nM and a Hill slope of 1.4 suggested cooperative binding. Doxorubicin, prazosin and daunomycin completely displaced the binding of radioligand, while mitoxantrone and Hoechst 33342 produced only a partial displacement. Analysis of the dissociation rates revealed that [3H]daunomycin and doxorubicin bind to multiple sites on the transporter. CONCLUSIONS: Both kinetic and equilibrium data support the presence of at least two symmetric drug binding sites on ABCG2, which is distinct from the asymmetry observed for P-gp. The data provide the first molecular details underlying the mechanism by which this transporter is capable of interacting with multiple substrates.

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0 RESEARCH PAPER Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter R Clark1 , ID Kerr2 and R Callaghan1 1 Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK and 2 Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, UK Background & Purpose: Drug-resistant cancer cells frequently display efflux pumps such as P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1) or the transporter ABCG2. Login to comment
42 The objective is to provide the mechanism underlying multidrug recognition by the R482G isoform (ABCG2R482G ) by examination of the number of drug-binding sites and whether competitive or allosteric interactions exist between substrates on the transporter. Login to comment
43 The R482G isoform was chosen for these investigations because of its wider pharmacological spectrum, thereby enabling a more exhaustive characterization of drug binding to the transporter. Login to comment
112 Membranes expressing the R482G isoform of ABCG2 containing an N-terminal hexahistidine tag were incubated in the presence of varying [3 H]daunomycin concentrations. Login to comment
161 This was achieved initially with heterologous displacement assays using compounds previously demonstrated to interact with the R482G isoform of ABCG2 (primarily through steady-state accumulation and ATPase assays). Login to comment
165 Mitoxantrone is one of the few compounds demonstrated to interact with both the wild-type and R482G mutant isoforms of ABCG2 (Mitomo et al., 2003; Sarkadi et al., 2004). Login to comment
213 How does our binding data compare to the existing data on transport of drugs by both wild-type and R482G ABCG2? Login to comment
252 Further studies will also be required to compare the binding of drugs to wild-type ABCG2 and the data obtained with the R482G isoform will be useful in eventually describing the location and molecular properties of drug interactions with this transporter. Login to comment

[hide] ATP-binding cassette, subfamily G (ABCG family). Pflugers Arch. 2007 Feb;453(5):735-44. Epub 2006 Sep 16. Kusuhara H, Sugiyama Y
ATP-binding cassette, subfamily G (ABCG family).
Pflugers Arch. 2007 Feb;453(5):735-44. Epub 2006 Sep 16., [PMID:16983557]

Abstract [show]
This review summarizes the characteristics of the ATP-binding cassette, subfamily G (ABCG family), which has five members: ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8. The members consist of a single ABC cassette in the amino terminal followed by six putative transmembrane domains, and to become functionally active, they form homo- or obligate heterodimers. Except for ABCG2, the members of the ABCG family play an important role in efflux transport of cholesterol. Mutations causing a loss of function of ABCG5 or ABCG8 are associated with sitosterolemia characterized by accumulation of phyto- and shellfish sterols. Unlike other members, ABCG2 is not involved in cholesterol efflux, but it exhibits broad substrate specificity to xenobiotic compounds. ABCG2 confers cancer cells resistance to anticancer drugs and plays a critical role in the pharmacokinetics of drugs in the clearance organs and tissue barriers. ABCG2 is also associated with a subpopulation phenotype of stem cells. Genetic polymorphisms of ABCG2 have been suggested to account for the interindividual differences in the pharmacokinetics of drugs.

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79 Attention should be paid to the acquired mutations of ABCG2 in some tumor cell lines (R482G and R482T), which have been shown to alter the spectrum of resistance to anticancer drugs. Login to comment

[hide] Human multidrug resistance ABCB and ABCG transport... Physiol Rev. 2006 Oct;86(4):1179-236. Sarkadi B, Homolya L, Szakacs G, Varadi A
Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.
Physiol Rev. 2006 Oct;86(4):1179-236., [PMID:17015488]

Abstract [show]
In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

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801 Mutant variants of ABCG2 The cloning variants first characterized in drug-selected mammalian cells (R482G and R482T) caused a lot of uncertainty regarding the substrate profile of ABCG2, but became also educative regarding the substrate handling of this protein. Login to comment
802 ABCG2 variants containing either R482G or R482T conferred increased mitoxantrone resistance to transfected cells; moreover, they introduced anthracyclin (doxorubicin) resistance and rhodamine-123 extrusion capacity, which were not found in the case of the wild-type protein (see Fig. 10, Refs. 127, 261). Login to comment
803 In contrast, the R482G and R482T mutants were not able to extrude methotrexate, which is a transported substrate of the wild-type ABCG2 (56, 242, 393). Login to comment
819 Transported substrates and inhibitors of the wild-type and R482G ABCG2 protein. Login to comment
820 The Venn diagram depicts some of the transported substrates and inhibitors (in a square) for the wild-type and the R482G mutant variant of ABCG2. Login to comment
1113 Because the substrate specificity of the R482G variant of ABCG2 differs from that of the wild-type protein, this mutant has a special advantage in gene therapy applications. Login to comment

[hide] Towards understanding the mechanism of action of t... J Mol Graph Model. 2007 Mar;25(6):837-51. Epub 2006 Aug 30. Li YF, Polgar O, Okada M, Esser L, Bates SE, Xia D
Towards understanding the mechanism of action of the multidrug resistance-linked half-ABC transporter ABCG2: a molecular modeling study.
J Mol Graph Model. 2007 Mar;25(6):837-51. Epub 2006 Aug 30., [PMID:17027309]

Abstract [show]
The ATP-binding cassette protein ABCG2 is a member of a broad family of ABC transporters with potential clinical importance as a mediator of multidrug resistance. We carried out a homology and knowledge-based, and mutationally improved molecular modeling study to establish a much needed structural framework for the protein, which could serve as guidance for further genetic, biochemical, and structural analyses. Based on homology with known structures of both full-length and nucleotide-binding domains (NBD) of ABC transporters and structural knowledge of integral membrane proteins, an initial model of ABCG2 was established. Subsequent refinement to conform to the lipophilic index distributions in the transmembrane domain (TMD) and to the results of site-directed mutagenesis experiments led to an improved model. The complete ABCG2 model consists of two identical subunits facing each other in a closed conformation. The dimeric interface in the nucleotide-binding domain (NBD) involves a characteristic nucleotide sandwich and the interface in the TMD consists of the TM helices 1-3 of one subunit and the helices 5 and 6 of the other. The interface between the NBD and the TMD is bridged by the conserved structural motif between TM2 and TM3, the intracellular domain 1 (ICD1), and the terminal beta-strand (S6) of the central beta-sheet in the NBD. The apparent flexibility of the ICD1 may play a role in transmitting conformational changes from the NBD to the TMD or from the TMD to the NBD.

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177 Exhibit reduced transport of mitoxantrone, pheophorbide and basal ATPase activity [17] G406A, G410A, G406A/G410A TM1 Fully functional b E446A,D,G,H,K,R,S TM2 Loss of drug resistance [21] R482G,T,M,S, N,D,K,Y TM3 T or G change produces gain of function mutant. Login to comment
245 It has been hypothesized that residue R482 in TM3 is likely to interact with substrates based on the effect of R482G/T mutations [19]; both are gain-of-function mutants, conferring resistance to a broader range of substrates than the wild-type transporter including rhodamine 123 and doxorubicin [19]. Login to comment

[hide] The effect of low pH on breast cancer resistance p... Mol Pharmacol. 2007 Jan;71(1):240-9. Epub 2006 Oct 10. Breedveld P, Pluim D, Cipriani G, Dahlhaus F, van Eijndhoven MA, de Wolf CJ, Kuil A, Beijnen JH, Scheffer GL, Jansen G, Borst P, Schellens JH
The effect of low pH on breast cancer resistance protein (ABCG2)-mediated transport of methotrexate, 7-hydroxymethotrexate, methotrexate diglutamate, folic acid, mitoxantrone, topotecan, and resveratrol in in vitro drug transport models.
Mol Pharmacol. 2007 Jan;71(1):240-9. Epub 2006 Oct 10., [PMID:17032904]

Abstract [show]
Some cellular uptake systems for (anti)folates function optimally at acidic pH. We have tested whether this also applies to efflux from cells by breast cancer resistance protein (BCRP; ABCG2), which has been reported to transport folic acid, methotrexate, and methotrexate di- and triglutamate at physiological pH. Using Spodoptera frugiperda-BCRP membrane vesicles, we showed that the ATP-dependent vesicular transport of 1 muM methotrexate by BCRP is 5-fold higher at pH 5.5 than at physiological pH. The transport of methotrexate was saturable at pH 5.5, with apparent Km and Vmax values of 1.3 +/- 0.2 mM and 44 +/- 2.5 nmol/mg of protein/min, respectively, but was linear with drug concentration at pH 7.3 up to 6 mM methotrexate. In contrast to recent reports, we did not detect transport of methotrexate diglutamate at physiological pH, but we did find transport at pH 5.5. We also found that 7-hydroxy-methotrexate, the major metabolite of methotrexate, is transported by BCRP both at physiological pH and (more efficiently) at low pH. The pH effect was also observed in intact BCRP-overexpressing cells: we found a 3-fold higher level of resistance to both methotrexate and the prototypical BCRP substrate mitoxantrone at pH 6.5 as at physiological pH. Furthermore, with MDCKII-BCRP monolayers, we found that resveratrol, which is a neutral compound at pH < or = 7.4, is efficiently transported by BCRP at pH 6.0, whereas we did not detect active transport at pH 7.4. We conclude that BCRP transports substrate drugs more efficiently at low pH, independent of the dissociation status of the substrate.

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249 For instance, Honjo et al. (2001) showed that cells with R482T and R482G BCRP displayed altered levels of resistance to anthracyclines, SN-38, and topotecan. Login to comment
250 For (anti)folates, vesicular studies have shown that folic acid, MTX, and MTX-glu2 are transported by wild-type BCRP (ABCG2-R482), used in our studies, but not by mutant BCRP (ABCG2-R482T and ABCG2-R482G) (Chen et al., 2003; Mitomo et al., 2003; Volk and Schneider, 2003), suggesting that changes in the protein structure indeed have consequences for the (anti)folate substrate specificity of BCRP. Login to comment
252 However, recent observations by Shafran et al. (2005) demonstrated that intact cells harboring the R482G and R482T amino acid replacements were markedly resistant to short-term exposures to antifolates, including MTX, in association with a poor accumulation of MTX-diglutamates. Login to comment

[hide] Purification and 3D structural analysis of oligome... Structure. 2006 Nov;14(11):1623-32. McDevitt CA, Collins RF, Conway M, Modok S, Storm J, Kerr ID, Ford RC, Callaghan R
Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2.
Structure. 2006 Nov;14(11):1623-32., [PMID:17098188]

Abstract [show]
ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a "half-transporter," and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2(R482G) isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2(R482G) from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2(R482G) provided 3D structural data at a resolution of approximately 18 A. Single-particle analysis revealed that the complex forms a tetrameric complex ( approximately 180 A in diameter x approximately 140 A high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.

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284 Multiple drug binding sites on the R482G isoform of the ABCG2 transporter. Login to comment

[hide] Genetic polymorphisms of human ABC transporter ABC... J Exp Ther Oncol. 2006;6(1):1-11. Tamura A, Wakabayashi K, Onishi Y, Nakagawa H, Tsuji M, Matsuda Y, Ishikawa T
Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system.
J Exp Ther Oncol. 2006;6(1):1-11., [PMID:17228519]

Abstract [show]
The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. However, integration of cDNA into the host's chromosomal DNA occurs randomly at unpredictable sites, and the number of integrated recombinant DNAs is not controllable. To investigate the effect of genetic polymorphisms of ABCG2 on the protein expression and the drug resistance profile, we developed the Flp-In method to integrate one single copy of ABCG2 variant-cDNA into FRT-tagged genomic DNA. More than 20 metaphase spreads were examined for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis, and it has been revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. Based on the currently available SNP data for human ABCG2, we have created a total of seven variants by site-directed mutagenesis and stably expressed them in Flp-In-293 cells. While mRNAs of those integrated ABCG2 variants and wild type were evenly expressed in Flp-In-293 cells, the protein expression levels of F208S and S441N variants were found to be markedly low. It is suggested that the protein instability due to enhanced degradation resulted in the low levels of their protein expression. Thus, the Flp recombinase system would provide a useful tool to validate the effect of nonsynonymous SNPs on the protein stability and post-translational modification of ABCG2.

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48 Standard method for functional validation of ABCG2 SNPs Journal of Experimental Therapeutics and Oncology Vol. 6 2006 3 Plasma Membrane inside outside S S S homodimer A B CH2N COOH V12M Q141K F208S S248P F431L S441N F489L R482G R482T Acquired mutation Figure 1. Login to comment
51 The variants R482G and R482T are acquired mutations. Login to comment
132 Figure 4 summarizes the characteristics of those Tamura et al. 8 Journal of Experimental Therapeutics and Oncology Vol. 6 2006 Class Class Class Class WT V12M Q141K F431L S248P F489L F208S S441N R482G R482T Protein expression + + + + + + - - + + SN-38 resistance + + + + + / - - - - + + MX resistance + + + + / - - - - - + + Doxorubicin resistance - - - - - - - - + + Daunorubicin resistance - - - - - - - - + + Figure 4. Login to comment
142 Finally, the acquired mutants R482G and R482T form another group, which is characteristic Standard method for functional validation of ABCG2 SNPs Journal of Experimental Therapeutics and Oncology Vol. 6 2006 9 Table 3 Remarks mRNA Protein Author Ref Host cell Vector Expression SNP expression expression Imai et al. (15) PA317 pHaL-IRES-DHFR bicistronic Stable V12M Similar to WT Similar to WT - - retrovirus vector plasmid - Q141K Similar to WT Lower than WT Mizuarai et al. (18) LLC-PK1 pcDNA3.1(+) Stable V12M Similar to WT N.D. - - - - Q141K Similar to WT N.D. Morisaki et al. (25) HEK293 pcDNA3.1 Stable V12M Vary among clones Vary among clones - - - - Q141K Vary among clones Vary among clones - - - - D620N Vary among clones Vary among clones Kondo et al. (26) LLC-PK1/ pcDNA3.1/ Stable/ V12M N.D. Similar to WT - HEK293 Adenovirus Transient Q141K N.D. 30 - 40% of WT - - - - A149P N.D. Similar to WT - - - - R163K N.D. Similar to WT - - - - Q166E N.D. Similar to WT - - - - P269S N.D. Similar to WT - - - - S441N N.D. Lower than WT Vethanayagam (27) HEK293 pcDNA3.1/myc-His(-) Stable I206L N.D. Vary among clones et al. - - - - N590Y N.D. Vary among clones - - - - D620N N.D. Vary among clones N.D.: No data Table 2. Login to comment

[hide] Re-evaluation and functional classification of non... Cancer Sci. 2007 Feb;98(2):231-9. Tamura A, Wakabayashi K, Onishi Y, Takeda M, Ikegami Y, Sawada S, Tsuji M, Matsuda Y, Ishikawa T
Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2.
Cancer Sci. 2007 Feb;98(2):231-9., [PMID:17297656]

Abstract [show]
Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.

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9 Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Login to comment
154 Because acquired mutants (i.e. R482G and R482T) are known to exhibit prazosin-stimulated ATPase activity,(35) we examined whether SNP variants carry such ATPase activity. Login to comment
155 For this purpose, we expressed WT ABCG2, V12M, Q141K, S248P, F431L, F489L, R482G and R482T in Sf9 insect cells and prepared plasma membranes as described previously,(16,35) as the plasma membrane of Sf9 cells has lower endogenous background ATPase activity than Flp-In-293 cells. Login to comment
157 Continued markedly stimulated with prazosin in those acquired mutants (R482G and R482T). Login to comment
203 As summarized in Fig. 5A, the functional properties of these variants were compared with acquired mutations of R482G and R482T. Login to comment
204 The drug resistance profiles and prazosin-stimulated ATPase activity of R482G and R482T were reported previously. Login to comment
212 In contrast, the acquired mutants R482G and R482T form another group, which is characterised by a positive contribution to drug resistance toward SN-38, mitoxantrone, doxorubicin and daurorubicin as well as prazosin-stimulated ATPase activity. Login to comment

[hide] Pharmacogenetics/genomics of membrane transporters... Cancer Metastasis Rev. 2007 Mar;26(1):183-201. Huang Y
Pharmacogenetics/genomics of membrane transporters in cancer chemotherapy.
Cancer Metastasis Rev. 2007 Mar;26(1):183-201., [PMID:17323126]

Abstract [show]
Inter-individual variability in drug response and the emergence of adverse drug reactions are main causes of treatment failure in cancer therapy. Recently, membrane transporters have been recognized as an important determinant of drug disposition, thereby affecting chemosensitivity and -resistance. Genetic factors contribute to inter-individual variability in drug transport and targeting. Therefore, pharmacogenetic studies of membrane transporters can lead to new approaches for optimizing cancer therapy. This review discusses genetic variations in efflux transporters of the ATP-binding cassette (ABC) family such as ABCB1 (MDR1, P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2) and ABCG2 (BCRP), and uptake transporters of the solute carrier (SLC) family such as SLC19A1 (RFC1) and SLCO1B1 (SLC21A6), and their relevance to cancer chemotherapy. Furthermore, a pharmacogenomic approach is outlined, which using correlations between the growth inhibitory potency of anticancer drugs and transporter gene expression in multiple human cancer cell lines, has shown promise for determining the relevant transporters for any given drugs and predicting anticancer drug response.

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192 Furthermore, a mutational hot spot located in amino acid codon 482 of ABCG2 has been identified in drug selected cancer cell lines where a single amino acid change (R482G or R482T) occurs [105]. Login to comment

[hide] Cholesterol potentiates ABCG2 activity in a hetero... J Pharmacol Exp Ther. 2007 Jun;321(3):1085-94. Epub 2007 Mar 8. Pal A, Mehn D, Molnar E, Gedey S, Meszaros P, Nagy T, Glavinas H, Janaky T, von Richter O, Bathori G, Szente L, Krajcsi P
Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2.
J Pharmacol Exp Ther. 2007 Jun;321(3):1085-94. Epub 2007 Mar 8., [PMID:17347325]

Abstract [show]
ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.

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30 The R482G/T mutants display a substrate specificity different from the wild-type (WT) protein (Honjo et al., 2001; Ozvegy et al., 2002). Login to comment
31 More strikingly, unlike the ATPase function of the R482G/T mutants, the ATPase activity of the WT protein could not be stimulated by prazosin, a known ABCG2 substrate (Xiao et al., 2006), when expressed in insect cell membranes (Ozvegy et al., 2001; Ishikawa et al., 2003). Login to comment

[hide] The emerging pharmacotherapeutic significance of t... Br J Pharmacol. 2007 May;151(2):163-74. Epub 2007 Mar 20. Hardwick LJ, Velamakanni S, van Veen HW
The emerging pharmacotherapeutic significance of the breast cancer resistance protein (ABCG2).
Br J Pharmacol. 2007 May;151(2):163-74. Epub 2007 Mar 20., [PMID:17375082]

Abstract [show]
The breast cancer resistance protein (also termed ABCG2) is an ATP-binding cassette transporter, which mediates the extrusion of toxic compounds from the cell, and which was originally identified in relation to the development of multidrug resistance of cancer cells. ABCG2 interacts with a range of substrates including clinical drugs but also substances such as sterols, porphyrins and a variety of dietary compounds. Physiological functions of ABCG2 at both cellular and systemic levels are reviewed. For example, ABCG2 expression in erythrocytes may function in porphyrin homeostasis. In addition, ABCG2 expression at apical membranes of cells such as hepatocytes, enterocytes, endothelial and syncytiotrophoblast cells may correlate to protective barrier or secretory functions against environmental or clinically administered substances. ABCG2 also appears influential in the inter-patient variation and generally poor oral bioavailability of certain chemotherapeutic drugs such as topotecan. As this often precludes an oral drug administration strategy, genotypic and environmental factors altering ABCG2 expression and activity are considered. Finally, clinical modulation of ABCG2 activity is discussed. Some of the more recent strategies include co-administered modulating agents, hammerhead ribozymes or antisense oligonucleotides, and with specificity in cell targeting, these may be used to reduce drug resistance and increase drug bioavailability to improve the profile of chemotherapeutic efficacy versus toxicity. While many such strategies remain in relative infancy at present, increased knowledge of modulators of ABCG2 could hold the key to novel approaches in medical treatment.

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47 Most recently, the structure of ABCG2 R482G (see below) was purified from Trichoplusia ni insect cells, and studied by cryonegatively stained electron microscopy and single-particle analysis. Login to comment

[hide] Expression of the ATP-binding cassette membrane tr... Pharm Res. 2007 Jul;24(7):1262-74. Epub 2007 Mar 23. Lee G, Babakhanian K, Ramaswamy M, Prat A, Wosik K, Bendayan R
Expression of the ATP-binding cassette membrane transporter, ABCG2, in human and rodent brain microvessel endothelial and glial cell culture systems.
Pharm Res. 2007 Jul;24(7):1262-74. Epub 2007 Mar 23., [PMID:17380269]

Abstract [show]
PURPOSE: The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments. MATERIALS AND METHODS: RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells. RESULTS: RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at approximately 72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of (3)H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems. CONCLUSIONS: Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.

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274 Although all three variants (R482, R482T, R482G) are able to transport mitoxantrone, wild-type ABCG2 is somewhat less effective in mitoxantrone extrusion than the other variants (37). Login to comment

[hide] Expression of breast cancer resistance protein (BC... Can J Physiol Pharmacol. 2006 Dec;84(12):1251-8. Yeboah D, Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, Gibb W
Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor.
Can J Physiol Pharmacol. 2006 Dec;84(12):1251-8., [PMID:17487233]

Abstract [show]
Breast cancer resistance protein, BCRP, is a multidrug resistance protein that is highly expressed in the human placenta. In cancer tissues, this protein actively extrudes a wide variety of chemically and structurally unrelated chemotherapeutic drugs and other compounds. Studies in mice have shown that in the absence of BCRP activity in the placenta, there is a 2-fold increase in the uptake in BCRP substrates into fetus. This suggests that in the placenta, BCRP extrudes compounds that would otherwise cross the syncytiotrophoblast cells into fetal circulation. The purpose of this study was to examine the expression and localization of BCRP in the human placenta throughout gestation. Tissues from 6-13, 16-19, 24-29, 32-35, and 38-41 weeks of gestation were used. Real time RT-PCR analysis demonstrated that the mRNA levels of BCRP in the placenta do not change significantly as gestation progressed. However, Western blot analysis revealed that the protein levels increased towards the end of gestation. We demonstrated that BCRP is localized to the syncytiotrophoblast of the placenta and in some fetal blood vessels within the placenta. Tissues from the early stages of pregnancy (6-13 weeks) showed fewer BCRP positive blood vessels than term tissues (38-41 weeks).

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52 For instance, the wild type BCRP is unable to transport anthracycline, doxorubicin, or rhodamine but mutant BCRP in which threonine or glycine replaces arginine at position 482 transports all 3 substrates (Allen and Schinkel 2002; Sarkadi et al. 2004; Volk et al. 2002). Login to comment

[hide] Evaluation of drug-transporter interactions using ... Curr Drug Metab. 2007 May;8(4):341-63. Xia CQ, Milton MN, Gan LS
Evaluation of drug-transporter interactions using in vitro and in vivo models.
Curr Drug Metab. 2007 May;8(4):341-63., [PMID:17504223]

Abstract [show]
Drug transporters, including efflux transporters (the ATP binding cassette (ABC) proteins) and uptake transporters (the solute carrier proteins (SLC)), have an important impact on drug disposition, efficacy, drug-drug interactions and toxicity. Identification of the interactions of chemical scaffolds with transporters at the early stages of drug development can assist in the optimization and selection of new drug candidates. In this review, we discuss current in vitro and in vivo models used to investigate the interactions between drugs and transporters such as P-gp, MRP, BCRP, BSEP, OAT, OATP, OCT, NTCP, PEPT1/2 and NT. In vitro models including cell-based, cell-free, and yeast systems as well as in vivo models such as genetic knockout, gene deficient and chemical knockout animals are discussed and compared. The applications, throughput, advantages and limitations of each model are also addressed in this review.

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124 Using a similar method, methotrexate has been demonstrated to be a substrate for wild type BCRP but not for the R482G BCRP mutant [44]. Login to comment

[hide] ABCG2 (breast cancer resistance protein/mitoxantro... Drug Metab Dispos. 2007 Sep;35(9):1533-42. Epub 2007 May 30. Glavinas H, Kis E, Pal A, Kovacs R, Jani M, Vagi E, Molnar E, Bansaghi S, Kele Z, Janaky T, Bathori G, von Richter O, Koomen GJ, Krajcsi P
ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.
Drug Metab Dispos. 2007 Sep;35(9):1533-42. Epub 2007 May 30., [PMID:17537873]

Abstract [show]
The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

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24 9 Copyright (c) 2007 by The American Society for Pharmacology and Experimental Therapeutics 14605/3238592 DMD 35:1533-1542, 2007 Printed in U.S.A. 1533 The first ABCG2 ATPase assays using Sf9 membranes were reported on the R482G version of the transporter (Ozvegy et al., 2001). Login to comment

[hide] A global drug inhibition pattern for the human ATP... J Pharmacol Exp Ther. 2007 Oct;323(1):19-30. Epub 2007 Jul 6. Matsson P, Englund G, Ahlin G, Bergstrom CA, Norinder U, Artursson P
A global drug inhibition pattern for the human ATP-binding cassette transporter breast cancer resistance protein (ABCG2).
J Pharmacol Exp Ther. 2007 Oct;323(1):19-30. Epub 2007 Jul 6., [PMID:17616561]

Abstract [show]
In this article, we explore the entire structural space of registered drugs to obtain a global model for the inhibition of the drug efflux transporter breast cancer resistance protein (BCRP; ABCG2). For this purpose, the inhibitory effect of 123 structurally diverse drugs and drug-like compounds on mitoxantrone efflux was studied in Saos-2 cells transfected with human wild-type (Arg482) BCRP. The search for BCRP inhibitors throughout the drug-like chemical space resulted in the identification of 29 previously unknown inhibitors. The frequency of BCRP inhibition was 3 times higher for compounds reported to interact with other ATP-binding cassette (ABC) transporters than for compounds without reported ABC transporter affinity. An easily interpreted computational model capable of discriminating inhibitors from noninhibitors using only two molecular descriptors, octanol-water partition coefficient at pH 7.4 and molecular polarizability, was constructed. The discriminating power of this two-descriptor model was 93% for the training set and 79% for the test set, respectively. The results were supported by a global pharmacophore model and are in agreement with a two-step mechanism for the inhibition of BCRP, where both the drug's capacity to insert into the cell membrane and to interact with the inhibitory binding site of the transporter are important.

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279 Biochemical data suggests as many as four distinct drug binding sites for P-gp (Shapiro and Ling, 1997; Ambudkar et al., 2006); likewise, the existence of two or three distinct but symmetrical binding sites has recently been suggested for the wild-type Arg482 BCRP and the R482G mutant isoform, respectively (Ejendal and Hrycyna, 2005; Clark et al., 2006). Login to comment

[hide] Membrane cholesterol selectively modulates the act... Biochim Biophys Acta. 2007 Nov;1768(11):2698-713. Epub 2007 Jul 10. Telbisz A, Muller M, Ozvegy-Laczka C, Homolya L, Szente L, Varadi A, Sarkadi B
Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter.
Biochim Biophys Acta. 2007 Nov;1768(11):2698-713. Epub 2007 Jul 10., [PMID:17662239]

Abstract [show]
The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.

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8 Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. Login to comment
27 The R482G and R482T mutant variants of ABCG2, found only in drug-selected tumor cells, show a significantly altered drug resistance pattern, as compared to the wild-type protein. Login to comment
29 In contrast, the R482G and R482T variants practically do not transport methotrexate or drug conjugates [14-17]. Login to comment
47 Interestingly, membrane cholesterol modulation under the same conditions had only a negligible effect on the activity of ABCG2-R482G and ABCG2-R482T mutant variants, or that of the MDR1 multidrug transporter. Login to comment
86 Confocal microscopy HEK cells stably transfected with ABCG2 (wt) or ABCG2-R482G were seeded onto eight-well Nunc Lab-Tek II Chambered Coverglass (Nalge Nunc International, Rochester, NY) at 3×104 per well cell density, and grown for 48 h in D-MEM containing 10% FCS. Login to comment
104 HEK cells, expressing the human wild-type ABCG2, the ABCG2-R482G variant, or the MDR1 protein, were pretreated in HPMI medium by 4 mM CD or 4 mM C-CD for 30 min at 37 °C, then washed twice to eliminate cyclodextrin. Login to comment
122 The Hoechst 33342 dye is a good substrate of both the wild-type and the R482G or R482T mutant variants of ABCG2 [1,3], as well as of the MDR1 transporter [4]. Login to comment
125 The expression of ABCG2, its R482G mutant form, as well as MDR1 strongly decrease Hoechst dye accumulation. Login to comment
131 As documented in Fig. 1A, cholesterol depletion of HEK cells by 4 mM CD for 30 min at 37 °C practically eliminated Hoechst dye extrusion by the wild-type ABCG2 (the activity factor decreased from 0.54 to 0.05), while this treatment had no effect on dye extrusion in ABCG2-R482G expressing, or MDR1 expressing HEK cells. Login to comment
144 In experiments not documented here in detail, we have repeated these cellular transport experiments by measuring the uptake of mitoxantrone (MX) and Pheophorbide A (PheA), both transported compounds for both ABCG2 and its R482G variant, by using flow cytometry. Login to comment
153 Again, dye extrusion activity by the ABCG2-R482G variant was practically unaffected by CD or C-CD treatment. Login to comment
154 These experiments confirm that Hoechst 33342 dye extrusion in HEK cells, expressing the wild-type ABCG2, is strongly modulated by membrane cholesterol, while Hoechst transport by the ABCG2-R482G variant is not affected by this membrane lipid. Login to comment
163 Experiments with isolated Sf9 cell membranes In order to explore the molecular details of the cholesterol effects observed in intact cells, ABCG2 and its R482G, R482T mutant variants were expressed in Sf9 cells. Login to comment
186 The R482G or R482T variants of ABCG2 have significantly different substrate handling properties than the wild-type protein. Login to comment
188 As shown in Fig. 3B, MTX transport by the ABCG2-R482G variant was very low both in the control Fig. 2. Login to comment
192 Hoechst dye accumulation was measured in cells expressing the human wild-type ABCG2 or the ABCG2-R482G variant at 37 °C, and cellular dye content was estimated based on the fluorescence in selected regions of interests. Login to comment
205 We found a similar lack of significant ESG and E3S transport by the R482G and R482T variants, irrespective of the cholesterol content of the membrane vesicles (not shown). Login to comment
214 The apparent Km of MTX uptake was about 0.5 mM in both cases, but the proper determination of the Km and Vmax values in these experiments was hindered by the low solubility of MTX at higher than 3 mM concentrations. Fig. 4A also documents that the R482G variant of ABCG2 had a very low MTX transport activity, irrespective of the MTX concentrations examined. Login to comment
217 Again, neither the R482G, nor the R482T variants showed any MTX transport activity, irrespective of the ATP concentration or the cholesterol content of the Sf9 cell membrane vesicles. Login to comment
228 MTX uptake was measured at 100 μM MTX concentration for 5 min at 37 °C in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment
233 MTX uptake was measured at 100 μM MTX concentration for 5 min at 37 °C in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment
244 The experiments shown in Fig. 4 demonstrate that increased membrane cholesterol did not convert the ABCG2-R482G mutant into an efficient MTX or ESG transporter. Login to comment
245 However, in order to further explore the effect of cholesterol on the substrate specificity of the mutant and wild-type ABCG2, we have also examined Rhodamine 123 (R123) uptake by the Sf9 membrane vesicles. R123 is a transported substrate of the ABCG2-R482G variant, while this compound is practically not transported by the wild-type protein. Login to comment
246 As shown in Fig. 5, MgATP-dependent, rapid vesicular R123 uptake was well measurable by flow cytometry in Sf9 membrane vesicles, expressing the human R482G-ABCG2 protein. Login to comment
250 These data indicate that cholesterol loading of the vesicles (in these experiments to 55-62 μg cholesterol/mg membrane protein) did not evoke R123 uptake in the vesicles containing the wild-type ABCG2, while slightly increased (as an average by 25-30%) both the initial rate and the maximum level of the R123 uptake in the R482G-ABCG2 vesicles. Login to comment
254 MTX and ESG uptake was measured for 5 min at 37 °C at 5 mM ATP in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment
259 ▪-MTX uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, •-MTX uptake in cholesterol-loaded vesicles (56 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, ▴-MTX uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter, ▵-MTX uptake in cholesterol-loaded vesicles (62 μg cholesterol/ mg membrane protein), containing the human R482G ABCG2 (G) transporter. Login to comment
271 In contrast, many substrates caused a strong activation for the ABCG2-ATPase of the R482G or R482T variants [9,14]. Login to comment
273 Fig. 6A shows the vanadate-sensitive ATPase activity of the wild-type ABCG2 as well as the R482G and the R482T variants, both in the absence and presence of two potential transported substrates. Login to comment
274 In these studies we selected prazosin, and the EKI-785 tyrosine kinase inhibitor (EKI), as these compounds were shown to be substrates both for the wild-type, as well as the R482G or R482T variants of ABCG2 [14,35]. Login to comment
279 However, cholesterol loading greatly increased the drug-stimulated ATPase activity of the wild-type ABCG2 in the presence of both substrates, while it had no such effect in the case of the R482G or R482T mutant variants. Login to comment
289 Effect of cholesterol loading on ATP-dependent Rhodamine 123 (R123) uptake in Sf9 membrane vesicles. R123 uptake was measured by flow cytometry (see Materials and methods) at 1 μM R123 concentration, by taking 30-s time points for 5 min at 22 °C in membrane vesicles containing the human wild-type ABCG2 (WT), or R482G-ABCG2 (G) transporter. Login to comment
292 ▪- R123 uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, •-R123 uptake in cholesterol-loaded vesicles (56 μg cholesterol/mg membrane protein), containing the human wild-type ABCG2 (WT) transporter, ▴-R123 uptake in the control vesicles (8 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter, ▾-R123 uptake in cholesterol-loaded vesicles (62 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter. Login to comment
293 ×-R123 uptake in cholesterol-loaded vesicles (62 μg cholesterol/mg membrane protein), containing the human R482G ABCG2 (G) transporter, in the presence of 1 μM Ko143. Login to comment
296 Fig. 6C also depicts the effect of membrane cholesterol on the prazosin and EKI stimulation of the ATPase activity of the ABCG2-R482G variant. Login to comment
306 In contrast, nucleotide trapping in the ABCG2-R482G variant was significantly increased by transported substrates. Login to comment
322 Panel A Effect of cholesterol loading on the vanadate-sensitive ATPase activity in isolated Sf9 membrane preparations. ATPase activity in the vesicles was measured for 20 min at 37 °C in membranes containing the human wild-type ABCG2 (WT), the R482G-ABCG2 (R482G), or the R482T-ABCG2 (R482T) transporter. Login to comment
327 Control membranes contained 8 μg cholesterol/mg membrane protein, while cholesterol-loaded membranes contained 56 μg cholesterol/mg membrane protein in the case of the wt ABCG2, 62 μg cholesterol/mg membrane protein in the case of the ABCG2-R482G, and 65 μg cholesterol/mg membrane protein in the case of the ABCG2-R482 T variant. Login to comment
335 ATPase activity was measured for 20 min at 37 °C in membranes containing the human wild-type ABCG2 (WT), or the R482G-ABCG2 (R482G) transporter. Login to comment
339 •-Per cent stimulation of the Ko143-sensitive ATPase activity by 20 μM Prazosin in membranes containing the human wild-type ABCG2 (WT) transporter, ▪- Per cent stimulation of the Ko143-sensitive ATPase activity by 1 μM EKI in membranes containing the human wild-type ABCG2 (WT) transporter, ○-Per cent stimulation of the Ko143-sensitive ATPase activity by 20 μM Prazosin in membranes containing the human R482G ABCG2 (G) transporter, □-Per cent stimulation of the Ko143-sensitive ATPase activity by 1 μM EKI in membranes containing the human R482G ABCG2 (G) transporter. Login to comment
343 In the case of the ABCG2-R482G mutant variant, prazosin and EKI stimulation of nucleotide trapping was already present in the control membranes [17], and in this case we did not find any significant difference by cholesterol enrichment of the Sf9 cell membranes in the present study (data not shown). Login to comment
349 This effect was fully reversible, and selective for the wild-type ABCG2, while the function of the highly active mutant variant ABCG2-R482G, found in drug-selected tumor cells, was not influenced by a similar cholesterol depletion. Login to comment
361 Similarly to that seen in intact cells, this effect of membrane cholesterol was absent in the case of the R482G mutant variant of the transporter. Login to comment
366 Interestingly, the ATPase activity of the R482G or R482T mutant variants of ABCG2 could be significantly enhanced by the respective substrate drugs both in the Sf9 and the mammalian cell membrane preparations [10,14]. Login to comment
374 In contrast, transported substrates significantly increased nucleotide trapping by the ABCG2-R482G variant [17]. Login to comment
383 Based on Sf9 membrane ATPase measurements, earlier we proposed that the R482G and R482T variants of ABCG2 may have "gain-of-function" properties [14]. Login to comment
161 Moreover, the ATP-dependent transport by ABCG2-R482G or MDR1 was also not significantly affected under these conditions. Login to comment

[hide] Mechanisms underlying the anticancer activities of... Biochem Pharmacol. 2007 Dec 15;74(12):1713-26. Epub 2007 Aug 25. Korynevska A, Heffeter P, Matselyukh B, Elbling L, Micksche M, Stoika R, Berger W
Mechanisms underlying the anticancer activities of the angucycline landomycin E.
Biochem Pharmacol. 2007 Dec 15;74(12):1713-26. Epub 2007 Aug 25., 2007-12-15 [PMID:17904109]

Abstract [show]
Anthracyline antibiotics, produced by Streptomyces sp., still rank among the most efficient anticancer drugs in clinical use. Aim of this study was to gain deeper insight into the anticancer properties of the anthracycline-related angucycline landomycin E (LE). The impact of LE on nuclear morphology was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining in the human carcinoma cell model KB-3-1. LE treatment led to the appearance of typical morphological signs of programmed cell death like cell shrinkage, chromatin condensation and formation of apoptotic bodies. Apoptotic cell death induced by LE was further characterised by caspase (substrate) cleavage and intense mitochondrial membrane depolarisation (JC-1 and rhodamine 123 staining) already after 1h drug incubation. Moreover, incubation with LE led to reduced intracellular ATP pools suggesting LE-induced apoptotic cell death as a consequence of rapid mitochondrial damage. Furthermore, LE treatment led to profound generation of intracellular oxidative stress, indicated by radical scavenger pre-treatment and dichlorofluorescin diacetate (DCF-DA) staining experiments. Since chemoresistance is a common problem in cancer therapy, we also investigated the influence of ABCB1 (P-glycoprotein, P-gp), ABCC1 (multidrug resistance-related protein, MRP1) and ABCG2 (breast cancer resistance protein, BCRP) overexpression on the anticancer activity of LE. Compared to anthracyclines, cytotoxic activity of LE was only weakly reduced by P-gp and MRP1 overexpression. Moreover, BCRP expression had no influence on LE anticancer activity. In summary, LE exerts anticancer activity via potent induction of apoptosis and has promising anticancer activity even against multidrug resistant (MDR) cells. Taken together, these data suggest further development of LE as a new anticancer drug.

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284 Interestingly, anthracyclines are not transported by wild-type BCRP but are substrates of the polymorphic BCRP mutants R482G and R482T only [4,40]. Login to comment

[hide] ABCG2/BCRP expression modulates D-Luciferin based ... Cancer Res. 2007 Oct 1;67(19):9389-97. Zhang Y, Bressler JP, Neal J, Lal B, Bhang HE, Laterra J, Pomper MG
ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.
Cancer Res. 2007 Oct 1;67(19):9389-97., 2007-10-01 [PMID:17909048]

Abstract [show]
Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of d-luciferin with firefly luciferase (fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate d-luciferin. In vitro studies show that d-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). d-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/BCRP expression and function within regions of interest substantially influence d-luciferin-dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during d-luciferin-based BLI and suggest novel high throughput methods for identifying new ABCG2/BCRP inhibitors.

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167 Cells engineered to express wild-type (wt) ABCG2/BCRP or mutant ABCG2/BCRP transporters T10 (R482T) and G2 (R482G) were transiently transfected with the CMV trifusion reporter and then seeded into 24-well plates for imaging in the presence or absence of transporter inhibitors (10). Login to comment
235 Whereas D-luciferin proved to be a substrate of wt ABCG2/BCRP, single amino acid changes (R482T and R482G) within the transporter change the BLI output enhancement in response to various inhibitors. Login to comment

[hide] Breast cancer resistance protein: mediating the tr... Placenta. 2008 Jan;29(1):39-43. Epub 2007 Oct 17. Gedeon C, Anger G, Piquette-Miller M, Koren G
Breast cancer resistance protein: mediating the trans-placental transfer of glyburide across the human placenta.
Placenta. 2008 Jan;29(1):39-43. Epub 2007 Oct 17., [PMID:17923155]

Abstract [show]
Members of the ATP-binding cassette (ABC) efflux transporter family, including P-glycoprotein (PGP), the multidrug resistance-associated proteins (MRPs) and the breast cancer resistance protein (BCRP) have been shown to be highly expressed in the human placenta. Recent studies documented that the oral hypoglycemic glyburide does not cross the human placenta to an appreciable extent. Furthermore, the trans-placental transfer of glyburide has been shown not to be affected by either the presence of PGP inhibitor, verapamil or MRP inhibitor, indomethacin. Therefore, our objective was to identify other human placental ABC transporters potentially involved in limiting the trans-placental transfer of glyburide to the fetus. [(3)H]-glyburide transport was examined in brush border human placental vesicles in the presence or absence of specific inhibitors. Prepared vesicles were 70% oriented right-side-out and demonstrated 25-27 fold enrichment as compared to whole placenta. Functional studies demonstrated significant increases in the intra-vesicular accumulation of [(3)H]-glyburide in vesicles treated with the BCRP inhibitor, novobiocin. In contrast, PGP inhibition as well as MRP inhibition did not affect [(3)H]-glyburide accumulation. This is the first evidence to clearly indicate that glyburide is preferentially transported by BCRP, in the brush border of the human placenta. Our study also indicates that BCRP likely effluxes substrates in the fetal to maternal direction in the human placenta.

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119 Other variants, such as Arg482Gly and Arg482Thr have been reported to have an important role in protein function and in altering the multidrug resistance phenotype by changing substrate specificity [22]. Login to comment

[hide] Effects of drug efflux proteins and topoisomerase ... Invest New Drugs. 2008 Jun;26(3):205-13. Epub 2007 Oct 18. Gounder MK, Nazar AS, Saleem A, Pungaliya P, Kulkarni D, Versace R, Rubin EH
Effects of drug efflux proteins and topoisomerase I mutations on the camptothecin analogue gimatecan.
Invest New Drugs. 2008 Jun;26(3):205-13. Epub 2007 Oct 18., [PMID:17943230]

Abstract [show]
Clinically relevant resistance to the currently approved camptothecins, irinotecan and topotecan, is poorly understood but may involve increased expression of ATP-dependent drug transporters such as ABCG2 (breast cancer resistant protein, BCRP). Gimatecan (ST1481) is a lipophilic 7-substituted camptothecin derivative that exhibits potent anti-tumor activity in a variety of preclinical cancer models and is under investigation in the clinic. Previous studies reported that gimatecan cytotoxicity was not affected by expression of ABCG2. To confirm and extend this finding, we assessed the cytotoxicity of gimatecan in pairs of isogenic cell lines consisting of transfectants expressing either ABCG2 (including wild-type, R482T, or R482G mutants), ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2), or ABCC4 (MRP4). Expression of wild-type or mutant ABCG2 in human cell lines conferred resistance to topotecan but not to gimatecan. Similarly, intracellular accumulation of gimatecan was unaffected by expression of wild-type ABCG2. Furthermore, expression of P-glycoprotein or MRP2 did not alter gimatecan cytotoxicity. Whereas expression of MRP1 had a minor effect on gimatecan cytotoxicity, expression of ABCC4 was found to significantly reduce the anti-proliferative effects of this drug. Cells containing resistance-conferring mutations in topoisomerase I were also resistant to gimatecan. These results suggest that gimatecan may be more effective than irinotecan or topotecan in cancers that express ABCG2, but not in cancers that express high levels of ABCC4 or contain certain topoisomerase I (TOP1) mutations.

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3 To confirm and extend this finding, we assessed the cytotoxicity of gimatecan in pairs of isogenic cell lines consisting of transfectants expressing either ABCG2 (including wild-type, R482T, or R482G mutants), ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2), or ABCC4 (MRP4). Login to comment
59 Data represent means of six replicate experiments. Bars represent standard errors Table 1 Cell lines Name Cell type/expression vector References MDCK II Madin-Darby canine kidney epithelial cells [25] MDCKII-ABCB1 MDCKII cells stably transfected with a vector expressing human ABCB1 (PgP) [26] MDCKII-ABCC1 MDCKII cells stably expressing human ABCC1 (MRP1) [27] MDCKII-ABCC2 MDCKII cells stably expressing human ABCC2 (MRP2) [28] HEK293-pcDNA3 Human embryonic kidney cells stably transfected with pcDNA3 control vector [17] HEK293-ABCG2 HEK293 cells stably transfected with pcDNA3-ABCG2 expressing wild-type ABCG2 [17] HEK293-ABCG2 R482T HEK293 cells stably transfected with pcDNA3-ABCG2 R482T expressing a mutant form of ABCG2 with threonine for arginine substitution at codon 482 [17] HEK293-ABCG2 R482G HEK293 cells stably transfected with pcDNA3-ABCG2 R482G expressing a mutant form of ABCG2 with Glycine for arginine substitution at codon 482 [17] Saos-2-pcDNA Human osteogenic sarcoma cells stably transfected with empty vector [29] Saos-2-ABCC4 Saos-2 cells stably transfected with a vector expressing ABCC4 (MRP4) [29] U-937 Human monoblastic leukemia cells [5] U-937-CR U-937 cells selected for resistance to 9-nitrocamptothecin [5] RPMI-8402 Human T-cell lymphoblastic leukemia cells [20] CPT-K5 RPMI-8402 cells selected for resistance to irinotecan [20] using the Bradford method [16], with the remaining sample subjected to liquid scintillation counting or HPLC analysis. Login to comment
66 HEK293 cells consisting of stable transfectants of vector alone (diamond), or vectors expressing wild-type ABCG2 (circle), or mutants R482T (square) or R482G (triangle), were incubated for 72 h with various concentrations of the indicated compounds. Cell survival was determined as described in the "Materials and methods." Login to comment
71 Overexpression Table 3 Anti-proliferative effects of camptothecin analogs in HEK293 cells expressing wild-type or mutant ABCG2 Drug Control ABCG2 RRa ABCG2 R482T RR ABCG2 R482G RR Gimatecan 0.005±0.001b 0.004±0.001 0.8 0.004±0.002 0.8 0.004±0.001 0.8 Topotecan 0.04±0.01 0.98±0.2* 25 0.39±0.25* 10 0.6±0.1* 15 9-NC 0.037±0.032 0.04±0.008 1.1 0.04±0.01 1.1 0.033±0.03 1 a Relative resistance of ABCG2-expressing cell line compared to control cell line b Results are expressed as mean ± standard deviations (μM) for calculated IC50s for six replicate experiments *Indicates statistically different compared to the parental cell line (p<0.05) Fig. 3 Effects of overexpression of ABCC4 on the cytotoxicity of gimatecan. Login to comment
75 Similar studies were done using HEK293 cell lines overexpressing wild-type ABCG2 or the R482T or R482G mutants [17]. Login to comment

[hide] High-speed screening and quantitative SAR analysis... Mini Rev Med Chem. 2007 Oct;7(10):1009-18. Saito H, Hirano H, Ishikawa T
High-speed screening and quantitative SAR analysis of human ABC transporter ABCG2 for molecular modeling of anticancer drugs to circumvent multidrug resistance.
Mini Rev Med Chem. 2007 Oct;7(10):1009-18., [PMID:17979803]

Abstract [show]
The transport mechanism-based molecular design strategy would provide an effective tool for rationalized chemotherapy against tumors. To develop a platform for molecular modeling to circumvent multidrug resistance, we established new methods of high-speed screening for human ABCG2-drug interactions, quantitative structure-activity relationship (QSAR) analysis, and quantum chemical calculation for lead optimization.

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50 The wild type of ABCG2 transports MTX, whereas acquired mutants, i.e., R482G and R482T, do not [15]. Login to comment

[hide] Erlotinib (Tarceva, OSI-774) antagonizes ATP-bindi... Cancer Res. 2007 Nov 15;67(22):11012-20. Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, Bates SE, Shen T, Ashby CR Jr, Fu LW, Ambudkar SV, Chen ZS
Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance.
Cancer Res. 2007 Nov 15;67(22):11012-20., 2007-11-15 [PMID:18006847]

Abstract [show]
It has been reported that gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has the ability to modulate the function of certain ATP-binding cassette (ABC) transporters and to reverse ABC subfamily B member 1 (ABCB1; P-glycoprotein)- and ABC subfamily G member 2 (ABCG2; breast cancer resistance protein/mitoxantrone resistance protein)-mediated multidrug resistance (MDR) in cancer cells. However, it is unknown whether other EGFR TKIs have effects similar to that of gefitinib. In the present study, we have investigated the interaction of another EGFR TKI, erlotinib, with selected ABC drug transporters. Our findings show that erlotinib significantly potentiated the sensitivity of established ABCB1 or ABCG2 substrates and increased the accumulation of paclitaxel or mitoxantrone in ABCB1- or ABCG2-overexpressing cells. Furthermore, erlotinib did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in all cells and was unable to reverse MRP1-mediated MDR and had no effect on the parental cells. However, erlotinib remarkably inhibited the transport of E(2)17 beta G and methotrexate by ABCG2. In addition, the results of ATPase assays show that erlotinib stimulated the ATPase activity of both ABCB1 and ABCG2. Interestingly, erlotinib slightly inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin (IAAP) at high concentration, but it did not inhibit the photolabeling of ABCG2 with IAAP. Overall, we conclude that erlotinib reverses ABCB1- and ABCG2-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1 and ABCG2. These findings may be useful for cancer combinational therapy with erlotinib in the clinic.

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121 Recent studies have shown that mutations at amino acid 482 in ABCG2 affect the substrate and antagonist specificity of ABCG2 (23, 34); therefore, we investigated the reversal effect of erlotinib on both wild-type (R482) and mutated (R482G and R482T) ABCG2-mediated MDR. Login to comment
137 These results suggest that erlotinib specifically potentiates the sensitivity of ABCB1 substrates in the ABCB1-overexpressing cells and also enhances the sensitivity of ABCG2 substrates in both wild-type and R482G/T mutant ABCG2-overexpressing cells we tested. Login to comment
211 In our results of transport by ABCG2 vesicles, E217hG and methotrexate are only transported by the wild-type ABCG2 and not by the two mutated R482G and R482T ABCG2. Login to comment

[hide] Evidence for dual mode of action of a thiosemicarb... Mol Cancer Ther. 2007 Dec;6(12 Pt 1):3287-96. Wu CP, Shukla S, Calcagno AM, Hall MD, Gottesman MM, Ambudkar SV
Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance linked ABCG2 transporter.
Mol Cancer Ther. 2007 Dec;6(12 Pt 1):3287-96., [PMID:18089722]

Abstract [show]
Multidrug resistance due to reduced drug accumulation is a phenomenon predominantly caused by the overexpression of members of the ATP-binding cassette (ABC) transporters, including ABCB1 (P-glycoprotein), ABCG2, and several ABCC family members [multidrug resistance-associated protein (MRP)]. We previously reported that a thiosemicarbazone derivative, NSC73306, is cytotoxic to carcinoma cells that overexpress functional P-glycoprotein, and it resensitizes these cells to chemotherapeutics. In this study, we investigated the effect of NSC73306 on cells overexpressing other ABC drug transporters, including ABCG2, MRP1, MRP4, and MRP5. Our findings showed that NSC73306 is not more toxic to cells that overexpress these transporters compared with their respective parental cells, and these transporters do not confer resistance to NSC73306 either. In spite of this, we observed that NSC73306 is a transport substrate for ABCG2 that can effectively inhibit ABCG2-mediated drug transport and reverse resistance to both mitoxantrone and topotecan in ABCG2-expressing cells. Interactions between NSC73306 and the ABCG2 drug-binding site(s) were confirmed by its stimulatory effect on ATPase activity (140-150 nmol/L concentration required for 50% stimulation) and by inhibition of [(125)I]iodoarylazidoprazosin photolabeling (50% inhibition at 250-400 nmol/L) of the substrate-binding site(s). Overall, NSC73306 seems to be a potent modulator of ABCG2 that does not interact with MRP1, MRP4, or MRP5. Collectively, these data suggest that NSC73306 can potentially be used, due to its dual mode of action, as an effective agent to overcome drug resistance by eliminating P-glycoprotein-overexpressing cells and by acting as a potent modulator that resensitizes ABCG2-expressing cancer cells to chemotherapeutics.

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341 Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter. Login to comment

[hide] ABC multidrug transporters: structure, function an... Pharmacogenomics. 2008 Jan;9(1):105-27. Sharom FJ
ABC multidrug transporters: structure, function and role in chemoresistance.
Pharmacogenomics. 2008 Jan;9(1):105-27., [PMID:18154452]

Abstract [show]
Three ATP-binding cassette (ABC)-superfamily multidrug efflux pumps are known to be responsible for chemoresistance; P-glycoprotein (ABCB1), MRP1 (ABCC1) and ABCG2 (BCRP). These transporters play an important role in normal physiology by protecting tissues from toxic xenobiotics and endogenous metabolites. Hydrophobic amphipathic compounds, including many clinically used drugs, interact with the substrate-binding pocket of these proteins via flexible hydrophobic and H-bonding interactions. These efflux pumps are expressed in many human tumors, where they likely contribute to resistance to chemotherapy treatment. However, the use of efflux-pump modulators in clinical cancer treatment has proved disappointing. Single nucleotide polymorphisms in ABC drug-efflux pumps may play a role in responses to drug therapy and disease susceptibility. The effect of various genotypes and haplotypes on the expression and function of these proteins is not yet clear, and their true impact remains controversial.

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372 The R482T and R482G variants were able to efflux rhodamine 123 and doxorubicin, whereas the wild-type was not, however, all three forms of ABCG2 transported mitoxantrone. Login to comment
648 Clark R, Kerr ID, Callaghan R: Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter. Login to comment
632 Clark R, Kerr ID, Callaghan R: Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter. Login to comment

[hide] Ubiquitin-mediated proteasomal degradation of non-... Biochem J. 2008 May 1;411(3):623-31. Nakagawa H, Tamura A, Wakabayashi K, Hoshijima K, Komada M, Yoshida T, Kometani S, Matsubara T, Mikuriya K, Ishikawa T
Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2.
Biochem J. 2008 May 1;411(3):623-31., 2008-05-01 [PMID:18237272]

Abstract [show]
Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.

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25 On the basis of our functional validation, the above-mentioned non-synonymous polymorphisms, as well as the acquired mutants (R482G and R482T) of ABCG2 were classified into four groups [33]. Login to comment

[hide] Homology modeling of breast cancer resistance prot... J Struct Biol. 2008 Apr;162(1):63-74. Epub 2007 Dec 15. Hazai E, Bikadi Z
Homology modeling of breast cancer resistance protein (ABCG2).
J Struct Biol. 2008 Apr;162(1):63-74. Epub 2007 Dec 15., [PMID:18249138]

Abstract [show]
BCRP (also known as ABCG2, MXR, and ABC-P) is a member of the ABC family that transports a wide variety of substrates. BCRP is known to play a key role as a xenobiotic transporter. Since discovering its role in multidrug resistance, considerable efforts have been made in order to gain deeper understanding of BCRP structure and function. The recent study was aimed at predicting BCRP structure by creating a homology model. Based on sequence similarity with known structures of full-length, NB and TM domain of ABC transporters, TM, NB, and linker regions of BCRP were defined. The NB domain of BCRP was modeled using MalK as a template. Based on secondary structure prediction of BCRP and comparison of the transmembrane connecting regions of known structures of ABC transporters, the TM domain arrangement of BCRP was established and was found to resemble to that of the recently published crystal structure of Sav1866. Thus, an initial alignment of TM domain of BCRP was established using Sav1866 as a template. This alignment was subsequently refined using constrains derived from secondary structure and TM predictions and the final model was built. Finally, the complete homodimer ABCG2 model was generated using Sav1866 as template. Furthermore, known ligands of BCRP were docked to our model in order to define possible binding sites. The results of molecular dockings of known BCRP substrates to the BCRP model were in agreement with recently published experimental data indicating multiple binding sites in BCRP.

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245 However, in our model, R482 cannot form interaction with rhodamine, but L484 is in interacting distance Table 3 Mutations on BCRP and their effect on its function Mutation Effect/results Reference V12M Did not effect Hemato and MTX transport Tamura et al. (2006) G51C Did not effect Hemato and MTX transport Tamura et al. (2006) K86M Inactivates transporter (dominant negative effect on ATPase activity); alters subcellular distribution Henriksen et al. (2005a) K86M Transporter inactive, but still able to bind ATP Ozvegy et al. (2002) Q126stop Defective porphyrin transport Tamura et al. (2006) Q141K Did not effect Hemato and MTX transport Tamura et al. (2006) T153M Did not effect Hemato and MTX transport Tamura et al. (2006) Q166E Did not effect Hemato and MTX transport Tamura et al. (2006) I206L Did not effect Hemato and MTX transport Tamura et al. (2006) F208S Defective porphyrin transport Tamura et al. (2006) S248P Defective porphyrin transport Tamura et al. (2006) E334stop Defective porphyrin transport Tamura et al. (2006) F431L Effects MTX transport Tamura et al. (2006) S441N Defective porphyrin transport Tamura et al. (2006) E446-mutants No drug resistance Miwa et al. (2003) R482G, R482T Effects MTX transport Tamura et al. (2006) R482T Substrate drug transport and inhibitor efficiency is not mediated by changes in drug-binding Pozza et al. (2006) R482G, R482T Substitution influence the substrate specificity of the transporter Ozvegy et al. (2002) R482G, R482T Altered substrate specificity Honjo et al. (2001) R482G Methotrexate not transported Chen et al. (2003b) Mitomo et al. (2003) R482G Resistance to hydrophilic antifolates in vitro, G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates Shafran et al., (2005) R482G Three distinct drug, binding sites Clark et al. (2006) R482G Altered substrate specificity, granulocyte maturation uneffected Ujhelly et al. (2003) R482 mutants Higher resistance to mitoxantrone and doxorubicin than wt Miwa et al. (2003) R482X Affects substrate transport and ATP hydrolysis but not substrate binding Ejendal et al. (2006) F489L Impaired porphyrin transport Tamura et al. (2006) G553L; G553E Impaired trafficing, expression, and N-linked glycosylation Polgar et al. (2006) L554P Dominant negative effect on drug sensitivity Kage et al. (2002) N557D Resistance to MTX, but decreased transport of SN-38; N557E no change in transport compared to wt Miwa et al. (2003) F571I Did not effect Hemato and MTX transport Tamura et al. (2006) N590Y Did not effect Hemato and MTX transport Tamura et al. (2006) C592A Impaired function and expression Henriksen et al. (2005b) C592A/C608A Restored plasma mb expression; MTX transport normal, BODIPY-prazosin impaired Henriksen et al. (2005b) C603A Disulfide bridge; no functional or membrane targeting change Henriksen et al. (2005b) C608A Impaired function and expression Henriksen et al. (2005b) D620N Did not effect Hemato and MTX transport Tamura et al. (2006) H630X No change in transport Miwa et al. (2003) Cand N-terminal truncated Impaired trafficing Takada et al. (2005) with the ligand. Login to comment

[hide] Drug transporters: recent advances concerning BCRP... Br J Cancer. 2008 Mar 11;98(5):857-62. Epub 2008 Feb 5. Lemos C, Jansen G, Peters GJ
Drug transporters: recent advances concerning BCRP and tyrosine kinase inhibitors.
Br J Cancer. 2008 Mar 11;98(5):857-62. Epub 2008 Feb 5., 2008-03-11 [PMID:18253130]

Abstract [show]
Multidrug resistance is often associated with the (over)expression of drug efflux transporters of the ATP-binding cassette (ABC) protein family. This minireview discusses the role of one selected ABC-transporter family member, the breast cancer resistance protein (BCRP/ABCG2), in the (pre)clinical efficacy of novel experimental anticancer drugs, in particular tyrosine kinase inhibitors.

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92 In contrast, cells carrying a glycine (R482G) or a threonine (R482T) at position 482 were able to transport rhodamine 123 and doxorubicin, while also maintaining their ability to transport mitoxantrone. Login to comment
93 The BCRP variants were found in drug-resistant S1-M1-80 (R482G) and MCF-7 AdVp3000 (R482T) but not in the parental S1 and MCF-7 cell lines, suggesting that these were acquired mutations resulting from drug selection. Login to comment
95 Hence these R482G cells were not resistant compared to cells with wild-type BCRP when continuously exposed to methotrexate (Chen et al, 2003). Login to comment
96 However, when a short 4 h exposure was used, the mutant R482G was highly resistant to methotrexate (Assaraf, 2006). Login to comment

[hide] Drug-induced phototoxicity evoked by inhibition of... Expert Opin Drug Metab Toxicol. 2008 Mar;4(3):255-72. Tamura A, An R, Hagiya Y, Hoshijima K, Yoshida T, Mikuriya K, Ishikawa T
Drug-induced phototoxicity evoked by inhibition of human ABC transporter ABCG2: development of in vitro high-speed screening systems.
Expert Opin Drug Metab Toxicol. 2008 Mar;4(3):255-72., [PMID:18363541]

Abstract [show]
BACKGROUND: Photosensitivity depends on both genetic and environmental factors. Pheophorbide a, present in various plant-derived foods and food supplements, can be absorbed by the small intestine. Accumulation of pheophorbide a and porphyrins in the systemic blood circulation can result in phototoxic lesions on light-exposed skin. OBJECTIVE: As the human ATP-binding cassette (ABC) transporter ABCG2 has been suggested to be critically involved in porphyrin-mediated photosensitivity, we aimed to develop in vitro screening systems for drug-induced phototoxicity. CONCLUSION: Functional impairment owing to inhibition of ABCG2 by drugs or its genetic polymorphisms can lead to the disruption of porphyrin homeostasis. This review article provides an overview on drug-induced photosensitivity, as well as our hypothesis on a potential role of ABCG2 in phototoxicity.

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230 Plasma membrane Outside Inside ATP-binding cassette H2 N COOH V12M G51C Q126stop Q141K T153M R160Q Q166E I206L F208S S248P E334stop F431L F489L S441N R482G R482T F571I R575stop N590Y D620N T542A A528T D296H P269S A. Login to comment
231 0.0 0.1 0.2 0.3 0.4 0.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T ATP-dependenthematoporphyrintransport (nmol/min/mgprotein) B. interactions should also take into consideration the presence of multiple flavonoids. Login to comment
245 Based on the presently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Login to comment

[hide] Effect of cysteine mutagenesis on the function and... J Pharmacol Exp Ther. 2008 Jul;326(1):33-40. Epub 2008 Apr 22. Liu Y, Yang Y, Qi J, Peng H, Zhang JT
Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2.
J Pharmacol Exp Ther. 2008 Jul;326(1):33-40. Epub 2008 Apr 22., [PMID:18430864]

Abstract [show]
ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. Its overexpression causes multidrug resistance in cancer chemotherapy. Based on its apparent half size in sequence when compared with other traditional ABC transporters, ABCG2 has been thought to exist and function as a homodimer linked by intermolecular disulfide bonds. However, recent evidence suggests that ABCG2 may exist as a higher form of oligomers due to noncovalent interactions. In this study, we attempted to create a cysless mutant ABCG2 as a tool for further characterization of this molecule. However, we found that the cysless mutant ABCG2 is well expressed but not functional. Mapping of the cysteine residues showed that three cysteine residues (Cys284, Cys374, and Cys438) are required concurrently for the function of ABCG2 and potentially for intramolecular disulfide bond formation. We also found that the cysteine residues (Cys592, Cys603, and Cys608) in the third extracellular loop are involved in forming intermolecular disulfide bonds and that mutation of these residues does not affect the expression or drug transport activity of human ABCG2. Thus, we conclude that Cys284, Cys374, and Cys438, which may be involved in intramolecular disulfide bond formation, are concurrently required for ABCG2 function, whereas Cys592, Cys603, and Cys608, potentially involved in intermolecular disulfide bond formation, are not required.

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37 It is interesting to note that two nonsynonymous mutations, R482G and R482T, resulted in the ability of ABCG2 to transport substrates, such as rhodamine 123, which cannot be transported by the wild-type isoform (Han and Zhang, 2004). Login to comment

[hide] Characterization of substrates and inhibitors for ... Pharm Res. 2008 Oct;25(10):2320-6. Epub 2008 Jun 4. Muenster U, Grieshop B, Ickenroth K, Gnoth MJ
Characterization of substrates and inhibitors for the in vitro assessment of Bcrp mediated drug-drug interactions.
Pharm Res. 2008 Oct;25(10):2320-6. Epub 2008 Jun 4., [PMID:18523872]

Abstract [show]
PURPOSE: In vitro assessment of drug candidates' affinity for multi-drug resistance proteins is of crucial importance for the prediction of in vivo pharmacokinetics and drug-drug interactions. To have well described experimental tools at hand, the objective of the study was to characterize substrates and inhibitors of Breast Cancer Resistance Protein (BCRP) and P-glycoprotein (P-gp). METHODS: Madin-Darbin canine kidney cells overexpressing mouse Bcrp (MDCKII-Bcrp) were incubated with various Bcrp substrates, or a mixture of substrate and inhibitor to either the apical (A) or basolateral (B) compartment of insert filter plates. Substrate concentrations in both compartments at time points t = 0 h and t = 2 h were determined by LC-MS/MS, and respective permeation coefficients (Papp) and efflux ratios were calculated. RESULTS: The Bcrp inhibitor Ko143 blocked topotecan and ABZSO transport in a concentration-dependent manner. P-gp inhibitors ivermectin, LY335979, PSC833, and the P-gp/Bcrp inhibitor ritonavir did not influence Bcrp mediated topotecan transport, however, blocked ABZSO transport. Additionally, neither was ABZSO transport influenced by topotecan, nor topotecan transport by ABZSO. CONCLUSIONS: Data suggest different modes of substrate and inhibitor binding to Bcrp. In order to not overlook potential drug-drug interactions when testing drug candidates for inhibitory potential towards Bcrp, distinct Bcrp probe substrates should be used.

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129 Recently strong evidence for the existence of at least two separate binding sites in the R482G BCRP mutant has been presented (31). Login to comment

[hide] Interaction with the 5D3 monoclonal antibody is re... J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21. Ozvegy-Laczka C, Laczko R, Hegedus C, Litman T, Varady G, Goda K, Hegedus T, Dokholyan NV, Sorrentino BP, Varadi A, Sarkadi B
Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.
J Biol Chem. 2008 Sep 19;283(38):26059-70. Epub 2008 Jul 21., 2008-09-19 [PMID:18644784]

Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

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52 Expression Vectors, Cell Lines, and Cell Culturing pCIN4 bicistronic mammalian expression vectors containing the cDNAs of ABCG2-R482G, or additional Cys to Ala mutations, were generated as described previously (10). Login to comment
113 To analyze whether there is a correlation between covalent dimer formation upon PFA fixation and increased 5D3 binding, and whether PFA cross-linking inhibits ABCG2 function, we treated intact HEK cells expressing ABCG2 (R482G) with increasing concentrations of PFA and analyzed 5D3 binding, ABCG2 function, and covalent dimer formation. Login to comment
114 Throughout this study we used both the wild-type and the R482G mutant variant of ABCG2 in HEK cells, because the background of the cysteine mutations was this latter variant (10). Login to comment
115 The R482G mutant also allowed the measurement of transport activity followed by rhodamine123 extrusion, characteristic of the mutant protein. Login to comment
116 In all functional experiments the R482G protein variant showed the same behavior regarding 5D3 binding as the wild-type ABCG2, that is PFA or Ko143 caused a significant 5D3 shift (Fig. 1B). Login to comment
121 Fig. 1C also shows rhodamine123 transport activity (activity factor) of the HEK-ABCG2-R482G cells treated with increasing concentrations of PFA. Login to comment
131 The experiments shown in Fig. 2 were performed in HEK-ABCG2-R482G cells, but experiments repeated in both PLB985 and A431 cells, expressing the wild-type ABCG2 protein, provided the same results (data not shown). Login to comment
140 HEK-ABCG2-R482G cells were lysed, dissolved in disaggregation buffer containing the reducing agent beta-mercaptoethanol or without it (as indicated on the figure), and subjected to 7.5% SDS-PAGE. ABCG2 in cells fixed with 0.5% PFA prior to cell lysis is also shown. Login to comment
144 HEK293 cells transfected with empty pCIN4 or pCIN4-ABCG2(R482G) were labeled with Alexa647-conjugated 5D3. Login to comment
149 Mock or pCIN4-ABCG2(R482G)-transfected HEK293 cells were fixed with increasing concentrations of PFA, washed, and then labeled with 5D3 antibody or mouse IgG2b (isotype control (IT)). Login to comment
171 We analyzed the transport of four different fluorescent ABCG2 substrate compounds, MX, pheophorbide A, Hoechst 33342, and rhodamine123 (rhodamine123 is transported only by the R482G ABCG2 mutant). Login to comment
173 Hoechst 33342 and pheophorbide A were transported by all of the mutants (except for C603A/C608A); however, the mitoxantrone transport capacity of the mutants, lacking the intramolecular or both disulfide bonds, was significantly weaker than that of the R482G or C603A variants. Login to comment
190 HEK-ABCG2-R482G cells were incubated with different protein cross-linkers, washed, and then labeled with 5D3-Alexa647 (left panel) or incubated with 2 ␮M rhodamine123 (right panel) in the presence or absence of the inhibitor Ko143. Fluorescence was determined by flow cytometry. Login to comment
213 In this study we have further analyzed how alterations in ABCG2 structure, covalent cross-linking, or changes in the S-S FIGURE3.EffectofDTTtreatmenton5D3binding.HEK293cellstransfected with empty pCIN4 (A) or pCIN4-ABCG2(R482G) (B) were incubated with or without 10 mM DTT, washed, and then labeled with 5D3 or mouse IgG2b and goat anti-mouse phycoerythrin-conjugated secondary antibody. Login to comment
218 HEK cells were transfected with pCIN4 vectors encoding different Cys-to-Ala ABCG2 mutants or R482G (indicated as ABCG2). Login to comment
221 B, mitoxantrone, pheophorbide A, rhodamine123, and Hoechst 33342 transport activity of Cys-to-Ala mutants. HEK cells expressing different ABCG2 mutants or R482G (indicated as ABCG2) were incubated with 5 ␮M mitoxantrone, 1 ␮M pheophorbide A, 2 ␮M rhodamine123, or 1 ␮M Hoechst 33342 in the absence or presence of 1 ␮M Ko143. Fluorescence of mitoxantrone, pheophorbide A, and rhodamine123 was detected in a FACSCalibur cytometer, and activity factors were calculated from mean fluorescence values as described under "Experimental Procedures." Login to comment

[hide] Is ATP binding responsible for initiating drug tra... FEBS J. 2008 Sep;275(17):4354-62. Epub 2008 Jul 24. McDevitt CA, Crowley E, Hobbs G, Starr KJ, Kerr ID, Callaghan R
Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2?
FEBS J. 2008 Sep;275(17):4354-62. Epub 2008 Jul 24., [PMID:18657189]

Abstract [show]
ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indicated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorientation to the low-affinity state to enable dissociation at the extracellular face. [3H]Daunomycin binding to the ABCG2 R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissociation of phosphate and/or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the 'power stroke' for translocation in ABCG2 R482G is the binding of nucleotide.

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27 Selection in mitoxantrone produced R482G or R482T point mutations that present considerably broader substrate selectivity [20,21]. Login to comment
28 For example, the R482G isoform is a gain-of-function mutation which mediates the transport of doxorubicin, daunomycin and rhodamine 123, whereas it has a loss of function with respect to methotrexate transport. Login to comment
31 In a departure from the drug-protein interactions with ABCB1, the R482G isoform also contains multiple sites of interaction for a single drug (daunomycin), which can manifest as homotropic allostery [22]. Login to comment
34 The best evidence for an interaction between the two domains is the ability of numerous substrates and modulators of ABCG2 (and the R482G isoform) to stimulate the rate of ATP hydrolysis [21,24,25], albeit to a lesser degree than that commonly encountered with ABCB1. Login to comment

[hide] Human ABC transporters ABCG2 (BCRP) and ABCG4. Xenobiotica. 2008 Jul;38(7-8):863-88. Koshiba S, An R, Saito H, Wakabayashi K, Tamura A, Ishikawa T
Human ABC transporters ABCG2 (BCRP) and ABCG4.
Xenobiotica. 2008 Jul;38(7-8):863-88., [PMID:18668433]

Abstract [show]
1. The human ABC transporter ABCG2 is regarded as a member of the phase III system for xenobiotic metabolism, and it has been suggested that this efflux pump is responsible for protecting the body from toxic xenobiotics and for removing metabolites. 2. This review paper will address the new aspects of ABCG2 in terms of post-translational modifications (i.e., disulfide bond formation, ubiquitination, and endoplasmic reticulum-associated degradation) of ABCG2 protein, high-speed screening, and quantitative structure-activity relationship (QSAR) analysis to evaluate ABCG2-drug interactions, and genetic polymorphisms potentially associated with photosensitivity. 3. In addition, new aspects of human ABCG4 and mouse Abcg4 are presented with respect to their molecular properties and potential physiological roles. Considering a high sequence similarity between ABCG1 and ABCG4, both Abcg4 and ABCG4 may be involved in the transport of cholesterol from neurons and astrocytes. Furthermore, high expression of the mouse Abcg4 protein in the testis implicates its involvement in transport of certain sex hormones.

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111 The wild-type of ABCG2 transports MTX, whereas acquired mutants, i.e., R482G and R482T, do not (Chen et al. 2003; Mitomo et al. 2003; Volk and Schneider 2003). Login to comment
225 Based on the currently available data on SNPs and acquired mutations, a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) were created by site-directed mutagenesis and expressed in Sf9 insect cells (Tamura et al. 2006, 2007). Login to comment
234 The F431L variant as well as the acquired mutants R482G and R482T transported haematoporphyrin (Figure 9a), although they did not transport methotrexate (Figure 9b). Login to comment

[hide] Pharmacogenetics of intestinal absorption. Curr Drug Deliv. 2008 Jul;5(3):153-69. Nakamura T, Yamamori M, Sakaeda T
Pharmacogenetics of intestinal absorption.
Curr Drug Deliv. 2008 Jul;5(3):153-69., [PMID:18673259]

Abstract [show]
The small intestine is the primary site of absorption for many drugs administered orally and so is the target tissue for pharmacotherapeutic strategies to control the oral absorption of drugs. Drug transporters, including the ATP-binding cassette (ABC) superfamily and the solute carrier (SLC) superfamily, have been considered to play a physiological role in regulating the absorption of xenobiotics, and variations in their expression level and function in the small intestine cause intra- and inter-individual variation in the oral absorption of drugs. Recent advances in molecular biology have suggested that genetic polymorphisms are associated with the expression level and function, and thereby inter-individual variation. In this review, the pharmacogenetics of these transporters is summarized, and their future significance in the clinical setting is discussed.

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69 Volk and co-workers reported that methotrexate resistance correlated with ABCG2 expression in cell lines expressing the wild-type transporter, whereas the Arg482Thr and Arg482Gly variants were more resistant to mitoxantrone and less resistant to methotrexate, than expected from their ABCG2 expression levels using drug-selected and ABCG2-transfected cell lines [78]. Login to comment

[hide] Natural allelic variants of bovine ATP-binding cas... Drug Metab Dispos. 2009 Jan;37(1):5-9. Epub 2008 Sep 29. Merino G, Real R, Baro MF, Gonzalez-Lobato L, Prieto JG, Alvarez AI, Marques MM
Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.
Drug Metab Dispos. 2009 Jan;37(1):5-9. Epub 2008 Sep 29., [PMID:18824523]

Abstract [show]
ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

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180 Clark R, Kerr ID, and Callaghan R (2006) Multiple drug binding sites on the R482G isoform of the ABCG2 transporter. Login to comment

[hide] Lapatinib (Tykerb, GW572016) reverses multidrug re... Cancer Res. 2008 Oct 1;68(19):7905-14. Dai CL, Tiwari AK, Wu CP, Su XD, Wang SR, Liu DG, Ashby CR Jr, Huang Y, Robey RW, Liang YJ, Chen LM, Shi CJ, Ambudkar SV, Chen ZS, Fu LW
Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2.
Cancer Res. 2008 Oct 1;68(19):7905-14., 2008-10-01 [PMID:18829547]

Abstract [show]
Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E(2)17betaG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.

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175 Therefore, we investigated whether lapatinib would reverse ABCG2-mediated resistance to mitoxantrone in cells transfected with either the wild-type (R482) or mutant (R482G and R482T) forms of ABCG2. Login to comment
181 These results suggest that lapatinib specifically enhances the sensitivity of ABCG2 substrates in cells expressing either wild-type or mutant R482G/T ABCG2. Login to comment
314 Our results showed that lapatinib significantly enhances the sensitivity of ABCG2 substrates not only in cells overexpressing wild-type but also in the R482G/T variants of ABCG2. Login to comment

[hide] Clinical pharmacogenetics and potential applicatio... Curr Drug Metab. 2008 Oct;9(8):738-84. Zhou SF, Di YM, Chan E, Du YM, Chow VD, Xue CC, Lai X, Wang JC, Li CG, Tian M, Duan W
Clinical pharmacogenetics and potential application in personalized medicine.
Curr Drug Metab. 2008 Oct;9(8):738-84., [PMID:18855611]

Abstract [show]
The current 'fixed-dosage strategy' approach to medicine, means there is much inter-individual variation in drug response. Pharmacogenetics is the study of how inter-individual variations in the DNA sequence of specific genes affect drug responses. This article will highlight current pharmacogenetic knowledge on important drug metabolizing enzymes, drug transporters and drug targets to understand interindividual variability in drug clearance and responses in clinical practice and potential use in personalized medicine. Polymorphisms in the cytochrome P450 (CYP) family may have had the most impact on the fate of pharmaceutical drugs. CYP2D6, CYP2C19 and CYP2C9 gene polymorphisms and gene duplications account for the most frequent variations in phase I metabolism of drugs since nearly 80% of drugs in use today are metabolised by these enzymes. Approximately 5% of Europeans and 1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers. CYP2C9 is another clinically significant drug metabolising enzyme that demonstrates genetic variants. Studies into CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and CYP2C9*3 alleles. Extensive polymorphism also occurs in a majority of Phase II drug metabolizing enzymes. One of the most important polymorphisms is thiopurine S-methyl transferases (TPMT) that catalyzes the S-methylation of thiopurine drugs. With respect to drug transport polymorphism, the most extensively studied drug transporter is P-glycoprotein (P-gp/MDR1), but the current data on the clinical impact is limited. Polymorphisms in drug transporters may change drug's distribution, excretion and response. Recent advances in molecular research have revealed many of the genes that encode drug targets demonstrate genetic polymorphism. These variations, in many cases, have altered the targets sensitivity to the specific drug molecule and thus have a profound effect on drug efficacy and toxicity. For example, the beta (2)-adrenoreceptor, which is encoded by the ADRB2 gene, illustrates a clinically significant genetic variation in drug targets. The variable number tandem repeat polymorphisms in serotonin transporter (SERT/SLC6A4) gene are associated with response to antidepressants. The distribution of the common variant alleles of genes that encode drug metabolizing enzymes, drug transporters and drug targets has been found to vary among different populations. The promise of pharmacogenetics lies in its potential to identify the right drug at the right dose for the right individual. Drugs with a narrow therapeutic index are thought to benefit more from pharmacogenetic studies. For example, warfarin serves as a good practical example of how pharmacogenetics can be utilized prior to commencement of therapy in order to achieve maximum efficacy and minimum toxicity. As such, pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and licensed drugs.

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614 It was discovered that the first cloned BCRP cDNA [265] encoded a mutant BCRP that differs from the wild-type BCRP at Arg482 (R482), which was substituted with either threonine (R482T) or glycine (R482G) [269]. Login to comment

[hide] Major SNP (Q141K) variant of human ABC transporter... Pharm Res. 2009 Feb;26(2):469-79. Epub 2008 Oct 29. Furukawa T, Wakabayashi K, Tamura A, Nakagawa H, Morishima Y, Osawa Y, Ishikawa T
Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations.
Pharm Res. 2009 Feb;26(2):469-79. Epub 2008 Oct 29., [PMID:18958403]

Abstract [show]
PURPOSE: Single nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients' responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations. METHODS: ABCG2 WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively. RESULTS: The protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A1, the protein levels of ABCG2 WT and Q141K increased 5- and 2-fold in Flp-In-293 cells, respectively. CONCLUSIONS: The results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.

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173 Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as two acquired mutants (R482G and R482T) of ABCG2 were classified into four groups (18). Login to comment
128 Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as two acquired mutants (R482G and R482T) of ABCG2 were classified into four groups (18). Login to comment

[hide] The 315-316 deletion determines the BXP-21 antibod... Mol Cell Biochem. 2009 Feb;322(1-2):63-71. Epub 2008 Nov 11. Polgar O, Deeken JF, Ediriwickrema LS, Tamaki A, Steinberg SM, Robey RW, Bates SE
The 315-316 deletion determines the BXP-21 antibody epitope but has no effect on the function of wild type ABCG2 or the Q141K variant.
Mol Cell Biochem. 2009 Feb;322(1-2):63-71. Epub 2008 Nov 11., [PMID:19002564]

Abstract [show]
ABCG2 is a half-transporter initially described in multidrug-resistant cancer cells and lately identified as an important factor in the pharmacokinetics of its substrates. Q141K is by far the most intensively studied single nucleotide polymorphism of ABCG2 with potential clinical relevance. Here we used stably transfected HEK cells to study the Q141K polymorphism together with the deletion of amino acids 315-316, which were recently reported to coexist in two cancer cell lines (A549 and SK-OV-3). Functional studies confirmed our previous report that when normalized to surface expression, Q141K has impaired transport of mitoxantrone. This result was extended to include the ABCG2-specific substrate pheophorbide a. While we found no functional consequence of deleting amino acids 315 and 316, we did find that the deletion mutant is no longer recognized by the BXP-21 antibody. We conclude that amino acids 315 and 316 form part of the epitope for the BXP-21 antibody.

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31 The wild type (R482), Q141K, R482G, and empty vector-transfected (pcDNA) cells were previously reported [13]. Login to comment
81 Figure 3 shows a representative example of surface expression, mitoxantrone and pheophorbide a efflux for each mutant compared to the wild type and the R482G mutant which is a previously characterized, so-called gain-of-function mutant with a wider substrate specificity than wild type ABCG2 [25]. Login to comment
90 In agreement with previous reports, the transport efficacy of the R482G mutant was found to be significantly higher than that of the wild type for mitoxantrone [13]. Login to comment
97 After normalizing the measured efflux values to ABCG2 expression by the 5D3 monoclonal antibody, we have found that the D315-316 mutant has equivalent efflux 0.331 0.289 0.261 0.256 0.520 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 482R Q141K 316 R482G p=0.028 p<0.0001 p=0.0099 Mitoxantroneefflux/5D3antibody n=14 n=15n=23n=23n=22 0.445 0.410 0.263 0.313 0.504 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 482R Q141K 316 R482G p=0.00075 p=0.0000052 p=0.013 p=0.039 Pheophorbideaefflux/5D3antibody n=6 n=10n=14n=15 n=12 A B ∆315-316 Q141K/∆315- ∆315-316 Q141K/∆315- Fig. 4 Efflux normalized to cell surface expression. Login to comment
98 The mean FTC-inhibitable mitoxantrone (panel A) and pheophorbide a (panel B) efflux was plotted for the wild type (482R) and each mutant (Q141K, D315-316, aQ141K/D315-316, and R482G). Login to comment
107 Finally, it should be noted that the gain-of-function feature of the R482G mutant was again demonstrated, although the latter was only significant when mitoxantrone was used. Login to comment
120 This could mean that similar to the R482G mutation [34] this splicing variant will be found only in cell culture. Login to comment
36 The wild type (R482), Q141K, R482G, and empty vector-transfected (pcDNA) cells were previously reported [13]. Login to comment
76 Figure 3 shows a representative example of surface expression, mitoxantrone and pheophorbide a efflux for each mutant compared to the wild type and the R482G mutant which is a previously characterized, so-called gain-of-function mutant with a wider substrate specificity than wild type ABCG2 [25]. Login to comment
85 In agreement with previous reports, the transport efficacy of the R482G mutant was found to be significantly higher than that of the wild type for mitoxantrone [13]. Login to comment
110 This could mean that similar to the R482G mutation [34] this splicing variant will be found only in cell culture. Login to comment

[hide] Substrate-dependent breast cancer resistance prote... Drug Metab Dispos. 2009 Mar;37(3):560-70. Epub 2008 Dec 4. Giri N, Agarwal S, Shaik N, Pan G, Chen Y, Elmquist WF
Substrate-dependent breast cancer resistance protein (Bcrp1/Abcg2)-mediated interactions: consideration of multiple binding sites in in vitro assay design.
Drug Metab Dispos. 2009 Mar;37(3):560-70. Epub 2008 Dec 4., [PMID:19056916]

Abstract [show]
In vitro assays are frequently used for the screening of substrates and inhibitors of transporter-mediated efflux. Examining directional flux across Madin-Darby canine kidney (MDCK) II cell monolayers that overexpress a transporter protein is particularly useful in identifying whether or not a candidate compound is an inhibitor or substrate for that transport system. Studies that use a single substrate or inhibitor in competition assays can be challenging to interpret because of the possible multiple mechanisms involved in substrate/inhibitor-protein interactions. During our previous studies of substrate-inhibitor-transporter interactions, we observed differences in breast cancer resistance protein (BCRP) inhibition, depending on the substrate and the inhibitor. Therefore, we investigated BCRP-mediated interactions with a 4 x 4 matrix of substrates and inhibitors using monolayers formed from MDCKII cells transfected with murine BCRP (Bcrp1/Abcg2). The selective BCRP inhibitor 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino [1',2':1,6] pyrido [3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143) effectively inhibited the Bcrp1-mediated transport of all substrates examined. However, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9 ,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), nelfinavir, and Pluronic P85 exhibited differences in inhibition depending on the substrate examined. Our findings support recent reports suggesting that the interactions of substrate molecules with BCRP involve multiple binding regions in the protein. The nucleoside substrates zidovudine and abacavir seem to bind to a region on BCRP that may have little or no overlap with the binding regions of either prazosin or imatinib. In conclusion, the choice of substrate or inhibitor molecules for an in vitro assay system can be crucial for the optimal design of experiments to evaluate transporter-mediated drug-drug interactions.

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185 Clark et al. (2006) reported that the binding of [3 H]daunomycin to the R482G isoform of BCRP was complete, partial, or not significant, depending on the competing BCRP substrate used to examine this binding interaction. Login to comment

[hide] Inhibiting the function of ABCB1 and ABCG2 by the ... Biochem Pharmacol. 2009 Mar 1;77(5):781-93. Epub 2008 Nov 18. Shi Z, Tiwari AK, Shukla S, Robey RW, Kim IW, Parmar S, Bates SE, Si QS, Goldblatt CS, Abraham I, Fu LW, Ambudkar SV, Chen ZS
Inhibiting the function of ABCB1 and ABCG2 by the EGFR tyrosine kinase inhibitor AG1478.
Biochem Pharmacol. 2009 Mar 1;77(5):781-93. Epub 2008 Nov 18., 2009-03-01 [PMID:19059384]

Abstract [show]
The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific EGFR tyrosine kinase inhibitor (TKI); its promising pre-clinical results have led to clinical trials. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCC1 and ABCG2 is one of the main causes of multidrug resistance (MDR) and usually results in the failure of cancer chemotherapy. However, the interaction of AG1478 with these ABC transporters is still unclear. In the present study, we have investigated this interaction and found that AG1478 has differential effects on these transporters. In ABCB1-overexpressing cells, non-toxic doses of AG1478 were found to partially inhibit resistance to ABCB1 substrate anticancer drugs as well as increase intracellular accumulation of [3H]-paclitaxel. Similarly, in ABCG2-overexpressing cells, AG1478 significantly reversed resistance to ABCG2 substrate anticancer drugs and increased intracellular accumulation of [3H]-mitoxantrone as well as fluorescent compound BODIPY-prazosin. AG1478 also profoundly inhibited the transport of [3H]-E(2)17betaG and [3H]-methotrexate by ABCG2. We also found that AG1478 slightly stimulated ABCB1 ATPase activity and significantly stimulated ABCG2 ATPase activity. Interestingly, AG1478 did not inhibit the photolabeling of ABCB1 or ABCG2 with [125I]-iodoarylazidoprazosin. Additionally, AG1478 did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drug and had no effect on the function of ABCC1. Overall, we conclude that AG1478 is able to inhibit the function of ABCB1 and ABCG2, with a more pronounced effect on ABCG2. Our findings provide valuable contributions to the development of safer and more effective EGFR TKIs for use as anticancer agents in the clinic.

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138 It has been reported that mutations at amino-acid 482 in ABCG2 alter the substrate and antagonist specificity of ABCG2 [16,25], therefore we examined the reversing effect of AG1478 on both wild-type (R482) and mutant (R482G and R482T) ABCG2. Login to comment

[hide] Functions of the breast cancer resistance protein ... Adv Drug Deliv Rev. 2009 Jan 31;61(1):26-33. Epub 2008 Dec 3. Noguchi K, Katayama K, Mitsuhashi J, Sugimoto Y
Functions of the breast cancer resistance protein (BCRP/ABCG2) in chemotherapy.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):26-33. Epub 2008 Dec 3., 2009-01-31 [PMID:19111841]

Abstract [show]
The breast cancer resistance protein, BCRP/ABCG2, is a half-molecule ATP-binding cassette transporter that facilitates the efflux of various anticancer agents from the cell, including 7-ethyl-10-hydroxycamptothecin, topotecan and mitoxantrone. The expression of BCRP can thus confer a multidrug resistance phenotype in cancer cells, and its transporter activity is involved in the in vivo efficacy of chemotherapeutic agents. Thus, the elucidation of the substrate preferences and structural relationships of BCRP is essential to understanding its in vivo functions during chemotherapeutic treatments. Single nucleotide polymorphisms (SNPs) have also been found to be key factors in determining the efficacy of chemotherapeutics, and those therapeutics that inhibit BCRP activity, such as the SNP that results in a C421A mutant, may result in unexpected side effects of the BCRP- anticancer drugs interaction even at normal dosages. In order to modulate the BCRP activity during chemotherapy, various compounds have been tested as inhibitors of this protein. Estrogenic compounds including estrone, several tamoxifen derivatives in addition to phytoestrogens and flavonoids have been shown to reverse BCRP-mediated drug resistance. Intriguingly, recently developed molecular targeted cancer drugs, such as the tyrosine kinase inhibitors imatinib mesylate, gefitinib and others, can also interact with BCRP. Since both functional SNPs and inhibitory agents of BCRP modulate the in vivo pharmacokinetics and pharmacodynamics of its substrate drugs, BCRP activity is an important consideration in the development of molecular targeted chemotherapeutics.

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826 MCF7/ AdVp3000 and S1-M1-80 cells expressing R482T and R482G variants of BCRP, respectively, are highly resistant to both mitoxantrone and doxorubicin. Login to comment
829 Moreover, the BCRP variants R482G and R482T lose their methotrexate-transporting activity but at the same time confer increased mitoxantrone resistance [18,42,44,45]. Login to comment

[hide] Quality control of human ABCG2 protein in the endo... Adv Drug Deliv Rev. 2009 Jan 31;61(1):66-72. Epub 2008 Dec 11. Wakabayashi-Nakao K, Tamura A, Furukawa T, Nakagawa H, Ishikawa T
Quality control of human ABCG2 protein in the endoplasmic reticulum: ubiquitination and proteasomal degradation.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):66-72. Epub 2008 Dec 11., 2009-01-31 [PMID:19111842]

Abstract [show]
Human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR/ABCP) is a plasma membrane protein carrying intra- and inter-molecular disulfide bonds and an N-linked glycan. Both disulfide bond formation and N-glycosylation are critical check points determining the stability and degradation fate of ABCG2 protein in the endoplasmic reticulum (ER). Misfolded ABCG2 protein without those post-translational modifications is removed from the ER by retrotranslocation to the cytosol compartment, ubiquitination by ubiquitin ligase, and finally degradation by proteasomes. Certain non-synonymous SNP variants of ABCG2 undergo such ER-associated degradation (ERAD).

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949 Based on our functional validation in vitro, the above-mentioned 17 non-synonymous polymorphisms [37] as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups [34]. Login to comment

[hide] Purification and structural analyses of ABCG2. Adv Drug Deliv Rev. 2009 Jan 31;61(1):57-65. Epub 2008 Dec 13. McDevitt CA, Collins R, Kerr ID, Callaghan R
Purification and structural analyses of ABCG2.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):57-65. Epub 2008 Dec 13., 2009-01-31 [PMID:19124053]

Abstract [show]
ABCG2 is best known as a multidrug transporter capable of conferring resistance to cancer cells. However, the protein is also inherently expressed in numerous barrier tissues and intriguingly within hematopoietic stem cells. Unlike its partners ABCB1 and ABCC1, there is considerably less information available on the molecular mechanism of ABCG2. The transporter has a distinct topology and is presumed to function as a homodimer. However, a number of biochemical studies have presented data to suggest that the protein adopts higher order oligomers. This review focuses on this controversial issue with particular reference to findings from low resolution structural data. In addition, a number of molecular models of ABCG2 based on high resolution structures of bacterial ABC transporters have recently become available and are critically assessed. ABCG2 is a structurally distinct member of the triumvirate of human multidrug transporters and continues to evade description of a unifying molecular mechanism.

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717 Perhaps the most significant variation in pharmacology arises with the R482G mutation, which dramatically alters the substrate specificity of ABCG2 and may provide clues into the site of drug interaction on theprotein [18,19]. Login to comment
722 The substrate specificities for the wild-type and R482G isoforms have been reasonably well characterised with respect to drug transport and the ability to confer resistance [3,19]. Login to comment
725 Table 1 Investigations reporting ATPase activity of various ABCG2 isoforms Isoform ATPase activity Drug effect Reference Vmax Km (ATP) R482G (B) 45 nmol min-1 mg-1 - Prazosin IC50 =5 μM [61] Vi inhibits at 50 μM R482G (B) 15 nmol min-1 mg-1 - [21] (S) 32 nmol min-1 mg-1 WT (B) 27 nmol min-1 mg-1 (S) 29 nmol min-1 mg-1 WT (pure) (B) 357 nmol min-1 mg-1 2 mM Vi inhibits 60-80% [20] R482T (pure) (B) 1111 nmol min-1 mg-1 1 mM R482G (B) 10 nmol min-1 mg-1 Stimulated activity at 100 μM prazosin [57] (S) 30 nmol min-1 mg-1 WT (B) 40 nmol min-1 mg-1 - Activities are Vi sensitive [19] (S) 41 nmol min-1 mg-1 Activities reported at a fixed [ATP] and not full Michaelis-Menten analysis R482G (B) 65 nmol min-1 mg-1 (S) 140 nmol min-1 mg-1 R482T (B) 42 nmol min-1 mg-1 (S) 81 nmol min-1 mg-1 R482G (B) 70 nmol min-1 mg-1 - Prazosin IC50 =1 μM and fold-stimulation was 2X [30] (S) 150 nmol min-1 mg-1 The Michaelis-Menten characteristics for ATPase activity by a number of isoforms of ABCG2 are tabulated from a number of source references. Login to comment
732 Wild-type and R482G ABCG2 isoforms are capable of ATP hydrolysis in the absence of any substrate although the Vmax for hydrolysis of 30-50 nmol Pi min-1 mg-1 is considerably lower than that shown for ABCB1. Login to comment
733 The most pronounced stimulation of ATP hydrolysis by substrate has been demonstrated for the R482G mutant in the presence of micromolar concentrations of prazosin. Login to comment
734 Unlike the R482G isoform, the wild-type protein was insensitive to stimulation by many drugs and increased nucleotide trapping in the presence of substrates could not be demonstrated [19]. Login to comment
745 For example, mitoxantrone was more efficient at displacing [125 I]-IAA-Rh123 binding to wild-type versus R482G, but the latter isoform was able to confer greater levels of resistance to the anticancer drug. Login to comment
747 An equilibrium and kinetic binding study on the R482G isoform of ABCG2 revealed the presence of multiple drug interaction sites on the protein [23]. Login to comment
760 The first studies of ABCG2, and its pharmacologically differing isoforms (R482G, R482T), were conducted in drug selected mammalian cell lines due to the relative simplicity of inducing its expression by drug treatment [26,27]. Login to comment
768 Janvilisri and co-workers utilised Lactococcus (L.) lactis as an expression system for wild-type and R482G ABCG2 [28,33] employing the nisin A-induced expression system that had previously been used for the expression of the prokaryotic ABC transporter LmrA [34]. Login to comment
800 Despite this lower affinity, a similar yield of ~1% was observed after IMAC for both the R482G isoform and the wild type (unpublished data). Login to comment
864 A large oligomeric complex (molecular weight of ~600 kDa) of the R482G isoform was identified by Blue-native PAGE and electron microscopy [37]. Login to comment

[hide] Ins and outs of the ABCG2 multidrug transporter: a... Adv Drug Deliv Rev. 2009 Jan 31;61(1):47-56. Epub 2008 Dec 24. Hegedus C, Szakacs G, Homolya L, Orban TI, Telbisz A, Jani M, Sarkadi B
Ins and outs of the ABCG2 multidrug transporter: an update on in vitro functional assays.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):47-56. Epub 2008 Dec 24., 2009-01-31 [PMID:19135105]

Abstract [show]
The major aim of this chapter is to provide a critical overview of the in vitro methods available for studying the function of the ABCG2 multidrug transporter protein. When describing the most applicable assay systems, in each case we present a short overview relevant to ABC multidrug transporters in general, and then we concentrate on the tools applicable to analysis of substrate-drug interactions, the effects of potential activators and inhibitors, and the role of polymorphisms of the ABCG2 transporter. Throughout this chapter we focus on recently developed assay systems, which may provide new possibilities for analyzing the pharmacological aspects of this medically important protein.

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975 WhileR482 is thewild-type form of ABCG2, R482G and R482Twere suggested to be gain-of-function mutations acquired during the course of drug selection. Login to comment
978 In contrast, doxorubicin and rhodamine123 are transported by the R482G and R482T protein variants, whereas they are not substrates for wtABCG2 [11,19,23,24]. Login to comment
1196 In contrast, Sf9 membranes engineered to overexpress the ABCG2 R482G or ABCG2 R482T mutants show much higher substrate stimulation. Login to comment
1210 Interestingly, the substrate-stimulated ATPase activity of the ABCG2 R482G and ABCG2 R482T mutant variants cannot be further increased by cholesterol loading. Login to comment

[hide] QSAR analysis and molecular modeling of ABCG2-spec... Adv Drug Deliv Rev. 2009 Jan 31;61(1):34-46. Epub 2008 Dec 16. Nicolle E, Boumendjel A, Macalou S, Genoux E, Ahmed-Belkacem A, Carrupt PA, Di Pietro A
QSAR analysis and molecular modeling of ABCG2-specific inhibitors.
Adv Drug Deliv Rev. 2009 Jan 31;61(1):34-46. Epub 2008 Dec 16., 2009-01-31 [PMID:19135106]

Abstract [show]
In addition to its critical role is controlling drug availability and protecting sensitive organs and stem cells through cellular detoxification, breast cancer resistance protein (BCRP/ABCG2) plays an important role in cancer cell resistance to chemotherapy, together with P-glycoprotein/ABCB1. A main approach to abolish multidrug resistance is to find out specific inhibitors of the drug-efflux activity, able to chemosensitize cancer cell proliferation. Many efforts have been primarily focused on ABCB1, discovered thirty years ago, whereas very few studies have concerned ABCG2, identified much more recently. This review describes the main types of inhibitors presently known for ABCG2, and how quantitative structure-activity relationship analysis among series of compounds may lead to build up molecular models and pharmacophores allowing to design lead inhibitors as future candidates for clinical trials. A special attention is drawn on flavonoids which constitute a structurally-diverse class of compounds, well suited to identify potent ABCG2-specific inhibitors.

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1252 In addition, the effects of the hotspot mutation R482G/T, which are well known to change the pattern of transported substrates, have not been systematically characterized for the inhibitors. Login to comment

[hide] BCRP expression does not result in resistance to S... Br J Cancer. 2009 Feb 10;100(3):476-86. Epub 2009 Jan 20. Day JM, Foster PA, Tutill HJ, Newman SP, Ho YT, Leese MP, Potter BV, Reed MJ, Purohit A
BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure.
Br J Cancer. 2009 Feb 10;100(3):476-86. Epub 2009 Jan 20., 2009-02-10 [PMID:19156141]

Abstract [show]
The anti-proliferative and anti-angiogenic properties of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2), are enhanced in a series of sulphamoylated derivatives of 2-MeOE2. To investigate possible mechanisms of resistance to these compounds, a cell line, A2780.140, eightfold less sensitive to the 3,17-O,O-bis-sulphamoylated derivative, STX140, was derived from the A2780 ovarian cancer cell line by dose escalation. Other cell lines tested did not develop STX140 resistance. RT-PCR and immunoblot analysis demonstrated that breast cancer resistance protein (BCRP) expression is dramatically increased in A2780.140 cells. The cells are cross-resistant to the most structurally similar bis-sulphamates, and to BCRP substrates, mitoxantrone and doxorubicin; but they remain sensitive to taxol, an MDR1 substrate, and to all other sulphamates tested. Sensitivity can be restored using a BCRP inhibitor, and this pattern of resistance is also seen in a BCRP-expressing MCF-7-derived cell line, MCF-7.MR. In mice bearing wild-type (wt) and BCRP-expressing tumours on either flank, both STX140 and mitoxantrone inhibited the growth of the MCF-7wt xenografts, but only STX140 inhibited growth of the MCF-7.MR tumours. In conclusion, STX140, a promising orally bioavailable anti-cancer agent in pre-clinical development, is highly efficacious in BCRP-expressing xenografts. This is despite an increase in BCRP expression in A2780 cells in vitro after chronic dosing with STX140.

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216 However, R482T and R482G mutations are found in the BCRP expressed by two well-characterised resistant cancer cell lines, MCF-7/AdrVp3000 (Lee et al, 1997) and S1-M1-80 (Miyake et al, 1999), respectively, selected by DOX and MXR treatment. Login to comment

[hide] Sensitization of ABCG2-overexpressing cells to con... Cancer Lett. 2009 Jun 28;279(1):74-83. Epub 2009 Feb 18. Dai CL, Liang YJ, Wang YS, Tiwari AK, Yan YY, Wang F, Chen ZS, Tong XZ, Fu LW
Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2.
Cancer Lett. 2009 Jun 28;279(1):74-83. Epub 2009 Feb 18., 2009-06-28 [PMID:19232821]

Abstract [show]
Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5muM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [(3)H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic.

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26 In contrast, cells carrying a glycine (R482G) or a threonine (R482T) at position 482 were able to transport rhodamine 123 and doxorubicin, while also maintained their ability to transport mitoxantrone. Login to comment
27 The ABCG2 variants were found in drug-resistant S1-M1-80 (R482G) and MCF-7 AdVp3000 (R482T) but not in the parental S1 and MCF-7 cell lines, suggesting that these were acquired mutations resulting from drug selection. Login to comment

[hide] Structural determinants of imidazoacridinones faci... Mol Pharmacol. 2009 May;75(5):1149-59. Epub 2009 Feb 27. Bram EE, Adar Y, Mesika N, Sabisz M, Skladanowski A, Assaraf YG
Structural determinants of imidazoacridinones facilitating antitumor activity are crucial for substrate recognition by ABCG2.
Mol Pharmacol. 2009 May;75(5):1149-59. Epub 2009 Feb 27., [PMID:19251825]

Abstract [show]
Symadex is the lead acridine compound of a novel class of imidazoacridinones (IAs) currently undergoing phase II clinical trials for the treatment of various cancers. Recently, we have shown that Symadex is extruded by ABCG2-overexpressing lung cancer A549/K1.5 cells, thereby resulting in a marked resistance to certain IAs. To identify the IA residues essential for substrate recognition by ABCG2, we here explored the ability of ABCG2 to extrude and confer resistance to a series of 23 IAs differing at defined residue(s) surrounding their common 10-azaanthracene structure. Taking advantage of the inherent fluorescent properties of IAs, ABCG2-dependent efflux and drug resistance were determined in A549/K1.5 cells using flow cytometry in the presence or absence of fumitremorgin C, a specific ABCG2 transport inhibitor. We find that a hydroxyl group at one of the R1, R2, or R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent IA resistance. Moreover, elongation of the common distal aliphatic side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Hence, the current study offers novel molecular insight into the structural determinants that facilitate ABCG2-mediated drug efflux and consequent drug resistance using a unique platform of fluorescent IAs. Moreover, these results establish that the IA determinants mediating cytotoxicity are precisely those that facilitate ABCG2-dependent drug efflux and IA resistance. The possible clinical implications for the future design of novel acridines that overcome ABCG2-dependent multidrug resistance are discussed.

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175 Mutant Arg482Gly and Arg482Thr ABCG2 Do Not Alter IA Substrate Recognition. Login to comment
176 Previous reports have established that the Arg482Gly and Arg482Thr ABCG2 mutations resulted in altered substrate specificity and augmented cellular drug resistance (Robey et al., 2003; Shafran et al., 2005; Bram et al., 2006). Login to comment
232 IA accumulation in HEK293 transfectant cells stably overexpressing wild-type Arg482 ABCG2 or mutant R482G/T ABCG2 in the presence or absence of fumitremorgin C. Login to comment

[hide] ABCG2-mediated DyeCycle Violet efflux defined side... Cell Cycle. 2009 Apr 1;8(7):1053-61. Epub 2009 Apr 30. Mathew G, Timm EA Jr, Sotomayor P, Godoy A, Montecinos VP, Smith GJ, Huss WJ
ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate.
Cell Cycle. 2009 Apr 1;8(7):1053-61. Epub 2009 Apr 30., 2009-04-01 [PMID:19270533]

Abstract [show]
The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the "side population" (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145 and RWPE-1, but not in the BPH-1, LAPC-4 or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP), human benign prostate tissue and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.

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27 To establish the optimal dye concentration for DCV labeling of cultured human cell lines, MCF-7, a breast cancer cell line that expresses an endogenous ABCG2, and HEK 293, a human embryonic kidney cell line that lacks all ABC-family members that was transfected with human ABCG2 (R482G) cloned into pcDNA (HEK 293 (R482G)), were used as ABCG2-mediated SP containing control cell lines.11,22 A distinct DCV-efflux mediated SP was observed in flow cytometric analyses of MCF-7 and HEK 293 (R482G) cells (Fig. 1C and E), and the SP was eliminated when the cells were pre-incubated with FTC (Fig. 1D and F). Login to comment
44 The SP is present in mouse bone marrow (A); in the MCF-7 cell line (C); and in the HEK 293 (R482G) cell line (E). Login to comment
45 The loss of the SP population due to inhibition of the ABCG2 pump by FTC is observed in mouse bone marrow (B); in the MCF-7 cell line (D); and in the HEK 293 (R482G) cell line (F). Login to comment
91 We confirmed that DCV is actively effluxed via ABCG2 by a subpopulation of mouse bone marrow cells that demonstrate the SP phenotype by the sensitivity of the efflux to inhibition by FTC.11 Analysis of the DCV profile in the HEK 293 (R482G) cell line pre-incubated with FTC confirmed that DCV efflux was mediated by the membrane transporter ABCG2. Login to comment
131 HEK 293 cells stably transfected with the pcDNA empty vector, or vector containing cDNA coding for ABCG2 (R482G), were maintained in Eagle`s Minimum Essential Medium (MEM) media (Mediatech Inc., Herndon, VA) supplemented with 10% FBS (Mediatech Inc., Herndon, VA), 2 mM L-glutamine (Mediatech Inc., Herndon, VA), penicillin (100 U/ml) (Mediatech Inc., Herndon, VA), streptomycin (100 mg/ml) (Mediatech Inc., Herndon, VA), and 0.5 mg/ml G418 (Mediatech Inc., Herndon, VA). Login to comment
264 Simon Hayward and HEK 293 (pcDNA) and HEK 293 (R482G) cells were a gift from Dr. Login to comment

[hide] Arginine 383 is a crucial residue in ABCG2 biogene... Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3. Polgar O, Ediriwickrema LS, Robey RW, Sharma A, Hegde RS, Li Y, Xia D, Ward Y, Dean M, Ozvegy-Laczka C, Sarkadi B, Bates SE
Arginine 383 is a crucial residue in ABCG2 biogenesis.
Biochim Biophys Acta. 2009 Jul;1788(7):1434-43. Epub 2009 May 3., [PMID:19406100]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.

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46 Cells previously transfected with wild-type ABCG2, R482G and pcDNA vector only were used as controls [27]. Login to comment

[hide] Nilotinib (AMN107, Tasigna) reverses multidrug res... Biochem Pharmacol. 2009 Jul 15;78(2):153-61. Epub 2009 Apr 11. Tiwari AK, Sodani K, Wang SR, Kuang YH, Ashby CR Jr, Chen X, Chen ZS
Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters.
Biochem Pharmacol. 2009 Jul 15;78(2):153-61. Epub 2009 Apr 11., 2009-07-15 [PMID:19427995]

Abstract [show]
Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E(2)17betaG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K(i) value of 0.69+/-0.083 microM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

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217 Robey et al. [34] have shown that the efficacy of ABCG2 transporters are also altered in these mutant cell lines, for e.g. novobiocin can only block wild-type ABCG2 and is nearly ineffective against mutant variants, however, FTC was shown to block both wild-type as well as mutant ABCG2. Our result shows that like FTC, nilotinib significantly enhanced the sensitivity of ABCG2 substrates not only in cells overexpressing wild-type R482 but also in the R482G/T variants of ABCG2. Our results, which indicate that specific TKIs interact with the ABCG2 and ABCB1 transporters, support the findings previously published by us and other groups. Login to comment

[hide] Human ABC transporter ABCG2 in cancer chemotherapy... J Exp Ther Oncol. 2009;8(1):5-24. Ishikawa T, Nakagawa H
Human ABC transporter ABCG2 in cancer chemotherapy and pharmacogenomics.
J Exp Ther Oncol. 2009;8(1):5-24., [PMID:19827267]

Abstract [show]
The ability of cancer cells to acquire resistance to multiple anticancer agents, termed multidrug resistance, is often mediated by overexpression of ATP-binding cassette (ABC) transporters that remove drugs out of the cell against a concentration gradient. ABCG2, or breast cancer resistance protein (BCRP), is an ABC transporter that has been the subject of intense study since its discovery a decade ago. While ABCG2 overexpression has been demonstrated in cancer cells after in vitro drug treatment, endogenous ABCG2 expression in certain cancers is considered as a reflection of the differentiated phenotype of the cell of origin and likely contributes to intrinsic drug resistance. Notably, ABCG2 is often expressed in stem cell populations, where it plays a critical role in cellular protection. ABCG2 exhibits a broad range of substrate specificity. New technologies of high-speed screening and quantitative structure-activity-relationship (QSAR) analysis have been developed to analyze the interactions of drugs with ABCG2. As ABCG2 reportedly transports porphyrins, its contribution to photodynamic therapy of human cancer is also implicated. Protein expression levels of ABCG2 in cancer cells are regulated by both transcriptional activation and protein degradation. The ABCG2 protein undergoes endosomal and/or ubiquitin-mediated proteasomal degradations. Furthermore, genetic polymorphisms in the ABCG2 gene are important factors in cancer chemotherapy to circumvent adverse effects and/or to enhance the efficacy of anticancer drugs. The present review article addresses recent advances in molecular pharmacology and pharmacogenomics of ABCG2 and provides novelideas to improve cancer chemotherapy.

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222 COOH H2N N590Y V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L F489L D620N R482G R482T S441N F571I OUT IN R160Q R575stop ATP-binding site Figure 7. Continued A 005-024 pp JETO-0900616-TI (Review).indd 8/7/2009 3:59:50 19 Q141K has been associated with lower levels of protein expression and impaired transport in vitro (Imai et al., 2002; Kobayashi et al., 2005; Misuarai et al., 2004; Zamber et al., 2003; Morisaki et al., 2008; Kondo et al., 2004). Login to comment
226 Impact of non-synonymous SNPs on function and protein stability Based on our functional validation in vitro, the above-mentioned 17 non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups (Tamura et al., 2007b) (Fig. 7B). Login to comment
228 Furthermore, the F208S, S248P, F431L, S441N, and F489L variants exhibited greatly altered protein expression levels and drug resistance profiles Figure 7. Continued WT V12M Q141K F208S S248P F431L S441N F489L R482G R482T Protein expression + + + - + + - + + + MTX transport + + + - - - - +/ - - Porphyrin transport + + + - - + - +/ + + SN-38 resistance + + + - +/ + - - + + MX resistance + + + - - - - - -- - - - - - - - +/ - - - - - - - - + + Doxorubicin resistance + + Daunorubicin resistance + + ATPase activity (Prazosin) + + WTV12M Q141K F431L F489L S248P F208S S441L R482G R482T ∆1.5 ∆3 ∆3.5 ∆5 ∆4 - - - - - - -- - - B 005-024 pp JETO-0900616-TI (Review).indd 8/7/2009 3:59:51 20 Journal of Experimental Therapeutics and Oncology Vol. 8 2009 (Tamura et al., 2007b). Login to comment
232 It is known that, in the ER, the N-linked glycans play pivotal roles in protein fold- 0.0 0.5 1.0 1.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T Methotrexatetransport (nmol/min/mgprotein) Methotrexate 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T Methotrexatetransport (nmol/min/mgprotein) MethotrexateMethotrexate Porphyrintransport (nmol/min/mgprotein) 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 Porphyrin Figure 7. Login to comment
235 The variants R482G and R482T are acquired mutations. Login to comment

[hide] Molecular mechanisms of drug resistance in single-... Methods Mol Biol. 2010;596:77-93. Calcagno AM, Ambudkar SV
Molecular mechanisms of drug resistance in single-step and multi-step drug-selected cancer cells.
Methods Mol Biol. 2010;596:77-93., [PMID:19949921]

Abstract [show]
Multidrug resistance (MDR) remains one of the key determinants in chemotherapeutic success of cancer patients. Often, acquired resistance is mediated by the overexpression of ATP-binding cassette (ABC) drug transporters. To study the mechanisms involved in the MDR phenotype, investigators have generated a variety of in vitro cell culture models using both multi-step and single-step drug selections. Sublines produced from multi-step selections have led to the discovery of several crucial drug transporters including ABCB1, ABCC1, and ABCG2. Additionally, a number of mechanisms causing gene overexpression have been elucidated. To more closely mimic in vivo conditions, investigators have also established MDR sublines with single-step drug selections. Here, we examine some of the multi-step and single-step selected cell lines generated to elucidate the mechanisms involved in the development of MDR in cancer cells.

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81 These S1 sublines also expressed a mutant R482G ABCG2 protein. Login to comment
85 In the case of ABCG2-overexpressing cell lines in vitro, two gain of function mutations have been identified (R482T and R482G). Login to comment

[hide] Impact of breast cancer resistance protein on canc... Methods Mol Biol. 2010;596:251-90. Ross DD, Nakanishi T
Impact of breast cancer resistance protein on cancer treatment outcomes.
Methods Mol Biol. 2010;596:251-90., [PMID:19949928]

Abstract [show]
Breast cancer resistance protein (BCRP/ABCG2) was discovered in multidrug resistant breast cancer cells having an ATP-dependent transport-based resistance phenotype. This ABC transporter functions (at least in part) as a xenobiotic protective mechanism for the organism: in the gut and biliary tract, it prevents absorption and enhances elimination of potentially toxic substances. As a placental barrier, it protects the fetus; similarly, it serves as a component of blood-brain and blood-testis barrier; BCRP is expressed in stem cells and may protect them from potentially harmful agents. Therefore, BCRP could influence cancer outcomes by (a) endogenous BCRP affecting the absorption, distribution, metabolism, and elimination of anticancer drugs; (b) BCRP expression in cancer cells may directly cause resistance by active efflux of anticancer drugs; (c) BCRP expression in cancer cells could be a manifestation of the activity of metabolic and signaling pathways that impart multiple mechanisms of drug resistance, self-renewal (stemness), and invasiveness (aggressiveness)--i.e. impart a poor prognosis--to cancers. This chapter presents a synopsis of translational clinical studies relating BCRP expression in leukemias, lymphomas, and a variety of solid tumors with clinical outcome. Data are emerging that expression of BCRP, like P-glycoprotein/ABCB1, is associated with adverse outcomes in a variety of human cancers. Whether this adverse prognostic effect results from resistance imparted to the cancer cells as the direct result of BCRP efflux of anticancer drugs, or whether BCRP expression (and also Pgp expression - coexpression of these transporters is common among poor risk cancers) serves as indicators of the activity of signaling pathways that enhance cancer cellular proliferation, metastases, genomic instability, enhance drug resistance, and oppose programmed cell death mechanisms is yet unknown.

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36 The ability of BCRP to efflux anthracyclines is greatly enhanced by the presence of a mutation at codon 482 (R482T or R482G), which has been observed in cells that overexpress BCRP following drug selection (61, 62). Login to comment
135 Furthermore, the R482T mutation conferred greater resistance to anthracyclines, and the R482G mutation appeared to cause less resistance to SN-38 and topotecan, compared with the wild-type arginine (113). Login to comment

[hide] Mutational analysis of threonine 402 adjacent to t... Biochemistry. 2010 Mar 16;49(10):2235-45. Polgar O, Ierano C, Tamaki A, Stanley B, Ward Y, Xia D, Tarasova N, Robey RW, Bates SE
Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2.
Biochemistry. 2010 Mar 16;49(10):2235-45., 2010-03-16 [PMID:20088606]

Abstract [show]
ABCG2 is an ATP-binding cassette half-transporter important in normal tissue protection, drug distribution, and excretion. ABCG2 requires homodimerization for function, though the mechanism for dimerization has not been elucidated. We conducted mutational analysis of threonine 402, three residues from the GXXXG motif in TM1, to study its potential role in ABCG2 dimerization (TXXXGXXXG). Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/G406L/G410L and T402R/G406L/G410L) resulted in a substantially reduced level of expression, altered glycosylation, degradation by a proteosome-independent pathway, and partial retention in the endoplasmic reticulum as suggested by immunostaining, Endo H sensitivity, and MG132 and bafilomycin failed effect. The T402L/G406L/G410L mutant when incubated with the ABCG2 substrate MX showed a shift on immunoblot analysis to the band representing the fully mature glycoprotein. The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. The same set of mutations also displayed impaired dimerization in the TOXCAT assay for TM1 compared to that of the wild type. Homology modeling of ABCG2 places the TXXXGXXXG motif at the dimer interface. These studies are consistent with a role for the extended TXXXGXXXG motif in ABCG2 folding, processing, and/or dimerization.

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47 The R482G control and G406L/G410L mutant were previously generated and characterized (19, 23). Login to comment
95 We used the previously generated G406L/G410L and R482G transfectants in HEK 293 cells as controls (19). Login to comment
96 The R482G mutation is considered a gain-of-function mutation, which enables the protein to transport a wider range of substrates (23). Login to comment
97 As in the previous study (19), all the GXXXG and threonine 402 mutants described here carry the R482G mutation. Login to comment
101 The T402L and T402R mutants exhibited levels comparable to the control (R482G), while the double (G406L/G410L) and triple mutants (T402L/G406L/G410L and T402R/G406L/G410L) had significantly decreased levels. Login to comment
106 As shown in the first column of Figure 1B, the control (R482G) and the T402L and T402R mutants displayed similar levels of surface expression. Login to comment
121 As shown in Figure 2A, a lower-molecular mass band was observed in the control (R482G) following digestion with N-glycosidase F to remove all N-linked glycans, consistent with removal of the polysaccharide chain from asparagine 596 of ABCG2 as shown following site-directed mutagenesis of N596 (31). Login to comment
130 (A) Cell lysates from stably transfected HEK 293 cells (20 μg for R482G, T402L, and T402R and 60 μg for G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L) were separated via SDS-PAGE, transferred onto a PVDF membrane, and probed with monoclonal anti-ABCG2 antibody BXP-21. Login to comment
155 Immunoblot analysis of cell lysates from the R482G, T402L, and T402R mutants [(A) 35 μg] and from the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L mutants [(B) 70 μg] with the BXP-21 antibody following overnight treatment with Endo H or N-glycosidase F. Login to comment
158 Cells were harvested after overnight incubation with or without 10 nM bafilomycin followed by immunoblot analysis with the BXP-21 and GAPDH antibodies for the R482G, T402L, and T402R mutants (40 μg/lane) and the G406L/G410L, T402L/G406L/G410L, and T402R/G406L/G410L mutants (100 μg/lane). Login to comment
166 (A) Following overnight incubation with or without 5 μM MX, cells were harvested, and the lysates (25 μg for R482G, T402R, and T402L and 50 μg for the other mutants) were subjected to immunoblot analysis with the BXP-21 and GAPDH antibodies as described in the legend of Figure 1. Login to comment
196 After overnight incubation with or without 5 μM MX, cells were harvested, and the lysates were subjected to overnight digestion with Endo H followed by immunoblot analysis with the BXP-21 antibody [(A) 35 μg for R482G, T402R, and T402L and (B) 70 μg for the double and triple mutants]. Login to comment

[hide] Role of basic residues within or near the predicte... J Pharmacol Exp Ther. 2010 Jun;333(3):670-81. Epub 2010 Mar 4. Cai X, Bikadi Z, Ni Z, Lee EW, Wang H, Rosenberg MF, Mao Q
Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.
J Pharmacol Exp Ther. 2010 Jun;333(3):670-81. Epub 2010 Mar 4., [PMID:20203106]

Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.

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312 For example, R482G and TABLE 3 Kinetic parameters of ATP hydrolysis by wild-type and mutant BCRP ATP hydrolysis activities of plasma membrane preparations from HEK293 cells expressing wild-type and mutant BCRP were determined as described under Materials and Methods. Login to comment

[hide] Pharmacological interaction with sunitinib is abol... Cancer Sci. 2010 Jun;101(6):1493-500. Epub 2010 Feb 22. Kawahara H, Noguchi K, Katayama K, Mitsuhashi J, Sugimoto Y
Pharmacological interaction with sunitinib is abolished by a germ-line mutation (1291T>C) of BCRP/ABCG2 gene.
Cancer Sci. 2010 Jun;101(6):1493-500. Epub 2010 Feb 22., [PMID:20345483]

Abstract [show]
Sunitinib malate (Sutent, SU11248) is a small-molecule multitargeted tyrosine kinase inhibitor (TKI) used for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. Some TKIs can overcome multidrug resistance conferred by ATP-binding cassette transporter, P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 1 (MRP1)/ABCC1, and breast cancer resistance protein (BCRP)/ABCG2. Here, we analyzed the effects of sunitinib on P-gp and on wild-type and germ-line mutant BCRPs. Sunitinib remarkably reversed BCRP-mediated and partially reversed P-gp-mediated drug resistance in the respective transfectants. The in vitro vesicle transport assay indicated that sunitinib competitively inhibited BCRP-mediated estrone 3-sulfate transport and P-gp-mediated vincristine transport. These inhibitory effects of sunitinib were further analyzed in Q141K-, R482G-, R482S-, and F431L-variant BCRPs. Intriguingly, the F431L-variant BCRP, which is expressed by a germ-line mutant allele 1291T>C, was almost insensitive to both sunitinib- and fumitremorgin C (FTC)-mediated inhibition in a cell proliferation assay. Sunitinib and FTC did not inhibit (125)I-iodoarylazidoprazosin-binding to F431L-BCRP. Thus, residue Phe-431 of BCRP is important for the pharmacological interaction with sunitinib and FTC. Collectively, this is the first report showing a differential effect of a germ-line variation of the BCRP/ABCG2 gene on the pharmacological interaction between small-molecule TKIs and BCRP. These findings would be useful for improving our understanding of the pharmaceutical effects of sunitinib in personalized chemotherapy.

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5 These inhibitory effects of sunitinib were further analyzed in Q141K-, R482G-, R482S-, and F431L-variant BCRPs. Login to comment
110 The R482T and R482G are BCRP variants identified after in vitro selection of culture cells and these variants confer DOX- and MXR-resistances. Login to comment
173 Reversal ratios are shown for wild-type (gray circles), and Q141K (open squares), F431L (filled circles), R482S (open diamonds), and R482G (open triangles) BCRP variant-expressing cells. Login to comment

[hide] Structure and function of the human breast cancer ... Curr Drug Metab. 2010 Sep;11(7):603-17. Ni Z, Bikadi Z, Rosenberg MF, Mao Q
Structure and function of the human breast cancer resistance protein (BCRP/ABCG2).
Curr Drug Metab. 2010 Sep;11(7):603-17., [PMID:20812902]

Abstract [show]
The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.

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87 Clark et al. examined the kinetics of association and dissociation of [3H]-daunomycin with the BCRP mutant R482G which can effectively transport the drug [67] using plasma membranes isolated from insect cells, and the ability of several other drugs to displace [3H]- daunomycin binding [64]. Login to comment
257 For example, wild-type BCRP does not transport daunorubicin, rhodamine 123, and LysoTracker Green; however, the mutants R482T and R482G do [108]. Login to comment

[hide] Apatinib (YN968D1) reverses multidrug resistance b... Cancer Res. 2010 Oct 15;70(20):7981-91. Epub 2010 Sep 28. Mi YJ, Liang YJ, Huang HB, Zhao HY, Wu CP, Wang F, Tao LY, Zhang CZ, Dai CL, Tiwari AK, Ma XX, To KK, Ambudkar SV, Chen ZS, Fu LW
Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters.
Cancer Res. 2010 Oct 15;70(20):7981-91. Epub 2010 Sep 28., 2010-10-15 [PMID:20876799]

Abstract [show]
Apatinib, a small-molecule multitargeted tyrosine kinase inhibitor, is in phase III clinical trial for the treatment of patients with non-small-cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr, and HEK293/ABCB1 cells overexpressing ABCB1 and in S1-M1-80, MCF-7/FLV1000, and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photoaffinity labeling of both ABCB1 and ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration that produced a reversal of MDR, did not significantly alter the ABCB1 or ABCG2 protein or mRNA expression levels or the phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1-resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking the AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs.

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116 It should be noted that S1-M1-80, but not the wild-type ABCG2, overexpressed the R482G variant, which can transport Rho 123 (39). Login to comment
117 It should be noted that S1-M1-80, but not the wild-type ABCG2, overexpressed the R482G variant, which can transport Rho 123 (39). Login to comment

[hide] In vitro and in vivo evidence for the importance o... Handb Exp Pharmacol. 2011;(201):325-71. Meyer zu Schwabedissen HE, Kroemer HK
In vitro and in vivo evidence for the importance of breast cancer resistance protein transporters (BCRP/MXR/ABCP/ABCG2).
Handb Exp Pharmacol. 2011;(201):325-71., [PMID:21103975]

Abstract [show]
The breast cancer resistance protein (BCRP/ABCG2) is a member of the G-subfamiliy of the ATP-binding cassette (ABC)-transporter superfamily. This half-transporter is assumed to function as an important mechanism limiting cellular accumulation of various compounds. In context of its tissue distribution with localization in the sinusoidal membrane of hepatocytes, and in the apical membrane of enterocytes ABCG2 is assumed to function as an important mechanism facilitating hepatobiliary excretion and limiting oral bioavailability, respectively. Indeed functional assessment performing mouse studies with genetic deletion or chemical inhibition of the transporter, or performing pharmacogenetic studies in humans support this assumption. Furthermore the efflux function of ABCG2 has been linked to sanctuary blood tissue barriers as described for placenta and the central nervous system. However, in lactating mammary glands ABCG2 increases the transfer of substrates into milk thereby increasing the exposure to potential noxes of a breastfed newborn. With regard to its broad substrate spectrum including various anticancer drugs and environmental carcinogens the function of ABCG2 has been associated with multidrug resistance and tumor development/progression. In terms of cancer biology current research is focusing on the expression and function of ABCG2 in immature stem cells. Recent findings support the notion that the physiological function of ABCG2 is involved in the elimination of uric acid resulting in higher risk for developing gout in male patients harboring genetic variants. Taken together ABCG2 is implicated in various pathophysiological and pharmacological processes.

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75 Indeed, following studies revealed that drug-selected cancer cells used to identify ABCG2-mediated anthracyclin-resistance carry an acquired mutation of amino acid 482 from arginine to glycine or threonine that changed substrate specificity of the transporter (Honjo et al. 2001; Nakanishi et al. 2003a; Robey et al. 2003). Login to comment
76 Accordingly, treatment of cancer cells has been demonstrated to result in allele (R482G or R482T) specific gene amplification (Bram et al. 2007), explaining the observed cross-resistance pattern for ABCG2 in previous studies. Login to comment
77 A similar "false-positive" substrate description has been assumed for the anti-folate drugs methotrexate, ralitrexate (ZD 1694) and GW1843, but the published results on the impact of the acquired ABCG2-mutations (p.ABCG2 R482G, R482T) are not conclusive (Bram et al. 2006; Breedveld et al. 2007; Chen et al. 2003; Mitomo et al. 2003; Shafran et al. 2005; Volk and Schneider 2003). Login to comment

[hide] The ABCG family of membrane-associated transporter... Br J Pharmacol. 2010 Dec 22. doi: 10.1111/j.1476-5381.2010.01177.x. Kerr ID, Haider AJ, Gelissen IC
The ABCG family of membrane-associated transporters: you don't have to be big to be mighty.
Br J Pharmacol. 2010 Dec 22. doi: 10.1111/j.1476-5381.2010.01177.x., 2010-12-22 [PMID:21175590]

Abstract [show]
Along with many other mammalian ATP binding cassette (ABC) transporters, members of the ABCG group are involved in the regulated transport of hydrophobic compounds across cellular membranes. In humans, 5 ABCG family members have been identified, encoding proteins ranging from 638 to 678 amino acids in length. All 5 have been the subject of intensive investigation to better understand their physiological roles, expression patterns, interactions with substrates and inhibitors, and regulation at both the transcript and protein level. The principal substrates for at least 4 of the ABCG proteins are endogenous and dietary lipids, with ABCG1 implicated in particular in the export of cholesterol, and ABCG5 and G8 forming a functional heterodimer responsible for plant sterol elimination from the body. ABCG2 has a much broader substrate specificity and its ability to transport numerous diverse pharmaceuticals has implications for the absorption, distribution, metabolism, excretion, and toxicity (ADMETox) profile of these compounds. ABCG2 is one of at least three so-called multidrug resistant (MDR) ABC-transporters expressed in humans, and its activity is associated with decreased efficacy of anticancer agents in several carcinomas. In addition to its role in cancer, ABCG2 also plays a role in the normal physiological transport of urate and haem, the implications of which are described below. We summarize here data on all 5 human ABCG transporters and provide a current perspective on their roles in human health and disease.

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33 Although performed with a mutant isoform of ABCG2 (Arg482Gly) this study remains the only direct determination of substrate binding affinities in a human ABCG transporter. Login to comment
38 Prazosin displayed much lower potency whilst methotrexate (which is not transported by the R482G isoform) was unable to displace the bound radioligand (Clark et al., 2006). Login to comment
51 Although performed with a mutant isoform of ABCG2 (Arg482Gly) this study remains the only direct determination of substrate binding affinities in a human ABCG transporter. Login to comment
56 Prazosin displayed much lower potency while methotrexate (which is not transported by the R482G isoform) was unable to displace the bound radioligand (Clark et al., 2006). Login to comment

[hide] Key Role of Human ABC Transporter ABCG2 in Photody... Adv Pharmacol Sci. 2010;2010:587306. Epub 2010 Jul 8. Ishikawa T, Nakagawa H, Hagiya Y, Nonoguchi N, Miyatake S, Kuroiwa T
Key Role of Human ABC Transporter ABCG2 in Photodynamic Therapy and Photodynamic Diagnosis.
Adv Pharmacol Sci. 2010;2010:587306. Epub 2010 Jul 8., [PMID:21188243]

Abstract [show]
Accumulating evidence indicates that ATP-binding cassette (ABC) transporter ABCG2 plays a key role in regulating the cellular accumulation of porphyrin derivatives in cancer cells and thereby affects the efficacy of photodynamic therapy and photodynamic diagnosis. The activity of porphyrin efflux can be affected by genetic polymorphisms in the ABCG2 gene. On the other hand, Nrf2, an NF-E2-related transcription factor, has been shown to be involved in oxidative stress-mediated induction of the ABCG2 gene. Since patients have demonstrated individual differences in their response to photodynamic therapy, transcriptional activation and/or genetic polymorphisms of the ABCG2 gene in cancer cells may affect patients' responses to photodynamic therapy. Protein kinase inhibitors, including imatinib mesylate and gefitinib, are suggested to potentially enhance the efficacy of photodynamic therapy by blocking ABCG2-mediated porphyrin efflux from cancer cells. This review article provides an overview on the role of human ABC transporter ABCG2 in photodynamic therapy and photodynamic diagnosis.

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167 Based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells [41, 90]. Login to comment
177 Gefitinib and imatinib are new anticancer drugs Outside Plasma membrane Inside H2N COOH V12M G51C Q126stop Q141K T153M R160Q Q166E I206L F208S S248P E334stop F431L F489L S441N R482G R482T F571I R575stop N590Y D620N T542A A528T D296H P269S ATP-binding cassette (a) 0 0.1 0.3 0.4 0.2 0.5 Mock WT V12M G51C Q126stop Q141K T153M Q166E I206L F208S S248P E334stop F431L S441N F489L F571I N590Y D620N R482G R482T ATP-dependenthematoporphyrin transport(nmol/min/mgprotein) (b) Figure 4: (a) Schematic illustration of human ABCG2 and its nonsynonymous polymorphisms. Login to comment
179 The variants R482G and R482T are acquired mutations. Login to comment

[hide] Sildenafil reverses ABCB1- and ABCG2-mediated chem... Cancer Res. 2011 Apr 15;71(8):3029-41. Epub 2011 Mar 14. Shi Z, Tiwari AK, Shukla S, Robey RW, Singh S, Kim IW, Bates SE, Peng X, Abraham I, Ambudkar SV, Talele TT, Fu LW, Chen ZS
Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.
Cancer Res. 2011 Apr 15;71(8):3029-41. Epub 2011 Mar 14., 2011-04-15 [PMID:21402712]

Abstract [show]
Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17betaG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions.

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110 It has been reported that mutations at amino acid 482 in ABCG2 alter the substrate and antagonist specificity of ABCG2 (16, 25); therefore, we examined the reversing effect of sildenafil on both wild-type (R482) and mutant (R482G and R482T) ABCG2-overexpressing cells. Login to comment
111 It has been reported that mutations at amino acid 482 in ABCG2 alter the substrate and antagonist specificity of ABCG2 (16, 25); therefore, we examined the reversing effect of sildenafil on both wild-type (R482) and mutant (R482G and R482T) ABCG2-overexpressing cells. Login to comment

[hide] Recombinant synthesis of human ABCG2 expressed in ... Protein J. 2011 Mar;30(3):201-11. Jacobs A, Emmert D, Wieschrath S, Hrycyna CA, Wiese M
Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study.
Protein J. 2011 Mar;30(3):201-11., [PMID:21424391]

Abstract [show]
Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS-PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-beta-D-glucopyranoside and n-dodecyl-beta-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.

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4 In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. Login to comment
5 We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Login to comment
22 Mutation of arginine-482 to threonine or glycine considerably extends the spectrum of transported substrates. Login to comment
23 The variants R482T or R482G transport additional substrates such as rhodamine 123 and doxorubicin, whereas the recognition of other substrates such as Hoechst 33342 and pheophorbide A remains unaffected [10, 17]. Login to comment
24 One of these variants (R482G) was chosen for purification experiments described in this manuscript showing the above mentioned broader substrate recognition. Login to comment
38 We have tried to apply the same system to human ABCG2 (R482G variant). Login to comment
41 We were able to show function by stimulation of ATPase activity with prazosin and other known substrates of ABCG2 (R482G) such as sulfasalazine and progesterone [15, 19]. Login to comment
44 2 Materials and Methods 2.1 Expression Vector pCHH10m3N-ABCG2 Human ABCG2 cDNA (R482G variant) was cloned into the pCHH10m3N plasmid [2], where the expression was under control of the constitutive phosphoglycerate kinase (PGK) promoter. Login to comment
100 3 Results and Discussion 3.1 Expression of ABCG2 (R482G) in S. cerevisiae The ABCG2 variant R482G was chosen, as it displays altered substrate specificity in comparison to wild-type (wt) ABCG2. Login to comment
143 Functionality of the purified ABCG2 (R482G) was analyzed by a drug-stimulated ATPase assay using the standard stimulator prazosin. Login to comment
166 Following solubilization trials, purification of ABCG2 from transformed LPY11-ABCG2 (R482G) cells was performed with 2% FC-16 as described for OG. Login to comment
234 Position in ABCG2 R482G Sequence 675.42 379-383 R.WVSKR.S 801.48 158-163 K.NERINR.V 820.48 178-184 K.VGTQFIR.G 863.48 359-365 K.KKITVFK.E 900.52 466-473 R.VSSYFLGK.L 970.58 324-331 K.QDKPLIEK.L 987.62 315-323 K.ATEIIEPSK.Q 1004.60 418-426 K.NDSTGIQNR.A 1014.62 164-172 R.VIQELGLDK.V 1020.58 48-56 K.LKSGFLPCR.K 1037.54 379-386 R.WVSKRSFK.L 1044.62 87-96 K.SSLLDVLAAR.K 1145.70 148-157 R.LATTMTNHEK.N 1148.64 138-147 R.ENLQFSAALR.L 1172.66 87-97 K.SSLLDVLAARK.D 1231.68 62-72 K.EILSNINGIMK.P 1292.78 252-263 K.LFDSLTLLASGR.L 1296.66 347-358 K.AELHQLSGGEKK.K 1358.74 315-326 K.ATEIIEPSKQDK.P 1360.68 50-61 K.SGFLPCRKPVEK.E 1397.74 161-172 R.INRVIQELGLDK.V 1399.76 617-628 K.QGIDLSPWGLWK.N 1424.68 347-359 K.AELHQLSGGEKKK.K 1433.70 332-343 K.LAEIYVNSSFYK.E 1462.78 178-191 K.VGTQFIRGVSGGER.K 1514.86 164-177 R.VIQELGLDKVADSK.V 1560.68 454-465 K.LFIHEYISGYYR.V 1601.88 48-61 K.LKSGFLPCRKPVEK.E 1791.82 332-346 K.LAEIYVNSSFYKETK.A 1962.91 173-191 K.VADSKVGTQFIRGVSGGER.K 2141.11 347-365 K.AELHQLSGGEKKKKITVFK.E 2247.19 173-193 K.VADSKVGTQFIRGVSGGERKR.T 2253.16 97-118 R.KDPSGLSGDVLINGAPRPANFK.C 2301.22 360-378 K.KITVFKEISYTTSFCHQLR.W 2464.36 62-86 K.EILSNINGIMKPGLNAILGPTGGGK.S 2537.14 231-251 R.MSKQGRTIIFSIHQPRYSIFK.L 2603.23 366-386 K.EISYTTSFCHQLRWVSKRSFK.L 2923.36 148-172 R.LATTMTNHEKNERINRVIQELGLDK.V 2941.39 332-357 K.LAEIYVNSSFYKETKAELHQLSGGEK.K 2957.35 360-383 K.KITVFKEISYTTSFCHQLRWVSKR.S 3194.56 387-417 K.NLLGNPQASIAQIIVTVVLGLVIGAIYFGLK.N 4862.00 315-357 K.ATEIIEPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEK.K Masses were matched employing a tryptic in-silico digest using the GPMAW 8.0 software (Lighthouse Data) standard compound prazosin, and other known stimulators of ATPase activity, such as progesterone and sulfasalazine, increased the liberation of inorganic phosphate from ATP. Login to comment

[hide] Characterization of rhodamine-123, calcein and 5(6... Eur J Pharm Sci. 2011 Aug 17;43(5):359-69. doi: 10.1016/j.ejps.2011.05.003. Epub 2011 May 14. Munic V, Hlevnjak M, Erakovic Haber V
Characterization of rhodamine-123, calcein and 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) export via MRP2 (ABCC2) in MES-SA and A549 cells.
Eur J Pharm Sci. 2011 Aug 17;43(5):359-69. doi: 10.1016/j.ejps.2011.05.003. Epub 2011 May 14., [PMID:21605668]

Abstract [show]
Based on our initial results on the effects of several ATP-binding cassette (ABC) transporter inhibitors on rhodamine-123 efflux from A549, a human lung carcinoma, and MES-SA, a human uterine sarcoma cell line, the aim of this study was to identify the transporter responsible for this export. Export of two fluorescent dyes, rhodamine-123 and calcein, was investigated in both cell lines by testing five commonly used inhibitors of ABC transporters: verapamil, cyclosporin A, MK571, GF129018 and fumitremorgin C. A very high degree of correlation (R(2)=0.91-0.99) between results obtained in the two cell lines suggested that the same transporter was involved in the export of tested fluorescent substrates in both cell lines. Expression analysis and gene silencing techniques, as well as transport of additional substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) on membrane vesicles revealed that the transporter was multidrug resistance related protein 2 (MRP2, ABCC2). Furthermore, it was found that the tested modulators showed very diverse effects on the export of three fluorescent substrates via MRP2, with some modulators being inhibitory in one, while having no effect or even stimulating the transport in the other fluorescent dye assay. Verapamil inhibited rhodamine-123, but stimulated CDCF transport and did not affect calcein export. GF129018 did not affect calcein and CDCF transport, but it inhibited rhodamine-123 transport. These results demonstrate the importance of studying various combinations of potential substrates and modulators of MRP2 in order to estimate possible drug-drug interactions in living organisms. In addition, A549 and MES-SA cells were shown to be good cell models for studying interactions of compounds with human MRP2.

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273 However, only BCRP with mutations R482T or R482G transports rhodamine-123, and this transport can be inhibited with 10 lM fumitremorgin C (Honjo et al., 2001). Login to comment

[hide] Intestinal ciprofloxacin efflux: the role of breas... Drug Metab Dispos. 2011 Dec;39(12):2321-8. Epub 2011 Sep 19. Haslam IS, Wright JA, O'Reilly DA, Sherlock DJ, Coleman T, Simmons NL
Intestinal ciprofloxacin efflux: the role of breast cancer resistance protein (ABCG2).
Drug Metab Dispos. 2011 Dec;39(12):2321-8. Epub 2011 Sep 19., [PMID:21930826]

Abstract [show]
Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)m ethylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human and rodent BCRP. An additional pathway for ciprofloxacin secretion exists in Caco-2 cells, which is unlikely to be MRP(4)-mediated. BCRP is likely to be the dominant transport mechanism for ciprofloxacin efflux in both rat and human jejunum.

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224 An R482G mutation is acquired during mitoxantrone selection and results in structural (transmembrane domain) and functional (substrate recognition) changes in the BCRP protein; other studies highlight the impact of the amino acid mutations at position 482 (Honjo et al., 2001; Volk et al., 2002), but this does not explain the differences noted in this and other studies. Login to comment

[hide] Zafirlukast antagonizes ATP-binding cassette subfa... Anticancer Drugs. 2012 Sep;23(8):865-73. Sun YL, Kathawala RJ, Singh S, Zheng K, Talele TT, Jiang WQ, Chen ZS
Zafirlukast antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.
Anticancer Drugs. 2012 Sep;23(8):865-73., [PMID:22614107]

Abstract [show]
ATP-binding cassette (ABC) transporters are present in the majority of human tumors and are involved in multidrug resistance (MDR). Therefore, compounds that inhibit the function of ABC transporters may improve the efficacy of anticancer agents. Previous research has shown that zafirlukast is a reversal drug for multidrug resistance protein (MRP) 1-mediated MDR. In the present study, we assessed whether zafirlukast could be a reversal agent for other ABC transporter-mediated MDR. Using the MTT assay, we found that zafirlukast enhanced the cytotoxicity of several anticancer drugs that are substrates of breast cancer resistance proteins (BCRP/ABCG2), including mitoxantrone and SN-38. Furthermore, zafirlukast could partially reverse P-glycoprotein-mediated (P-gp/ABCB1) and MRP7 (ABCC10)-mediated MDR at nontoxic doses. Studies on [(3)H]-mitoxantrone accumulation and efflux have shown that zafirlukast increases the intracellular accumulation of [(3)H]-mitoxantrone by directly inhibiting ABCG2-mediated drug efflux. Western blot analysis indicated that zafirlukast did not alter the expression of ABCG2. In addition, a docking model predicted the binding conformation of zafirlukast within the transmembrane region of homology-modeled human ABCG2. Our findings suggest a possible strategy to potentially enhance the activity of anticancer drugs using a clinically approved drug with known side effects and drug-drug interactions.

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102 Therefore, we also examined the reversal effect of zafirlukast on mutant (R482G and R482T) ABCG2-overexpressing cells. Login to comment

[hide] Interaction of the EGFR inhibitors gefitinib, vand... Biochem Pharmacol. 2012 Aug 1;84(3):260-7. Epub 2012 Apr 21. Hegedus C, Truta-Feles K, Antalffy G, Varady G, Nemet K, Ozvegy-Laczka C, Keri G, Orfi L, Szakacs G, Settleman J, Varadi A, Sarkadi B
Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance.
Biochem Pharmacol. 2012 Aug 1;84(3):260-7. Epub 2012 Apr 21., [PMID:22548830]

Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies.

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174 In contrast, until now the issue of conferring vandetanib resistance has only been addressed using the clinically irrelevant ABCG2 R482G variant [42], and to the best of our knowledge, this is also the first report analyzing ABCG2- interaction profiles of the other two novel EGFR inhibitors, pelitinib or neratinib. Login to comment
180 In contrast, until now the issue of conferring vandetanib resistance has only been addressed using the clinically irrelevant ABCG2 R482G variant [42], and to the best of our knowledge, this is also the first report analyzing ABCG2- interaction profiles of the other two novel EGFR inhibitors, pelitinib or neratinib. Login to comment

[hide] New use for an old drug: inhibiting ABCG2 with sor... Mol Cancer Ther. 2012 Aug;11(8):1693-702. Epub 2012 May 16. Wei Y, Ma Y, Zhao Q, Ren Z, Li Y, Hou T, Peng H
New use for an old drug: inhibiting ABCG2 with sorafenib.
Mol Cancer Ther. 2012 Aug;11(8):1693-702. Epub 2012 May 16., [PMID:22593228]

Abstract [show]
Human ABCG2, a member of the ATP-binding cassette transporter superfamily, represents a promising target for sensitizing MDR in cancer chemotherapy. Although lots of ABCG2 inhibitors were identified, none of them has been tested clinically, maybe because of several problems such as toxicity or safety and pharmacokinetic uncertainty of compounds with novel chemical structures. One efficient solution is to rediscover new uses for existing drugs with known pharmacokinetics and safety profiles. Here, we found the new use for sorafenib, which has a dual-mode action by inducing ABCG2 degradation in lysosome in addition to inhibiting its function. Previously, we reported some novel dual-acting ABCG2 inhibitors that showed closer similarity to degradation-induced mechanism of action. On the basis of these ABCG2 inhibitors with diverse chemical structures, we developed a pharmacophore model for identifying the critical pharmacophore features necessary for dual-acting ABCG2 inhibitors. Sorafenib forms impressive alignment with the pharmacophore hypothesis, supporting the argument that sorafenib is a potential ABCG2 inhibitor. This is the first article that sorafenib may be a good candidate for chemosensitizing agent targeting ABCG2-mediated MDR. This study may facilitate the rediscovery of new functions of structurally diverse old drugs and provide a more effective and safe way of sensitizing MDR in cancer chemotherapy.

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77 Intracellular mitoxantrone accumulation in ABCG2 R482G-transfected stable HEK293 (HEK293/ ABCG2) cells and its corresponding ABCG2-negative vector-transfected control (HEK293/Vec) cells were measured in the presence or absence of sorafenib or Ko143 at 2.5 mmol/L. Login to comment
76 Intracellular mitoxantrone accumulation in ABCG2 R482G-transfected stable HEK293 (HEK293/ ABCG2) cells and its corresponding ABCG2-negative vector-transfected control (HEK293/Vec) cells were measured in the presence or absence of sorafenib or Ko143 at 2.5 mmol/L. Login to comment

[hide] Axitinib targeted cancer stemlike cells to enhance... Mol Med. 2012 Jul 18;18(1):887-98. doi: 10.2119/molmed.2011.00444. Wang F, Mi YJ, Chen XG, Wu XP, Liu Z, Chen SP, Liang YJ, Cheng C, To KK, Fu LW
Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2.
Mol Med. 2012 Jul 18;18(1):887-98. doi: 10.2119/molmed.2011.00444., [PMID:22549112]

Abstract [show]
Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.

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29 MCF7/AdVp3000 and S1-M1-80 cells expressing R482T and R482G variants of BCRP/ABCG2, respectively, transported rhodamine 123 and Dox while also maintaining their ability to transport mitoxantrone (16,17). Login to comment

[hide] Different roles of TM5, TM6, and ECL3 in the oligo... Biochemistry. 2012 May 1;51(17):3634-41. Epub 2012 Apr 19. Mo W, Qi J, Zhang JT
Different roles of TM5, TM6, and ECL3 in the oligomerization and function of human ABCG2.
Biochemistry. 2012 May 1;51(17):3634-41. Epub 2012 Apr 19., [PMID:22497316]

Abstract [show]
ABCG2 is a member of the ATP-binding cassette transporter superfamily, and its overexpression causes multidrug resistance (MDR) in cancer chemotherapy. ABCG2 may also protect cancer stem cells by extruding cytotoxic materials. ABCG2 has previously been shown to exist as a high-order homo-oligomer consisting of possibly 8-12 subunits, and the oligomerization domain was mapped to the C-terminal domain, including TM5, ECL3, and TM6. In this study, we further investigate this domain in detail for the role of each segment in the oligomerization and drug transport function of ABCG2 using domain swapping and site-directed mutagenesis. We found that none of the three segments (TM5, TM6, and ECL3) is essential for the oligomerization activity of ABCG2 and that any one of these three segments in the full-length context is sufficient to support ABCG2 oligomerization. While TM5 plays an important role in the drug transport function of ABCG2, TM6 and ECL3 are replaceable. Thus, each segment in the TM5-ECL3-TM6 domain plays a distinctive role in the oligomerization and function of ABCG2.

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175 However, their oligomerization activities do not appear to depend on the hydrophobicity of these TM segments, which is different from the TM segments involved in ABCC1 dimerization.15 While the exact drug-binding sites in ABCG2 are unknown, early studies have suggested that the interactions of the substrate with ABCG2 involve multiple binding sites in the protein.25 For example, Arg482 has been shown to affect substrate specificity.26 All our constructs have the same R482G mutation that provides a gain of function in recognition of more substrates. Login to comment

[hide] ABC transporters and their role in nucleoside and ... Biochem Pharmacol. 2012 Apr 15;83(8):1073-83. Epub 2012 Jan 20. Fukuda Y, Schuetz JD
ABC transporters and their role in nucleoside and nucleotide drug resistance.
Biochem Pharmacol. 2012 Apr 15;83(8):1073-83. Epub 2012 Jan 20., [PMID:22285911]

Abstract [show]
ATP-binding cassette (ABC) transporters confer drug resistance against a wide range of chemotherapeutic agents, including nucleoside and nucleotide based drugs. While nucleoside based drugs have been used for many years in the treatment of solid and hematological malignancies as well as viral and autoimmune diseases, the potential contribution of ABC transporters has only recently been recognized. This neglect is likely because activation of nucleoside derivatives require an initial carrier-mediated uptake step followed by phosphorylation by nucleoside kinases, and defects in uptake or kinase activation were considered the primary mechanisms of nucleoside drug resistance. However, recent studies demonstrate that members of the ABCC transporter subfamily reduce the intracellular concentration of monophosphorylated nucleoside drugs. In addition to the ABCC subfamily members, ABCG2 has been shown to transport nucleoside drugs and nucleoside-monophosphate derivatives of clinically relevant nucleoside drugs such as cytarabine, cladribine, and clofarabine to name a few. This review will discuss ABC transporters and how they interact with other processes affecting the efficacy of nucleoside based drugs.

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859 One such non-synonymous change is well known for ABCG2, the Arg482 to Gly substitution [124]. Login to comment

[hide] Antibody binding shift assay for rapid screening o... Eur J Pharm Sci. 2012 Jan 23;45(1-2):101-9. Epub 2011 Nov 17. Telbisz A, Hegedus C, Ozvegy-Laczka C, Goda K, Varady G, Takats Z, Szabo E, Sorrentino BP, Varadi A, Sarkadi B
Antibody binding shift assay for rapid screening of drug interactions with the human ABCG2 multidrug transporter.
Eur J Pharm Sci. 2012 Jan 23;45(1-2):101-9. Epub 2011 Nov 17., [PMID:22115866]

Abstract [show]
The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided.

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18 The mutants, containing R482G, T (or R482M or S in the mouse Abcg2), showed altered substrate specificities. Login to comment
57 Cytotoxicity assays Cytotoxicity assays were performed using PLB cells expressing the wild-type or the mutant (R482G, R482T) ABCG2 proteins. Login to comment
64 Measurement of imatinib accumulation by HPLC-MS Imatinib accumulation was measured in intact parental and wild-type or R482G mutant ABCG2 expressing PLB cells. Login to comment
135 Therefore, in the next set of experiments, we performed comparative studies for several compounds by using isolated membranes for measuring their modulation of ABCG2 ATPase activity and intact cells expressing the wild type or mutant (R482G and R482T) ABCG2 variants for antibody binding studies. Login to comment
140 In contrast, elacridar greatly stimulated the ATPase activities of both ABCG2-R482G and ABCG2-R482T. Login to comment
143 Namely, while this compound inhibited the ATPase activity of the wild type protein, it exerted an ATPase stimulatory effect on ABCG2-R482G and ABCG2-R482T (Fig. 3E). Login to comment
148 Concentration-dependent modulation of the ATPase activity and 5D3 immunoreactivity of the wild type (j), R482G (d) and R482T (.) Login to comment
155 These types of drugs seem to be mutant selective, they can be potent inhibitors of the wild type ABCG2 while still be transported efficiently by ABCG2-R482G and ABCG2-R482T mutants in the same concentration range. Login to comment
157 PLB/ABCG2-wild-type, PLB/ABCG2-R482G and PLB/ABCG2-R482T cells were treated with the cytotoxic topoisomerase inhibitor compounds, topotecan and mitoxantrone. Login to comment
166 Both in the case of mitoxantrone (Fig. 4A) and topotecan (Fig. 4B), much higher concentrations of elacridar were required to produce a 50% decrease of the IC50 values measured in PLB/ ABCG2-R482G or PLB/ABCG2-R482T cells than in PLB/ABCG2- wild-type cells. Login to comment
167 These results confirm that ABCG2-R482G and ABCG2-R482T have decreased sensitivity to the ABCG2 inhibitor drug elacridar, as compared to that of the wild type protein. Login to comment
170 Detailed examination of imatinib accumulation in PLB/ABCG2-wild-type and PLB/ABCG2-R482G at different imatinib concentrations revealed that the R482G variant can lower the accumulation of imatinib with significantly higher capacity, than the wild-type ABCG2 (at similar expression level). Login to comment
171 These data confirm that although imatinib is a transported substrate at low nanomolar concentrations of both ABCG2-types, when imatinib is applied at higher doses (0.2-1 lM), it is not as effectively extruded by wild-type ABCG2 than by the R482G variant. Login to comment
174 Reversal of (A) mitoxantrone and (B) topotecan resistance by elacridar in PLB/ABCG2-wild-type (j), PLB/ABCG2-R482G (d) and PLB/ABCG2-R482T (.) Login to comment
181 Effect of the wild type ABCG2 (j) and the ABCG2/R482G (d) proteins on the intracellular accumulation of imatinib in the absence (-) or in the presence (---) of 1 lM Ko143. Login to comment
185 The star denotes statistically significant difference (95% confidence interval) between imatinib accumulation measured in PLB/ABCG2-wild-type and PLB/ ABCG2-R482G cells. Login to comment

[hide] Human ABCG2: structure, function, and its role in ... Int J Biochem Mol Biol. 2012;3(1):1-27. Epub 2011 Mar 30. Mo W, Zhang JT
Human ABCG2: structure, function, and its role in multidrug resistance.
Int J Biochem Mol Biol. 2012;3(1):1-27. Epub 2011 Mar 30., [PMID:22509477]

Abstract [show]
Human ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily and is known to contribute to multidrug resistance (MDR) in cancer chemotherapy. Among ABC transporters that are known to cause MDR, ABCG2 is particularly interesting for its potential role in protecting cancer stem cells and its complex oligomeric structure. Recent studies have also revealed that the biogenesis of ABCG2 could be modulated by small molecule compounds. These modulators, upon binding to ABCG2, accelerate the endocytosis and trafficking to lysosome for degradation and effectively reduce the half-life of ABCG2. Hence, targeting ABCG2 stability could be a new venue for therapeutic discovery to sensitize drug resistant human cancers. In this report, we review recent progress on understanding the structure, function, biogenesis, as well as physiological and pathophysiological functions of ABCG2.

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204 This inconsistency led to discovery of a gain of function ABCG2 mutant with R482G/T mutation [109, 110] Both the R482G and R482T mutants and the wild-type ABCG2 are able to efflux mitoxantrone, topotecan, SN-38, Hoechst 33342 [111] and BODIPY-prazosin [112]. Login to comment
205 However, the R482G and R482T mutants have higher affinity with anthracyclines, including doxorubicin, daunorubicin, epirubicin, as well as bisantrene, fluorescence dye rhodamine 123 and lysotracker green [112]. Login to comment
208 Nevertheless, a study using IAARh123, the photoreactive analogue of rhodamine 123, has surprisingly shown that the wild type ABCG2 along with the two R482G and R482T mutants can all bind directly to IAARh123 although the wild type ABCG2 could not transport rhodamine [116]. Login to comment

[hide] ABCG2 is a direct transcriptional target of hedgeh... Oncogene. 2011 Dec 8;30(49):4874-86. doi: 10.1038/onc.2011.195. Epub 2011 May 30. Singh RR, Kunkalla K, Qu C, Schlette E, Neelapu SS, Samaniego F, Vega F
ABCG2 is a direct transcriptional target of hedgehog signaling and involved in stroma-induced drug tolerance in diffuse large B-cell lymphoma.
Oncogene. 2011 Dec 8;30(49):4874-86. doi: 10.1038/onc.2011.195. Epub 2011 May 30., [PMID:21625222]

Abstract [show]
Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by the development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of antiapoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. Previously, our studies showed that high expression of adenosine triphosphate-binding cassette drug transporter ABCG2 in DLBCL correlated inversely with disease- and failure-free survival. In this study, we have implicated activated hedgehog (Hh) signaling pathway as a key factor behind high ABCG2 expression in DLBCL through direct upregulation of ABCG2 gene transcription. We have identified a single binding site for GLI transcription factors in the ABCG2 promoter and established its functionality using luciferase reporter, site-directed mutagenesis and chromatin-immunoprecipitation assays. Furthermore, in DLBCL tumor samples, significantly high ABCG2 and GLI1 levels were found in DLBCL tumors with lymph node involvement in comparison with DLBCL tumor cells collected from pleural and/or peritoneal effusions. This suggests a role for the stromal microenvironment in maintaining high levels of ABCG2 and GLI1. Accordingly, in vitro co-culture of DLBCL cells with HS-5 stromal cells increased ABCG2 mRNA and protein levels by paracrine activation of Hh signaling. In addition to ABCG2, co-culture of DLBCL cells with HS-5 cells also resulted in increase expression of the antiapoptotic proteins BCL2, BCL-xL and BCL2A1 and in induced chemotolerance to doxorubicin and methotrexate, drugs routinely used for the treatment of DLBCL. Similarly, activation of Hh signaling in DLBCL cell lines with recombinant Shh N-terminal peptide resulted in increased expression of BCL2 and ABCG2 associated with increased chemotolerance. Finally, functional inhibition of ABCG2 drug efflux activity with fumitremorgin C or inhibition of Hh signaling with cyclopamine-KAAD abrogated the stroma-induced chemotolerance suggesting that targeting ABCG2 and Hh signaling may have therapeutic value in overcoming chemoresistance in DLBCL.

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90 DLBCL cell lines and tumors express wild-type ABCG2 (R482) but not the R482G mutant A point mutation of adenine (A) to guanine (G) at 1443 position of ABCG2 coding sequence results in replacement of an arginine residue at position 482 with glycine or threonine (R482G/T), changing the substrate specificity of ABCG2. Login to comment
170 Moreover, it has been also shown that cell lines selected with doxorubicin frequently show enrichment of cells with the R482G/T ABCG2 mutant form (Robey et al., 2003). Login to comment
171 A similar scenario is also plausible in DLBCL patients, where initial treatment with doxorubicin may result in selection of cells with a mutant ABCG2 gene carrying the R482G/T mutation that could contribute to chemoresistance. Login to comment

[hide] pH-Dependent transport of pemetrexed by breast can... Drug Metab Dispos. 2011 Sep;39(9):1478-85. Epub 2011 May 31. Li L, Sham YY, Bikadi Z, Elmquist WF
pH-Dependent transport of pemetrexed by breast cancer resistance protein.
Drug Metab Dispos. 2011 Sep;39(9):1478-85. Epub 2011 May 31., [PMID:21628496]

Abstract [show]
Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment.

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43 HEK293 cells transfected with ABCG2-Arg482, ABCG2- R482G, and ABCG2-R482T and plasmid vectors were obtained from Dr. Susan E. Bates (National Cancer Institute, Bethesda, MD). Login to comment
46 Comparable protein expression in the wild-type (Arg482) and mutant (R482G, R482T) ABCG2-transfected cells has been demonstrated by the Bates group (Robey et al., 2003). Login to comment
153 As shown in Fig. 9A, the extent of pH dependence, calculated as the ratio of uptake at pH 5.5 to the uptake at pH 7.4, was approximately 61, 12, and 6 for Arg482 (wild-type BCRP), R482G (mutant BCRP), and R482T (mutant BCRP), respectively. Login to comment
154 A similar trend was observed for MTX; that is, pH-dependent transport was significantly disturbed when positively charged arginine at 482 (Arg482) was replaced by nonionized amino acids (R482G and R482T) at lower pH (Fig. 9B). Login to comment
203 For estrone sulfate, mutant (R482G, R482T) and wild-type (Arg482) BCRP exhibited a similar extent of increase in the transport activity, suggesting that Arg482 was not involved in the pH-dependent transport of estrone sulfate. This is reasonable because the intrinsic pKa of arginine is approximately 12. Login to comment

[hide] Prognostic value of the multidrug resistance trans... Eur J Cancer. 2011 Sep;47(13):1990-9. Epub 2011 Apr 29. Wang F, Liang YJ, Wu XP, Chen LM, To KK, Dai CL, Yan YY, Wang YS, Tong XZ, Fu LW
Prognostic value of the multidrug resistance transporter ABCG2 gene polymorphisms in Chinese patients with de novo acute leukaemia.
Eur J Cancer. 2011 Sep;47(13):1990-9. Epub 2011 Apr 29., [PMID:21531129]

Abstract [show]
BACKGROUND: Functional polymorphisms of the ABCG2 gene may contribute to individual variability in drug response and the prognosis of patients. METHODS: In the present study, the genetic polymorphisms and expression of ABCG2 were analysed in blasts cells obtained from 184 Chinese patients with de novo acute leukaemia to investigate their possible association with clinical outcomes. RESULTS: A novel synonymous ABCG2-single nucleotide polymorphism (SNP) at exon 16 (13561218 C/T) and five known SNPs at exon 2 (13608835 G/A), exon 5 (13600044 C/A), intron 10 (13576005 C/T), intron 13 (13564503 C/T) and intron 14 (13563578 A/G) were identified with occurrence rates of 1.1%, 64.1%, 30.4%, 21.2%, 39.7% and 28.8%, respectively. We found that patients with the ABCG2 34GG genotype displayed longer disease free survival (DFS) (P<0.001) and overall survival (OS) (P<0.001) than those with the 34GA/AA genotypes. Furthermore, the DFS and OS were significantly diminished in bone marrow transplantation (BMT) patients with the 34GA/AA genotypes relative to those with the 34GG genotype. CONCLUSIONS: These results suggest that these highly prevalent ABCG2 34GA/AA genotypes are associated with poor prognosis of Chinese patients with acute leukaemia and BMT patients.

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18 The C421A polymorphism was associated with decreased protein expression and transport activity and altered pharmacokinetic parameters of some ABCG2 substrates in vitro and in vivo.24-28 Polarised LLC-PK1 cells transfected with the ABCG2 34AA variant displayed a dramatic increase in cytotoxic effect of ABCG2 substrate anticancer drugs such as mitoxantrone, doxorubicin, vincristine and topoisomerase I inhibitors compared with the wild-type ABCG2 (34GG) transfected cells.29 Interestingly, two other ABCG2 variants R482G and R482T identified in drug-resistant human cancer cell lines, which demonstrated altered substrate specificity, have not been detected in human individuals.30,31 In the present study, we sought to identify the positions and frequencies of ABCG2 SNPs and assess their possible prognostic impact on Chinese patients with acute leukaemia. Login to comment

[hide] Drug transport by breast cancer resistance protein... Expert Opin Drug Metab Toxicol. 2010 Nov;6(11):1363-84. Epub 2010 Sep 27. Poguntke M, Hazai E, Fromm MF, Zolk O
Drug transport by breast cancer resistance protein.
Expert Opin Drug Metab Toxicol. 2010 Nov;6(11):1363-84. Epub 2010 Sep 27., [PMID:20873966]

Abstract [show]
IMPORTANCE OF THE FIELD: The ATP-binding cassette transporter ABCG2 is a well-known major mediator of multi-drug resistance in cancers. Beyond multi-drug resistance, experimental and recent clinical studies demonstrate a role for ABCG2 as a determinant of drug pharmacokinetic, safety and efficacy profiles. AREAS COVERED IN THIS REVIEW: The clinical evidence of the role of ABCG2 in pharmacokinetics and pharmacodynamics is reviewed. Key questions that arise from the perspective of preclinical drug evaluation are addressed, including the structure of ABCG2 and mechanisms of drug-transporter interactions, mechanisms responsible for the polyspecificity of ABCG2, methods suitable for studying drug-ABCG2 interactions in vitro and in silico prediction of ABCG2 substrates and inhibitors. WHAT THE READER WILL GAIN: An update on current knowledge of the importance of ABCG2 in drug disposition with special emphasis on drug development. TAKE HOME MESSAGE: The field of drug-ABCG2 interaction is rapidly advancing and beginning to expand into clinical practice. However, the structural understanding of drug binding and transport by ABCG2 is still incomplete. Incorporation of novel concepts of drug-transporter interactions such as electrostatic funneling might help explain the multispecificity of ABCG2 and enable in silico predictions.

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391 Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter. Login to comment

[hide] Structure, function, expression, genomic organizat... Int J Toxicol. 2006 Jul-Aug;25(4):231-59. Choudhuri S, Klaassen CD
Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters.
Int J Toxicol. 2006 Jul-Aug;25(4):231-59., [PMID:16815813]

Abstract [show]
The ATP-binding cassette (ABC) transporters constitute a large family of membrane proteins, which transport a variety of compounds through the membrane against a concentration gradient at the cost of ATP hydrolysis. Substrates of the ABC transporters include lipids, bile acids, xenobiotics, and peptides for antigen presentation. As they transport exogenous and endogenous compounds, they reduce the body load of potentially harmful substances. One by-product of such protective function is that they also eliminate various useful drugs from the body, causing drug resistance. This review is a brief summary of the structure, function, and expression of the important drug resistance-conferring members belonging to three subfamilies of the human ABC family; these are ABCB1 (MDR1/P-glycoprotein of subfamily ABCB), subfamily ABCC (MRPs), and ABCG2 (BCRP of subfamily ABCG), which are expressed in various organs. In the text, the transporter symbol that carries the subfamily name (such as ABCB1, ABCC1, etc.) is used interchangeably with the corresponding original names, such as MDR1P-glycoprotein, MRP1, etc., respectively. Both nomenclatures are maintained in the text because both are still used in the transporter literature. This helps readers relate various names that they encounter in the literature. It now appears that P-glycoprotein, MRP1, MRP2, and BCRP can explain the phenomenon of multidrug resistance in all cell lines analyzed thus far. Also discussed are the gene structure, regulation of expression, and various polymorphisms in these genes. Because genetic polymorphism is thought to underlie interindividual differences, including their response to drugs and other xenobiotics, the importance of polymorphism in these genes is also discussed.

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594 Replacement of Arg482 by Gly (Arg482Gly) or Thr (Arg482Thr) was found to be associated with rhodamine transport ability and higher doxorubicin resistance. Login to comment
595 However, during SNP analysis of ABCG2 gene from 90 ethnically diverse individuals, the same group (Honjo et al. 2002) found no such SNPs at amino acid 482 (Arg482Gly or Arg482Thr) that they had previously reported in two cell lines. Login to comment
616 In MCF-7/AdrVp3000 cell line, this has been mutated to Thr (R482T) and in S1-M1-80 cell line this has been mutated to Gly (R482G). Login to comment

[hide] Zosuquidar restores drug sensitivity in P-glycopro... BMC Cancer. 2008 Feb 13;8:51. Tang R, Faussat AM, Perrot JY, Marjanovic Z, Cohen S, Storme T, Morjani H, Legrand O, Marie JP
Zosuquidar restores drug sensitivity in P-glycoprotein expressing acute myeloid leukemia (AML).
BMC Cancer. 2008 Feb 13;8:51., [PMID:18271955]

Abstract [show]
BACKGROUND: Chemotherapeutic drug efflux via the P-glycoprotein (P-gp) transporter encoded by the MDR1/ABCB1 gene is a significant cause of drug resistance in numerous malignancies, including acute leukemias, especially in older patients with acute myeloid leukemia (AML). Therefore, the P-gp modulators that block P-gp-mediated drug efflux have been developed, and used in combination with standard chemotherapy. In this paper, the capacity of zosuquidar, a specific P-gp modulator, to reverse chemoresistance was examined in both leukemia cell lines and primary AML blasts. METHODS: The transporter protein expressions were analyzed by flow cytometry using their specific antibodies. The protein functionalities were assessed by the uptake of their fluorescence substrates in presence or absence their specific modulators. The drug cytotoxicity was evaluated by MTT test. RESULTS: Zosuquidar completely or partially restored drug sensitivity in all P-gp-expressing leukemia cell lines tested and enhanced the cytotoxicity of anthracyclines (daunorubicin, idarubicin, mitoxantrone) and gemtuzumab ozogamicin (Mylotarg) in primary AML blasts with active P-gp. In addition, P-gp inhibition by zosuquidar was found to be more potent than cyclosporine A in cells with highly active P-gp. CONCLUSION: These in vitro studies suggest that zosuquidar may be an effective adjunct to cytotoxic chemotherapy for AML patients whose blasts express P-gp, especially for older patients.

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88 Lack of effect of zosuquidar on wild type BCRP-expressing cells Daunorubicin and idarubicin are transported by mutant BCRP (R482T or R482G) and not by wild type BCRP (R482), while mitoxantrone is transported by all BCRP variants [20]. Login to comment

[hide] A functional steroid-binding element in an ATP-bin... Mol Pharmacol. 2008 Jan;73(1):12-7. Velamakanni S, Janvilisri T, Shahi S, van Veen HW
A functional steroid-binding element in an ATP-binding cassette multidrug transporter.
Mol Pharmacol. 2008 Jan;73(1):12-7., [PMID:18094074]

Abstract [show]
The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding and mediates modulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new therapeutic ligands for ABCG2.

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18 The R482G replacement does not significantly affect the interactions of ABCG2 with Hoechst 33342 and steroid hormones (Robey et al., 2003; Janvilisri et al., 2005; Ozvegy-Laczka et al., 2005). Login to comment
22 ABCG2 mutants were generated with the QuikChange method (Stratagene, La Jolla, CA) using pGEM-BCRP R482G This study was supported by the Medical Research Council and Association for International Cancer Research. Login to comment
86 B, ABCG2 R482G -ATPase activity in inside-out membrane vesicles without substrate (basal) or with 25 òe;M ED, 10 òe;M PG, 10 òe;M daunomycin, or 10 òe;M cholesterol. Login to comment

[hide] The epidermal growth factor tyrosine kinase inhibi... Oncol Rep. 2009 Feb;21(2):483-9. Shi Z, Parmar S, Peng XX, Shen T, Robey RW, Bates SE, Fu LW, Shao Y, Chen YM, Zang F, Chen ZS
The epidermal growth factor tyrosine kinase inhibitor AG1478 and erlotinib reverse ABCG2-mediated drug resistance.
Oncol Rep. 2009 Feb;21(2):483-9., [PMID:19148526]

Abstract [show]
ABCG2 is an important member of ATP-binding cassette (ABC) transporter shown to confer drug resistance in cancer cells. Recent studies show that an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), gefitinib, is able to modulate the function of ABCG2 and reverse ABCG2-mediated multidrug resistance (MDR) in cancer cells. Additionally, ABCG2 expression has been shown to impact treatment efficacy and development of side-effects in patients receiving gefitinib. However, it is unclear whether other EGFR TKIs interact with ABCG2 in a similar manner. In the present study, we investigated the interaction of two other EGFR TKIs, AG1478 and erlotinib, with ABCG2. Our data show that AG1478 and erlotinib potently sensitized drug-resistant cells overexpressing either wild-type or mutated ABCG2 to the ABCG2 substrate anti-cancer drugs flavopiridol and mitoxantrone. Neither AG1478 nor erlotinib sensitized ABCG2-overexpressing cells to drugs that are not substrates of ABCG2 nor did they impact drug sensitivity of parental cells. Furthermore, AG1478 and erlotinib were able to significantly enhance the intracellular accumulation of mitoxantrone in cells expressing either wild-type or mutated ABCG2. Additionally, they did not alter the protein expression of ABCG2 in the ABCG2-overexpressing cells. Taken together, we conclude that AG1478 and erlotinib potently reverse ABCG2-mediated MDR through directly inhibiting the drug efflux function of ABCG2 in the ABCG2-overexpressing cells. These results will be useful in the development of novel and more effective EGFR TKIs as well as the development of combinational chemotherapeutic strategies.

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67 Since mutations at amino acid 482 of ABCG2 could alter the substrate and antagonist specificity of ABCG2 (26,27), we examined the potential impact of mutation at this site on the effects of AG1478 and erlotinib by performing studies on cells overexpressing wild-type (R482) ABCG2-overexpressing MCF-7/FLV1000, mutant ABCG2-overexpressing MCF-7/AdVp3000 (R482T) and S1-M1-80 (R482G). Login to comment

[hide] Analysis of the effect of the bovine adenosine tri... J Anim Sci. 2011 Dec;89(12):4325-38. doi: 10.2527/jas.2011-3841. Epub 2011 Aug 5. Real R, Gonzalez-Lobato L, Baro MF, Valbuena S, de la Fuente A, Prieto JG, Alvarez AI, Marques MM, Merino G
Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models.
J Anim Sci. 2011 Dec;89(12):4325-38. doi: 10.2527/jas.2011-3841. Epub 2011 Aug 5., [PMID:21821808]

Abstract [show]
In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 muM, P = 0.017; 10 muM, P = 0.001; 25 muM, P = 0.008; and 50 muM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.

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255 In this way, the R482G and R482T variants show increased transport and ATP hydrolytic-activity for various compounds (Ozvegy et al., 2002). Login to comment

[hide] Arginine 482 to glycine mutation in ABCG2/BCRP inc... Oncol Rep. 2012 Jan;27(1):232-7. doi: 10.3892/or.2011.1468. Epub 2011 Sep 20. Eddabra L, Wenner T, El Btaouri H, Baranek T, Madoulet C, Cornillet-Lefebvre P, Morjani H
Arginine 482 to glycine mutation in ABCG2/BCRP increases etoposide transport and resistance to the drug in HEK-293 cells.
Oncol Rep. 2012 Jan;27(1):232-7. doi: 10.3892/or.2011.1468. Epub 2011 Sep 20., [PMID:21935580]

Abstract [show]
Resistance to etoposide has been associated with the overexpression of P-glycoprotein and MRP1 in human tumor cells. However, the role of BCRP in resistance to etoposide has not been clearly established, especially the significance of arginine 482 mutations in drug transport (cellular uptake and efflux). Different levels of resistance to etoposide have been recently observed in cells expressing BCRP in terms of cytotoxicity. The aim of this work was to study the effects of these mutations on the functional involvement of BCRP in etoposide transport. HEK293 cells were transfected with an empty vector (HEK/V), the vector bearing the wild-type BCRP (HEK/R482), the mutant arginine-482-glycine (HEK/R482G) or the mutant arginine-482-threonine (HEK/R482T). MTT assay was used to study the cytotoxic effect of etoposide and [3H]-etoposide was used to determine cellular drug uptake and efflux. Data show that HEK/R482G cells displayed the highest levels of resistance to etoposide. Cellular [3H]-etoposide uptake was lower in HEK/R482, HEK/R482G and HEK/R482T cells compared to HEK/V cells. In addition, cellular [3H]-etoposide uptake in HEK/R482G was the lowest. Drug efflux measurements showed that fumitremorgin C was able to increase the residual cellular [3H]-etoposide uptake in BCRP-transfected cells and especially in HEK/R482G ones. Our data show that the R482G mutation in BCRP is able to increase efflux of etoposide and that mutation analysis at codon 482 may be of clinical importance in cancers treated with etoposide.

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5 HEK293 cells were transfected with an empty vector (HEK/V), the vector bearing the wild-type BCRP (HEK/R482), the mutant arginine-482-glycine (HEK/R482G) or the mutant arginine-482-threonine (HEK/R482T). Login to comment
7 Data show that HEK/R482G cells displayed the highest levels of resistance to etoposide. Login to comment
8 Cellular [3 H]-etoposide uptake was lower in HEK/R482, HEK/R482G and HEK/R482T cells compared to HEK/V cells. Login to comment
9 In addition, cellular [3 H]-etoposide uptake in HEK/R482G was the lowest. Login to comment
10 Drug efflux measurements showed that fumitremorgin C was able to increase the residual cellular [3 H]-etoposide uptake in BCRP-transfected cells and especially in HEK/R482G ones. Login to comment
11 Our data show that the R482G mutation in BCRP is able to increase efflux of etoposide and that mutation analysis at codon 482 may be of clinical importance in cancers treated with etoposide. Login to comment
20 The same amino acid is also mutated in another cell line but substituted for a glycine (R482G) (9,10). Login to comment
23 We studied the resistance to etoposide and its cellular transport in HEK293 cells transfected by the human wild-type BCRP or its R482G and R482T mutants. Login to comment
24 We show that cells expressing the R482G mutant display higher resistance level to etoposide than the wild-type BCRP or the mutant R482T genes. Login to comment
29 Fumitremorgin C (FTC) Arginine 482 to glycine mutation in ABCG2/BCRP increases etoposide transport and resistance to the drug in HEK-293 cells LAHCEN EDDABRA1 , THOMAS WENNER2 , HASSAN EL BTAOURI1 , THOMAS BARANEK3 , CLAUDIE MADOULET1 , PASCALE CORNILLET-LEFEBVRE4 and HAMID MORJANI1 1 MEDyC Unit&#e9; CNRS UMR6237, UFR de Pharmacie, IFR53, 51096 Reims, France; 2 CRP-Sant&#e9;, Luxembourg; 3 IIER EA 4303, UFR M&#e9;decine, IFR53; 4 EA3801, Laboratoire d'H&#e9;matologie, CHU de Reims, 51096 Reims, France Received July 13, 2011; Accepted August 16, 2011 DOI: 10.3892/or.2011.1468 Correspondence to: Dr Hamid Morjani, MEDyC Unit&#e9; CNRS UMR6237, UFR de Pharmacie, IFR53, 51096 Reims, France E-mail: hamid.morjani@univ-reims.fr Key words: ABCG2/BCRP, mutation, etoposide transport, resistance was kindly provided by R.W. Robey and Professor S. Bates (National Cancer Institute, NIH, Bethesda, MD, USA). Login to comment
31 The human embryonic kidney HEK293 cells transfected with empty vector (HEK/V) or BCRP (HEK/R482 and HEK/R482G and HEK/R482T) were kindly provided by R.W. RobeyandS.Bates(12).Cells weregrownas monolayerin MEM (Invitrogen, Paris, France) supplemented with 10% fetal calf serum and 100 &#b5;g/ml penicillin, and 100 &#b5;g/ml streptomycin. Login to comment
79 The relative expression level of BCRP mRNA in HEK/R482, HEK/R482G and HEK/R482T cell lines was 347-, 247- and 315-fold and is higher than in HEK/V cells, respectively. Login to comment
90 Interestingly, the resistance factor was significantly increased to 30-fold in the glycine mutant (HEK/R482G). Login to comment
93 Relative mRNA expression levels of BCRP and MRP1 as estimated by real-time RT-PCR in HEK293 and 2008 transfected cells.a HEK/V HEK/R482 HEK/R482G HEK/R482T 2008 2008/MRP1 Ct TBP 21.20 20.59 20.43 20.07 25.13 25.31 Ct BCRP 28.41 19.36 19.69 18.98 - - Ct MRP1 - - - - 27.20 22.23 ࢞Ct 7.21 -1.23 -0.74 -1.09 2.07 -3.08 ࢞࢞Ct - -8.44 -7.95 -8.30 - -5.15 2-࢞࢞Ct - 347.29 247.28 315.17 - 35.51 a The fold change of BCRP or MRP1 expression in the BCRPand MRP1-transfected cell lines was determined as described in Materials and methods. Login to comment
98 HEK/V HEK/R482 HEK/R482G HEK/R482T 2008 2008/MRP1 BCRP 1.56 20 25 21 - - MRP1 - - - - 1.02 4.44 Figure 1. Login to comment
102 (A) Cytotoxic effect of etoposide in HEK293 (c6;), HEK/R482 (fc;), HEK/R482T (cf;) and HEK/R482G (b2;) cell lines. Login to comment
109 By contrast, FTC was able to reverse resistance to etoposide moderately in HEK/R482G cell line as compared to novobiocin. Login to comment
115 The [3 H]-etoposide uptake represented 63 (p<0.02), 40 (p<0.01) and 54% (p<0.02), respectively, in HEK/R482, HEK/ R482G and HEK/R482T, of the value measured in HEK/V cells (Fig. 3A and B). Login to comment
116 Moreover, the [3 H]-etoposide uptake is significantly reduced in HEK/R482G cells as compared to HEK/ R482 cells (p<0.05). Login to comment
118 IC50 of mitoxantrone and etoposide in HEK293 and 2008 cells in the presence or not of novobiocin or FTC.a Cell line -------------------------------------------------------------------------- HEK/V HEK/R482 HEK/R482G HEK/R482T 2008 2008/MRP1 ------ ----------- ------------ ----------- ---- ---------- Drug Inhibitor IC50 IC50 RF IC50 RF IC50 RF IC50 IC50 RF Mitoxantrone - 0.15 1.17 11.33 3.10 20.67 3.00 20.00 Novobiocin 0.08 0.08 1.00 0.50 6.25 0.90 11.25 FTC 0.12 0.11 0.92 0.21 1.75 0.32 2.67 Etoposide - 1.00 5.00 5.00 30.00 30.00 8.00 8.00 0.70 6.00 8.57 Novobiocin 1.00 2.00 2.00 4.50 4.50 8.20 8.20 FTC 1.00 1.30 1.30 8.20 8.20 3.20 3.20 a IC50 values are the means of three independent experiments, each performed in triplicate. Login to comment
138 [3 H]-etoposide uptake reached 92 (p<0.05), 77 (p<0.02) and 81% (p<0.05) in HEK/R482, HEK/R482G and HEK/R482T cells, respectively, when compared to the values observed in the absence of novobiocin. Login to comment
146 However, in transfected cells it represented 44 (p<0.05), 35 (p<0.01) and 41% (p<0.02) of the initial concentration in HEK/ R482, HEK/R482G and HEK/R482T cells, respectively (Fig. 4). Login to comment
147 The intracellular [3 H]-etoposide concentration is significantly lower in HEK/R482G cells as compared to HEK/R482 cells (p<0.05). Login to comment
150 However, in HEK/R482, HEK/R482G and HEK/R482T cells treated with FTC, the cellular residual [3 H]-etoposide was increased to 73 (p<0.02), 77 (p<0.01) and 68% (p<0.01) of the initial concentration, and was significantly higher than that observed in the absence of FTC (Fig. 4). Login to comment
152 In drug-selected cell lines, two different mutations leading to a transition from arginine 482 to threonine (R482T) and glycine (R482G) respectively, have been observed (9,10). Login to comment
153 R482T mutation confers high-level resistance to anthracyclines (21), and cells with R482G or R482T mutation are able to efflux more efficiently rhodamine 123 (9). Login to comment
154 Moreover, R482G mutation seems to confer relatively less resistance to camptothecins. Login to comment
158 In order to study the role of this protein in resistance to etoposide in human, we used the embryonic HEK293 cells transfected with the wild-type BCRP (HEK/ R482) or its two mutants R482G (HEK/R482G) and R482T (HEK/R482T) (5). Login to comment
160 Determination of rhodamine 123 uptake confirmed that HEK/R482G and HEK/R482T cells transported efficiently this probe when compared with HEK/R482 cells (data not shown). Login to comment
161 In our study, we have shown that wild-type BCRP, R482T and especially R482G mutant can confer significant resistance to etoposide, the HEK/R482G cells showing an IC50 value six-fold higher than in HEK/R482 cells (Table III). Login to comment
164 This suggest that R482G mutation confer to the BCRP protein a better affinity for etoposide than the mutation R482T, or a better efficiency in drug efflux, as suggested by our results (Fig. 3). Login to comment
166 In the presence of FTC, cellular uptake of etoposide was significantly increased and particularly in HEK/R482 and HEK/R482G cells. Login to comment
170 Our data show clearly that R482G mutation is able to increase resistance to etoposide. Login to comment

[hide] Influence of bioluminescence imaging dynamics by D... Mol Imaging. 2012 Nov-Dec;11(6):499-506. Zhang Y, Pullambhatla M, Laterra J, Pomper MG
Influence of bioluminescence imaging dynamics by D-luciferin uptake and efflux mechanisms.
Mol Imaging. 2012 Nov-Dec;11(6):499-506., [PMID:23084250]

Abstract [show]
Bioluminescence imaging (BLI) detects light generated by luciferase-mediated oxidation of substrate and is used widely for evaluating transgene expression in cell-based assays and in vivo in relevant preclinical models. The most commonly used luciferase for in vivo applications is firefly luciferase (fLuc), for which D-luciferin serves as the substrate. We demonstrated previously that the expression of the ABCG2 efflux transporter can significantly reduce BLI signal output and that HhAntag-691 can inhibit the efflux of D-luciferin, thereby enhancing BLI signal. Here we show that an HhAntag-691-sensitive uptake mechanism facilitates the intracellular concentration of D-luciferin and that the BLI dynamics of different cell lines are coregulated by this uptake mechanism in conjunction with ABCG2-mediated efflux. After administration of D-luciferin, the HhAntag-691-sensitive uptake mechanism generates a rapid increase in BLI signal that decreases over time, whereas ABCG2-mediated efflux stably reduces signal output. We implicate SLC22A4 (OCTN1), a member of the organic cation/zwitterion uptake transporter family, as one potential mediator of the HhAntag-691-sensitive D-luciferin uptake. These findings provide insight into mechanisms that contribute to the cellular uptake kinetics and in vivo biodistribution of D-luciferin.

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48 On the other hand, expressing either of two mutant forms of ABCG2, T10 (R482T) and G2 (R482G), each of which has altered substrate specificity and does not transport D-luciferin as effectively as wt ABCG2,5,13 failed to restore the signal enhancement by HhAntag-691 (Figure 2, C and D). Login to comment

[hide] Overexpression of ATP-binding cassette transporter... Biochem Pharmacol. 2013 Feb 1;85(3):325-34. doi: 10.1016/j.bcp.2012.11.003. Epub 2012 Nov 12. Wu CP, Sim HM, Huang YH, Liu YC, Hsiao SH, Cheng HW, Li YQ, Ambudkar SV, Hsu SC
Overexpression of ATP-binding cassette transporter ABCG2 as a potential mechanism of acquired resistance to vemurafenib in BRAF(V600E) mutant cancer cells.
Biochem Pharmacol. 2013 Feb 1;85(3):325-34. doi: 10.1016/j.bcp.2012.11.003. Epub 2012 Nov 12., [PMID:23153455]

Abstract [show]
Melanoma is the most serious type of skin cancer with a high potential for metastasis and very low survival rates. The discovery of constitutive activation of the BRAF kinase caused by activating BRAF(V600E) kinase mutation in most melanoma patients led to the discovery of the first potent BRAF(V600E) signaling inhibitor, vemurafenib. Vemurafenib was effective in treating advanced melanoma patients and was proposed for the treatment of other BRAF(V600E) mutant cancers as well. Unfortunately, the success of vemurafenib was hampered by the rapid development of acquired resistance in different types of BRAF(V600E) mutant cancer cells. It becomes important to identify and evaluate all of the potential mechanisms of cellular resistance to vemurafenib. In this study, we characterized the interactions of vemurafenib with three major ATP-binding cassette (ABC) transporters, ABCB1, ABCC1 and ABCG2. We found that vemurafenib stimulated the ATPase activity and potently inhibited drug efflux mediated by ABCB1 and ABCG2. Vemurafenib also restored drug sensitivity in ABCG2-overexpressing cells. Moreover, we revealed that in the presence of functional ABCG2, BRAF kinase inhibition by vemurafenib is reduced in BRAF(V600E) mutant A375 cells. Taken together, our findings indicate that ABCG2 confers resistance to vemurafenib in A375 cells, suggesting involvement of this transporter in acquired resistance to vemurafenib. Thus, combination chemotherapy targeting multiple pathways could be an effective therapeutic strategy to overcome acquired resistance to vemurafenib for cancers harboring the BRAF(V600E) mutation.

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78 ABCG2-mediated efflux of PhA was inhibited significantly by vemurafenib (2 mM) in ABCG2-overexpressing HEK293 (Fig. 1A), MCF7-FLV1000 (wild type R482) (Fig. 1B, middle panel),MCF7-AdVp3000(R482 T)(Fig1B, rightpanel) and S1-M1-80 (R482G) (Fig. 1 C) MDR cancer cell lines. Login to comment
98 The levels of accumulated fluorescent PhA in ABCG2-overexpressing (A) R482-HEK293, (B) MCF7-FLV1000 (wild-type), MCF7-AdVp3000 (R482T), (C) S1-M1-80 (R482G) cells and drug-sensitive parental cells or (D) concentration-dependent inhibition of ABCG2-mediated efflux of mitoxantrone by increasing concentrations of vemurafenib in MCF7-FLV1000 (*), MCF7-AdVp3000 (*) and S1-M1-80 (&) cells. Login to comment

[hide] Effects of the lipid environment, cholesterol and ... Biochem J. 2013 Mar 1;450(2):387-95. doi: 10.1042/BJ20121485. Telbisz A, Ozvegy-Laczka C, Hegedus T, Varadi A, Sarkadi B
Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein.
Biochem J. 2013 Mar 1;450(2):387-95. doi: 10.1042/BJ20121485., [PMID:23205634]

Abstract [show]
The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein. We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators of ABCG2 activity. Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cell membrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter.

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4 Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Login to comment
21 We have reconstituted the purified wild-type and the ABCG2-R482G variant into liposomes with different lipid compositions, and evaluated the effects of mutations on the cholesterol and bile acid sensitivity of these transporter variants. Login to comment
29 The His6-tagged ABCG2-R482G mutant was created by cloning the PstI/SacI fragment of pAcUW21-L/ABCG2-R482G into the pAcUW21-L/His6-wtABCG2 vector. Login to comment
163 Characterization of the purified ABCG2-R482G variant ABCG2 Arg482 variants have grossly altered substrate, cholesterol and bile acid interactions when expressed in Sf9 cells ([8,13,20] and &#b4; A. Telbisz and Cs. &#a8; Ozvegy-Laczka, unpublished work). Login to comment
164 In the present study we also generated the N-terminally His6-tagged version of ABCG2-R482G and characterized its activity in reconstituted proteoliposomes. Login to comment
165 The His6-ABCG2-R482G variant was successfully expressed in Sf9 cells, at a similar level to the wild-type ABCG2 protein (results not shown). Login to comment
167 By examining the ATPase activity of the purified and reconstituted ABCG2-R482G protein, we used E. coli lipids and cholesterol-containing liposomes and the simplified reconstitution protocol. Login to comment
168 As shown in Supplementary Figure S5 (at http://www.biochemj.org/bj/450/bj4500387add.htm), we found that, in accordance with results obtained in cholesterol-loaded Sf9 cell membrane preparations, the calculated turnover of the ATPase activity of the purified R482G variant was similar to or higher than that of the wild-type protein. Login to comment
170 In contrast with the wild-type protein, the ATPase activity of the R482G variant could be significantly stimulated by elacridar or imatinib [21], inhibitors of the wild-type ABCG2, whereas quercetin or nilotinib had a similar stimulatory effect as in the case of wild-type ABCG2 (see Supplementary Figure S5). Login to comment
171 Figure 6 summarizes the data obtained for the sterol modulation of the activity of the purified and reconstituted ABCG2-R482G protein. Login to comment
173 As depicted in Figure 6(A), when reconstituted in E. coli lipids in the absence of cholesterol, the ATPase activity of the ABCG2-R482G protein was low and practically not stimulated by quercetin (or by other activating agents, results not shown). Login to comment
176 We also compared the effects of various cholesterol concentrations in the reconstituting lipid mixture on the ATPase activities of the wild-type ABCG2 and the R482G variant (Figure 6B). Login to comment
178 Although the wild-type protein requires 30-40 mol% of cholesterol to reach full drug-stimulated activity, the R482G variant is already maximally active at 20 mol% of cholesterol. Login to comment
179 This difference may explain the findings in the insect cell membranes, where low levels of endogenous sterols may provide full activity for the R482G variant, but not for the wild-type ABCG2 protein. Login to comment
180 The activity of the purified and reconstituted ABCG2-R482G protein was also modulated by the addition of bile acids (cholic acid, taurocholate or deoxycholate). Login to comment
181 The results obtained in the presence of 1 mM cholic acid are presented on Figure 6(C), demonstrating the effect of bile acids on the R482G mutant of ABCG2. Login to comment
182 The basal ATPase activity of the R482G variant reconstituted in brain lipid with 40 mol% cholesterol was reduced by cholic acid, although to a lesser extent than that of the wild-type protein. Login to comment
188 These Figure 6 Comparison of the sterol and bile acid dependence of the ATPase activity of the purified and reconstituted wild-type and R482G variant of ABCG2 (A) ATPase activity of ABCG2-R482G was determined in control (cont, pure E. coli lipid based) liposomes or in liposomes prepared from E. coli lipid with 20 or 40 mol% of the indicated sterol. Login to comment
191 (B) Comparison of the ATPase activity of the wild-type (wt) and the ABCG2-R482G proteins reconstituted in E. coli lipid-based liposomes with increasing amounts of cholesterol. Login to comment
192 Basal (wild-type, Ƭf;; and R482G, Ⴢ) and drug-stimulated (5 bc;M quercetin: wild-type, Ⴡ; and R482G, Ƭa;) ATPase activities were examined. Login to comment
193 (C) Effect of cholic acid on the ATPase activity of reconstituted wild-type ABCG2 and ABCG2-R482G protein. Login to comment
217 An interesting finding, which could not be derived from studies on ABCG2 in cholesterol-loaded Sf9 cell membrane preparations, was the absolute cholesterol dependence of the activity of the ABCG2 protein, even in the case of the R482G variant, which was found to be insensitive to cholesterol loading of the Sf9 membranes [8]. Login to comment
218 We suggest that the explanation for this discrepancy is that even very low membrane sterol levels may fully activate the R482G transporter variant in the Sf9 membrane. Login to comment
219 Thus the R482G protein is not insensitive to cholesterol, but instead has a high affinity for cholesterol, exceeding that of the wild-type ABCG2 protein. Login to comment
229 This basal activity is somewhat different in the R482G mutant variant, depending on the lipid environment and cholesterol, but may not be coupled to any transport activity. Login to comment

[hide] Sequence mutations of the substrate binding pocket... Oncol Rep. 2013 May;29(5):1697-700. doi: 10.3892/or.2013.2324. Epub 2013 Mar 5. Zoernig I, Ziegelmeier C, Lahrmann B, Grabe N, Jager D, Halama N
Sequence mutations of the substrate binding pocket of stem cell factor and multidrug resistance protein ABCG2 in renal cell cancer: a possible link to treatment resistance.
Oncol Rep. 2013 May;29(5):1697-700. doi: 10.3892/or.2013.2324. Epub 2013 Mar 5., [PMID:23467750]

Abstract [show]
ABCG2 is a multidrug cellular transport protein that is associated with resistance to certain treatments in patients, particularly anticancer treatment. The tumor-protective properties of ABCG2 expression are reported to be a feature of a subset of stem cell-like tumor cells. While protection against chemotherapy has been well analyzed, the role of ABCG2 in the treatment with tyrosine kinase inhibitors is only partially understood. Tyrosine kinase inhibitors are currently the main treatment option in irresectable renal cell carcinomas. To investigate possible underlying sequence variations in the ABCG2 gene with relevance to the functional properties of the protein, 36 patient samples were analyzed. Using sequence analysis and single-nucleotide polymorphism databases, sequence variations in the highly conserved domains of the binding pocket of ABCG2 were analyzed. The resulting variations were used for computational protein prediction algorithms to identify conformational alterations. A relevant shift from A to G at position 1376 (resulting in Y-->C at 459 aa) was identified and found to be present in 8.3% of the patients. These patients are currently in follow-up after resection, thus, further analysis will reveal whether this mutation has relevance to treatment efficacy.

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88 It has been hypothesized that residue R482 in the transmembrane domain 3 (TM3) is likely to interact with substrates based on the effect of R482G/T mutations (29). Login to comment

[hide] Fluorescent substrates for flow cytometric evaluat... Anal Biochem. 2013 Jun 1;437(1):77-87. doi: 10.1016/j.ab.2013.02.018. Epub 2013 Mar 5. Strouse JJ, Ivnitski-Steele I, Waller A, Young SM, Perez D, Evangelisti AM, Ursu O, Bologa CG, Carter MB, Salas VM, Tegos G, Larson RS, Oprea TI, Edwards BS, Sklar LA
Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters.
Anal Biochem. 2013 Jun 1;437(1):77-87. doi: 10.1016/j.ab.2013.02.018. Epub 2013 Mar 5., [PMID:23470221]

Abstract [show]
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

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319 [7] R. Clark, I.D. Kerr, R. Callaghan, Multiple drug-binding sites on the R482G isoform of the ABCG2 transporter, Br. J. Pharmacol. 149 (2006) 506-515. Login to comment

[hide] Predicting substrates of the human breast cancer r... BMC Bioinformatics. 2013 Apr 15;14:130. doi: 10.1186/1471-2105-14-130. Hazai E, Hazai I, Ragueneau-Majlessi I, Chung SP, Bikadi Z, Mao Q
Predicting substrates of the human breast cancer resistance protein using a support vector machine method.
BMC Bioinformatics. 2013 Apr 15;14:130. doi: 10.1186/1471-2105-14-130., [PMID:23586520]

Abstract [show]
BACKGROUND: Human breast cancer resistance protein (BCRP) is an ATP-binding cassette (ABC) efflux transporter that confers multidrug resistance in cancers and also plays an important role in the absorption, distribution and elimination of drugs. Prediction as to if drugs or new molecular entities are BCRP substrates should afford a cost-effective means that can help evaluate the pharmacokinetic properties, efficacy, and safety of these drugs or drug candidates. At present, limited studies have been done to develop in silico prediction models for BCRP substrates. In this study, we developed support vector machine (SVM) models to predict wild-type BCRP substrates based on a total of 263 known BCRP substrates and non-substrates collected from literature. The final SVM model was integrated to a free web server. RESULTS: We showed that the final SVM model had an overall prediction accuracy of ~73% for an independent external validation data set of 40 compounds. The prediction accuracy for wild-type BCRP substrates was ~76%, which is higher than that for non-substrates. The free web server (http://bcrp.althotas.com) allows the users to predict whether a query compound is a wild-type BCRP substrate and calculate its physicochemical properties such as molecular weight, logP value, and polarizability. CONCLUSIONS: We have developed an SVM prediction model for wild-type BCRP substrates based on a relatively large number of known wild-type BCRP substrates and non-substrates. This model may prove valuable for screening substrates and non-substrates of BCRP, a clinically important ABC efflux drug transporter.

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55 For example, doxorubicin, rhodamine 123 and LysoTracker Green are substrates of the mutant R482G or R482T, but cannot be efficiently transported by wild-type BCRP [23-25]. Login to comment

[hide] Molecular pharmacology of ABCG2 and its role in ch... Mol Pharmacol. 2013 Nov;84(5):655-69. doi: 10.1124/mol.113.088609. Epub 2013 Sep 10. Stacy AE, Jansson PJ, Richardson DR
Molecular pharmacology of ABCG2 and its role in chemoresistance.
Mol Pharmacol. 2013 Nov;84(5):655-69. doi: 10.1124/mol.113.088609. Epub 2013 Sep 10., [PMID:24021215]

Abstract [show]
The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) is an important member of the ABC transporter superfamily, which has been suggested to be involved in multidrug resistance (MDR) in cancer. Its diverse range of substrates includes many common chemotherapeutics such as imatinib, doxorubicin, and mitoxantrone. Physiologically, ABCG2 is highly expressed in areas such as the blood-brain barrier and gastrointestinal tract, where it is thought to play a role in protection against xenobiotic exposure. High ABCG2 expression has also been found in a variety of solid tumors and in hematologic malignancies and has been correlated with poorer clinical outcomes. Furthermore, ABCG2 expression is a characteristic feature of cancer stem cells, which are able to self-renew and differentiate. These cancer stem cells have been postulated to play an important role in MDR, where their inherent ABCG2 expression may allow them to survive chemotherapy and repopulate the tumor after exposure to chemotherapeutics. This observation raises the exciting possibility that by inhibiting ABCG2, cancer stem cells and other cancers may be targeted and eradicated, at which point conventional chemotherapeutics would be sufficient to eliminate the remaining tumor cells. Inhibitors of ABCG2, such as tyrosine kinase inhibitors, phosphodiesterase-5 inhibitors, and the fumitremorgin-type indolyl diketopiperazine, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxo pyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], could potentially be used for this purpose. However, these agents are still awaiting comprehensive clinical assessment.

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44 *Substrates transported by ABCG2 mutant R482G. Login to comment
64 Additionally, electron microscopy has shown an octameric complex organized as a tetramer of dimers in the mutant R482G isoform of ABCG2 (McDevitt et al., 2006). Login to comment
81 The R482G isoform of ABCG2 appears in drug-selected cell lines and displays altered substrate specificity to the wild-type transporter. Login to comment
117 This observation could explain why resistance to methotrexate (which interacts with Arg482) is decreased by the R482G and R482T mutations (Chen et al., 2003b), whereas efflux of prazosin (which binds to a separate and distinct binding site) was not affected (Giri et al., 2009). Login to comment
123 Some drug-selected cell lines that express mutant forms of ABCG2, R482G, and R482T (discussed in "Homology Modeling") are considered to be gain-of-function mutants, as their altered substrate specificity increases resistance to anthracyclines (doxorubicin, daunorubicin) and rhodamine 123 (Fig. 6) (Chen et al., 1990; Honjo et al., 2001; Allen et al., 2002a). Login to comment

[hide] Regulation of the function of the human ABCG2 mult... Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2. Telbisz A, Hegedus C, Varadi A, Sarkadi B, Ozvegy-Laczka C
Regulation of the function of the human ABCG2 multidrug transporter by cholesterol and bile acids: effects of mutations in potential substrate and steroid binding sites.
Drug Metab Dispos. 2014 Apr;42(4):575-85. doi: 10.1124/dmd.113.055731. Epub 2014 Jan 2., [PMID:24384916]

Abstract [show]
ABCG2 (ATP-binding cassette, subfamily G, member 2) is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity, and by examining purified ABCG2 reconstituted in proteoliposomes we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol dependence, but changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Regarding the effect of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, which did not significantly affect substrate-stimulated ATPase activity. These ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate were not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacologic modulation of ABCG2 function.

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26 Still, in isolated ABCG2 preparations even the R482G mutant required low levels of cholesterol for full function (Telbisz et al., 2013). Login to comment
52 The R482G/L555/ 558A triple mutant was created by replacing the DNA fragment between the PstI-NcoI sites of the pAcUW21-L/R482G with that of derived from the pAcUW21-L/L555/558A vector. Login to comment
91 In contrast to the wild-type protein, in Sf9 cell membranes increasing the membrane cholesterol levels did not significantly influence the activity of the R482G and R482T mutants (Telbisz et al., 2007), although experiments on purified ABCG2 reconstituted in proteoliposomes revealed that cholesterol is also essential for the function of the R482G variant (Telbisz et al., 2013). Login to comment
145 Effect of Cholesterol on the Function of Isolated and Reconstituted ABCG2-SBE Mutants It was shown earlier that an increase in the cholesterol levels of the insect membranes did not significantly modulate the function of the ABCG2 R482G mutant, which would imply its apparent cholesterol insensitivity. Login to comment
146 However, reconstitution of the purified ABCG2 R482G variant revealed that the presence of cholesterol was also essential for the function of this mutant variant; however, lower cholesterol levels (amounts that are most probably present in native insect membranes) were sufficient to achieve its full activity as compared with wtABCG2 (Telbisz et al., 2013). Login to comment
225 These were the R482G and R482S variants, which are fully active already at low membrane cholesterol levels, and the R482K and R482I mutants, which show similar cholesterol-sensing capability to the wtABCG2 (see earlier). Login to comment
237 4, C and D, and 6A), in the case of the R482G or S variants low concentrations of CA did not significantly alter ABCG2-ATPase activity; however, when we used higher bile acid concentrations (above 0.5 mM), both baseline and substrate-stimulated ATPase activities decreased (see Fig. 6B for the R482G mutant; R482S is not shown). Login to comment
243 In the case of the L558A mutant, a similar effect of bile acids was observed as in the case of the R482G variant: CA and TC inhibited both basal and substrate-stimulated ATPase activity (Supplemental Fig. 5), but the relative substrate activation was practically unchanged (Fig. 6D). Login to comment
271 mutant contradicted the results described by Velamakanni et al. (2008), which may be due to the fact that they investigated a triple mutant of ABCG2, which had R482G besides the L555A/L558A mutation, whereas we performed our experiments using the wild-type ABCG2 (482R) as a background. Login to comment
272 As demonstrated elsewhere and in this report as well, the R482G variant is already fully active in cholesterol-deficient Sf9 membranes. Login to comment
273 To solve this contradiction, we also generated the triple mutant R482G/L555A/L558A of ABCG2 and expressed this protein in insect cells. Login to comment
295 Membranes were incubated with or without 1 mM bile acid; the ratio of ATP hydrolysis in the presence of 1 mM quercetin (wt, R482G, S, K, and I) or 1 mM nilotinib (SBE mutants) and the baseline ATPase activity were calculated. Login to comment

[hide] Impact of genetic variability in the ABCG2 gene on... Biochem Biophys Res Commun. 2014 Jan 24;443(4):1211-7. doi: 10.1016/j.bbrc.2013.12.119. Epub 2014 Jan 3. Deppe S, Ripperger A, Weiss J, Ergun S, Benndorf RA
Impact of genetic variability in the ABCG2 gene on ABCG2 expression, function, and interaction with AT1 receptor antagonist telmisartan.
Biochem Biophys Res Commun. 2014 Jan 24;443(4):1211-7. doi: 10.1016/j.bbrc.2013.12.119. Epub 2014 Jan 3., [PMID:24388985]

Abstract [show]
The ATP-binding cassette transporter ABCG2 plays a prominent role in cardiovascular and cancer pathophysiology, is involved in the pathogenesis of gout, and affects pharmacokinetics of numerous drugs. Telmisartan, a widely used AT1 receptor antagonist, inhibits the transport capacity of ABCG2 and may cause drug-drug interactions, especially in individuals carrying polymorphism that facilitate the telmisartan-ABCG2 interaction. Thus, the aim of this study was to identify ABCG2 polymorphisms and somatic mutations with relevance for the telmisartan-ABCG2 interaction. For this purpose, a cellular system for the conditional expression of ABCG2 was established. ABCG2 variants were generated via site-directed mutagenesis. Interaction of telmisartan with these ABCG2 variants was investigated in HEK293-Tet-On cells using the pheophorbide A efflux assay. Moreover, expression of ABCG2 variants was studied in these cells. Importantly, protein levels of the Q141K and F489L variant were significantly reduced, a phenomenon that was partly reversed by pharmacological proteasome inhibition. Moreover, basal pheophorbide A efflux capacity of S248P, F431L, and F489L variants was significantly impaired. Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. We conclude that the arginine residue at position 482 of the ABCG2 molecule is of major importance for the interaction of telmisartan with this ABC transporter. Furthermore, individuals carrying the F489L polymorphism may be at increased risk of developing adverse drug reactions in multi-drug regimens involving ABCG2 substrates and telmisartan.

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8 Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. Login to comment
37 Site-directed mutagenesis Non-synonymous ABCG2 single nucleotide polymorphisms (SNPs) G34A (V12M), C421A (Q141K), T742C (S248P), T1291C (F431L), T1465C (F489L) as well as somatic mutation A1444G (R482G) were inserted into the ABCG2 cDNA sequence in the pTRE-Tight-BI-AcGFP1-ABCG2 plasmid using the QuickChange&#d2; Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Waldbronn, Germany) with specific primers according to the manufacturer`s instructions (Supplemental Fig. 1). Login to comment
91 The average PhA-associated fluorescence in non-induced HEK293-Tet-On cells transiently transfected with the various ABCG2 variants was not significantly different as compared with that observed in HEK293-Tet-On cells transfected with ABCG2 wild-type (wild-type (100 &#b1; 12.1%), V12M (106.7 &#b1; 2.0%), Q141K (97.1 &#b1; 9.3%), S248P (99.1 &#b1; 9.8%), F431L (104.7% &#b1; 10.9%), R482G A B C D Fig. 2. Login to comment
99 PhA-associated fluorescence was similar in doxycycline-induced AcGFP1-positive HEK293-Tet-On cells transfected with the ABCG2 variants V12M (11.8 &#b1; 0.9%), Q141K (17.9 &#b1; 5.8%), and R482G (17.0 &#b1; 2.8%). Login to comment
105 Interestingly, the inhibitory effect of telmisartan on ABCG2-mediated PhA efflux was almost abolished by the R482G mutation (Fig. 4B and C), thereby indicating that the arginine residue at position 482 of the ABCG2 molecule may be of major importance for the interaction of telmisartan with the ABC transporter. Login to comment
131 Interestingly, the somatic mutation R482G, which frequently occurs in drug-resistant cancer cell lines and has been associated with an altered ABCG2 substrate spectrum with regard to cytotoxic drugs [1], almost completely abolished telmisartan-induced inhibition of PhA transport in our experimental system. These data suggest that the arginine residue at position 482 of the ABCG2 molecule is of major relevance for the interaction of telmisartan with ABCG2 and that removal of the positive charge at this position in ABCG2 may largely reduce the ability of the ABC transporter to interact with telmisartan. Login to comment
132 Indeed, experimental evidence points to a prominent role of Arg-482 in ABCG2-drug interaction. For instance, the R482G mutation abolished ABCG2-mediated transport of the dihydrofolate reductase inhibitor methotrexate, whereas it acted as a gain-of-function mutation with respect to doxorubicin or rhodamine 123 [1,24]. Login to comment
133 Hence, our findings add to the available evidence on the important role of the R482G mutation for A B C Fig. 4. Login to comment

[hide] Baicalein decreases side population proportion via... Fitoterapia. 2014 Apr;94:21-8. doi: 10.1016/j.fitote.2014.01.019. Epub 2014 Jan 25. Gu YY, Liu LP, Qin J, Zhang M, Chen Y, Wang D, Li Z, Tang JZ, Mo SL
Baicalein decreases side population proportion via inhibition of ABCG2 in multiple myeloma cell line RPMI 8226 in vitro.
Fitoterapia. 2014 Apr;94:21-8. doi: 10.1016/j.fitote.2014.01.019. Epub 2014 Jan 25., [PMID:24468191]

Abstract [show]
OBJECTIVE: To investigate the effect of baicalein on side population in human multiple myeloma cell line RPMI 8226 and the underlying molecular mechanisms in vitro and in silico. METHODS: MTT assay was applied to detect the anti-proliferation effect of baicalein. The detection of side population cells is based on the Hoechst 33342 exclusion assay technique and flow cytometric analysis. Western blotting assay was used to explore the expression of ABCG2 protein. Homology modeling and molecular docking were performed with Discovery Studio 2.1. RESULTS: Baicalein decreased both cell viability with IC50=168.5 muM and the proportion of SP cells in a dose-dependent manner. Correspondingly, it significantly decreased the expression level of ABCG2 protein. Baicalein also shared similar binding sites and modes with fumitremorgin C to the protein. CONCLUSIONS: Baicalein possessed novel anticancer properties, such as anti-proliferation and drug efflux inhibition in side population cells, which suggested its potential feature of targeting cancer stem cells of multiple myeloma.

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251 Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter. Login to comment

[hide] Masitinib antagonizes ATP-binding cassette subfami... Int J Oncol. 2014 May;44(5):1634-42. doi: 10.3892/ijo.2014.2341. Epub 2014 Mar 13. Kathawala RJ, Chen JJ, Zhang YK, Wang YJ, Patel A, Wang DS, Talele TT, Ashby CR Jr, Chen ZS
Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.
Int J Oncol. 2014 May;44(5):1634-42. doi: 10.3892/ijo.2014.2341. Epub 2014 Mar 13., [PMID:24626598]

Abstract [show]
In this in vitro study, we determined whether masitinib could reverse multidrug resistance (MDR) in cells overexpressing the ATP binding cassette subfamily G member 2 (ABCG2) transporter. Masitinib (1.25 and 2.5 microM) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 microM) significantly increased the intracellular accumulation of [(3)H]-MX, a substrate for ABCG2, by inhibiting the function of ABCG2 and significantly decreased the efflux of [(3)H]-MX. However, masitinib (2.5 microM) did not significantly alter the expression of the ABCG2 protein. In addition, a docking model suggested that masitinib binds within the transmembrane region of a homology-modeled human ABCG2 transporter. Overall, our in vitro findings suggest that masitinib reverses MDR to various anti-neoplastic drugs in HEK293 and H460 cells overexpressing ABCG2 by inhibiting their transport activity as opposed to altering their levels of expression.

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106 Therefore, in the present study, both wild-type (R482) and two mutant forms (R482T and R482G) of ABCG2 were used. Login to comment

[hide] Linsitinib (OSI-906) antagonizes ATP-binding casse... Int J Biochem Cell Biol. 2014 Jun;51:111-9. doi: 10.1016/j.biocel.2014.03.026. Epub 2014 Apr 12. Zhang H, Kathawala RJ, Wang YJ, Zhang YK, Patel A, Shukla S, Robey RW, Talele TT, Ashby CR Jr, Ambudkar SV, Bates SE, Fu LW, Chen ZS
Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance.
Int J Biochem Cell Biol. 2014 Jun;51:111-9. doi: 10.1016/j.biocel.2014.03.026. Epub 2014 Apr 12., [PMID:24726739]

Abstract [show]
In this study we investigated the effect of linsitinib on the reversal of multidrug resistance (MDR) mediated by the overexpression of the ATP-binding cassette (ABC) subfamily members ABCB1, ABCG2, ABCC1 and ABCC10. Our results indicate for the first time that linsitinib significantly potentiate the effect of anti-neoplastic drugs mitoxantrone (MX) and SN-38 in ABCG2-overexpressing cells; paclitaxel, docetaxel and vinblastine in ABCC10-overexpressing cells. Linsitinib moderately enhanced the cytotoxicity of vincristine in cell lines overexpressing ABCB1, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, linsitinib significantly increased the intracellular accumulation and decreased the efflux of [(3)H]-MX in ABCG2-overexpressing cells and [(3)H]-paclitaxel in ABCC10-overexpressing cells. However, linsitinib, at a concentration that reversed MDR, did not significantly alter the expression levels of either the ABCG2 or ABCC10 transporter proteins. Furthermore, linsitinib did not significantly alter the intracellular localization of ABCG2 or ABCC10. Moreover, linsitinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner. Overall, our study suggests that linsitinib attenuates ABCG2- and ABCC10-mediated MDR by directly inhibiting their function as opposed to altering ABCG2 or ABCC10 protein expression.

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134 Thus, in order to determine whether linsitinib increases the drug sensitivity in both wild-type and mutant ABCG2-overexpressing cells, we used HEK293 transfected wild-type ABCG2-482-R2 (Arg482), mutant ABCG2-482-G2 (Arg482Gly) and mutant ABCG2-482-T7 (Arg482Thr) cell lines. Login to comment
144 These results indicated that linsitinib inhibits the activity of both wild-type and mutant Arg482Gly/Thr ABCG2. Login to comment

[hide] AST1306, a potent EGFR inhibitor, antagonizes ATP-... Cancer Lett. 2014 Aug 1;350(1-2):61-8. doi: 10.1016/j.canlet.2014.04.008. Epub 2014 Apr 18. Zhang H, Wang YJ, Zhang YK, Wang DS, Kathawala RJ, Patel A, Talele TT, Chen ZS, Fu LW
AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.
Cancer Lett. 2014 Aug 1;350(1-2):61-8. doi: 10.1016/j.canlet.2014.04.008. Epub 2014 Apr 18., [PMID:24747122]

Abstract [show]
AST1306, an inhibitor of EGFR and ErbB2, is currently in phase I of clinical trials. We evaluated the effect of AST306 on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters. We found that AST1306 significantly sensitized the ABC subfamily G member 2 (ABCG2)-overexpressing cells to ABCG2 substrate chemotherapeutics. AST1306 significantly increased intracellular accumulation of [(3)H]-mitoxantrone in ABCG2-overexpressing cells by blocking ABCG2 efflux function. Moreover, AST1306 stimulated the ATPase activity of ABCG2. Homology modeling predicted the binding conformation of AST1306 to be within the transmembrane region of ABCG2. In conclusion, AST1306 could notably reverse ABCG2-mediated MDR.

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86 Therefore, we investigated whether AST1306 could reverse ABCG2-mediated resistance to its substrates in cells transfected with either the wild-type (Arg482) or mutant (Arg482Gly and Arg482Thr) forms of ABCG2. Login to comment
87 As shown in Table 2, AST1306 could significantly increase sensitivity to MX and SN-38 in ABCG2-482-R2, ABCG2-482-G2 and ABCG2-482-T7 cells, respectively, and this effect was similar to that obtained with 1 lM FTC. These results indicated that AST1306 can reverse ABCG2-mediated MDR in cells expressing either wild-type or mutant Arg482Gly/Thr ABCG2 (Table 2). Login to comment

[hide] Structure and function of BCRP, a broad specificit... Arch Toxicol. 2014 Jun;88(6):1205-48. doi: 10.1007/s00204-014-1224-8. Epub 2014 Apr 29. Jani M, Ambrus C, Magnan R, Jakab KT, Beery E, Zolnerciks JK, Krajcsi P
Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics.
Arch Toxicol. 2014 Jun;88(6):1205-48. doi: 10.1007/s00204-014-1224-8. Epub 2014 Apr 29., [PMID:24777822]

Abstract [show]
The discovery and characterization of breast cancer resistance protein (BCRP) as an efflux transporter conferring multidrug resistance has set off a remarkable trajectory in the understanding of its role in physiology and disease. While the relevance in drug resistance and general pharmacokinetic properties quickly became apparent, the lack of a characteristic phenotype in genetically impaired animals and humans cast doubt on the physiological importance of this ATP-binding cassette family member, similarly to fellow multidrug transporters, despite well-known endogenous substrates. Later, high-performance genetic analyses and fine resolution tissue expression data forayed into unexpected territories concerning BCRP relevance, and ultimately, the rise of quantitative proteomics allows putting observed interactions into absolute frameworks for modeling and insight into interindividual and species differences. This overview summarizes existing knowledge on the BCRP transporter on molecular, tissue and system level, both in physiology and disease, and describes a selection of experimental procedures that are the most widely applied for the identification and characterization of substrate and inhibitor-type interactions.

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69 Mutants Arg482Thr and Arg482Gly transport rhodamine 123, lysoTracker Green, and daunorubicin, whereas wild-type BCRP does not (Honjo et al. 2001). Login to comment

[hide] Determinants of the activity and substrate recogni... Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18. Szafraniec MJ, Szczygiel M, Urbanska K, Fiedor L
Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2).
Drug Metab Rev. 2014 Nov;46(4):459-74. doi: 10.3109/03602532.2014.942037. Epub 2014 Jul 18., [PMID:25036722]

Abstract [show]
The xenobiotic transporters are among the most important constituents of detoxification system in living organisms. Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of xenobiotics. To understand its role in chemotherapeutic and multidrug resistance, it is crucial to establish the determinants of its substrate specificity, which obviously is of high relevance for successful therapy of many diseases. This article summarizes the current knowledge about the substrate preferences of BCRP. We overview the factors which determine its activity, inhibition and substrate recognition, focusing on the structural features of the transporter. BCRP substrate specificity is quite low as it interacts with a spectrum of substances with only a few common features: hydrophobic and aromatic regions, possibly a flat conformation and the metal ion-, oxygen- and nitrogen-containing functionalities, most of which may be the donors/acceptors of H-bonds. Several amino acid residues and structural motifs are responsible for BCRP activity and substrate recognition. Thus, the active form of BCRP, at least a dimer or a larger oligomer is maintained by intramolecular disulfide bridge that involves Cys(603) residues. The GXXXG motif in transmembrane helix 1, Cys residues, Arg(482) and Lys(86) are responsible for maintaining the protein structure, which confers transport activity, and the His(457) or Arg(456) residues are directly involved in substrate binding. Arg(482) does not directly bind substrates, but electrostatically interacts with charged molecules, which initiates the conformational changes that transmit the signal from the transmembrane regions to the ABC domain.

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68 This effect was observed only for Arg482 -BCRP (WT protein), with no such influence on the Arg482 Gly or Arg482 Thr variants. Login to comment
163 The role of residue 482 The substitution of Arg482 by Thr or Gly results in the capability of BCRP-overexpressing cells to efflux rhodamine 123 and doxorubicin (Honjo et al., 2001), but irrespective of residue 482, the cells were able to transport mitoxantrone. Login to comment
164 Moreover, the Arg482 Thr variant seemed to enhance the resistance of BCRP-transfected HEK-293 cells to anthracyclines, whereas the Arg482 Gly variant passed on diminished resistance in this cell line to SN-38 and topotecan. Login to comment
183 There were also significant differences in ATPase activity among the three BCRP variants, WT form, Arg482 Gly and Arg482 Thr, expressed in the Sf9 cell membranes and stimulated by several substrates. Login to comment
186 The ATPase activity of WT BCRP stimulated by mitoxantrone or estradiol-17b-glucuronide was greatly enhanced in cholesterol-enriched membranes, but no such an effect was observed in the Arg482 Gly or Arg482 Thr variants (Telbisz et al., 2007). Login to comment
201 To elucidate the significance of this polymorphism for porphyrin transport, a set of 18 variants of BCRP (Val12 Met, Gly51 Cys, Gln126 stop, Gln141 Lys, Thr153 Met, Gln166 Glu, Ile206 Leu, Phe208 Ser, Ser248 Pro, Glu334 stop, Phe431 Leu, Ser441 Asn, Arg482 Gly, Arg482 Thr, Phe489 Leu, Phe571 Ile, Asn590 Tyr and Asp620 Asn) have been expressed in insect cells. Login to comment
209 Position Type of mutation Effect on the transporter References NBD Lys 86 Met (i) No stimulation of the ATPase activity by prazosin; (ii) no influence on the transport of mitoxantrone Henriksen et al. (2005b) Glu 126 stop, Phe 208 Ser, Ser 248 Phe, Glu 334 stop Inability to transport hematoporphyrin Tamura et al. (2006) Glu 211 Gln Complete abolishment of the ATPase activity and methotrexate transport Hou et al. (2009) Pro 392 Ala Significant reduction in the efflux activity of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Ni et al. (2011) TM1 Gly 406 Ala Gly 410 Ala No influence on the activity of the transporter Polgar et al. (2004) Gly 406 Leu Gly 410 Leu (i) Loss of the ability to transport rhodamine123; (ii) impaired transport of mitoxantrone, Pheide and BODIPY-prazosin Polgar et al. (2004) Extracellular loop 1 Phe 431 Leu (i) Loss of the ability to transport methotrexate; (ii) 10% level of hematoporphyrin transport compared to the WT protein Tamura et al. (2006) Ser 441 Asn Inability to transport hematoporphyrin Tamura et al. (2006) Ser 441 Asn Loss of the ability to transport methotrexate Tamura et al. (2006) TM2 Lys 452 Ala His 457 Ala Increase in transport of mitoxantrone, BODIPY-prazosin and Hoechst 33342 Cai et al. (2010) Lys 453 Ala Arg 465 Ala Decrease in transport of mitoxantrone, BODIPY-prazosin, Hoechst 33342, doxorubicin, SN-38 and rhodamine 123 Cai et al. (2010) TM3 Arg 482 Gly Arg 482 Thr (i) No change in the inhibitory activity of lapatinib; (ii) about two times greater inhibition by ritonavir, saquinavir and nalfinavir than in the WT variant; (iii) gaining the ability to transport rhodamine123 and doxorubicin; (iv) no influence on the transport of mitoxantrone; (v) loss of the ability to transport methotrexate Dai et al. (2008), Gupta et al. (2004), Honjo et al. (2001), Mitomo et al. (2003) Arg 482 Thr (i) Lower IC 50 of cyclosporine A for mutant than for WT variant; (ii) lower elacridar inhibition potency Xia et al. (2007) Arg 482 Lys Complete loss of transport activity Ejendal et al. (2006) Phe 489 Leu Impaired transport of porphyrins, no transport of methotrexate Tamura et al. (2006) Extracellular loop 3 Asn 590 Tyr Over twice reduced transport of mitoxantrone, topotecan, daunorubicin and rhodamine 123 Vethanayagam et al. (2005) Cys 592 Ala/Cys 608 Ala (i) Transport of mitoxantrone almost unchanged; (ii) transport of BODIPY-prazosin significantly impaired Henriksen et al. (2005a) Extracellular loop 3 Cys 603 Ser Cys 592 Ser/Cys 608 Ser Cys 592 Ser/Cys 603 Ser/Cys 608 Ser Diminished susceptibility to the inhibitory activity of fumitremorgin C Shigeta et al. (2010) Cys-less Arg 482 Gly-BCRP Complete loss of the ability to efflux mitoxantrone Liu et al. (2008b) The positions of the amino acid residues refer to the topological model of BCRP proposed by Wang et al. (2009). Login to comment

[hide] Role of the breast cancer resistance protein (BCRP... AAPS J. 2015 Jan;17(1):65-82. doi: 10.1208/s12248-014-9668-6. Epub 2014 Sep 19. Mao Q, Unadkat JD
Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update.
AAPS J. 2015 Jan;17(1):65-82. doi: 10.1208/s12248-014-9668-6. Epub 2014 Sep 19., [PMID:25236865]

Abstract [show]
The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is an ATP-binding cassette (ABC) efflux transporter. It was so named because it was initially cloned from a multidrug-resistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents such as mitoxantrone and topotecan. Since its discovery in 1998, the substrates of BCRP have been rapidly expanding to include not only therapeutic agents but also physiological substances such as estrone-3-sulfate, 17beta-estradiol 17-(beta-D-glucuronide) and uric acid. Likewise, at least hundreds of BCRP inhibitors have been identified. Among normal human tissues, BCRP is highly expressed on the apical membranes of the placental syncytiotrophoblasts, the intestinal epithelium, the liver hepatocytes, the endothelial cells of brain microvessels, and the renal proximal tubular cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds as well as tissue protection against xenobiotic exposure. As a result, BCRP has now been recognized by the FDA to be one of the key drug transporters involved in clinically relevant drug disposition. We published a highly-accessed review article on BCRP in 2005, and much progress has been made since then. In this review, we provide an update of current knowledge on basic biochemistry and pharmacological functions of BCRP as well as its relevance to drug resistance and drug disposition.

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60 Rhodamine 123 and LysoTracker Green are substrates of the mutants, R482G and R482T, but not substrates of wild-type BCRP (15). Login to comment
175 Wild-type BCRP with Arg482 does not transport daunorubicin, rhodamine 123, and Lyso-Tracker Green; however, these compounds are excellent substrates of the BCRP mutants R482T and R482G (16,91). Login to comment

[hide] Mutations of the central tyrosines of putative cho... Biochim Biophys Acta. 2015 Feb;1848(2):477-87. doi: 10.1016/j.bbamem.2014.11.006. Epub 2014 Nov 14. Gal Z, Hegedus C, Szakacs G, Varadi A, Sarkadi B, Ozvegy-Laczka C
Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.
Biochim Biophys Acta. 2015 Feb;1848(2):477-87. doi: 10.1016/j.bbamem.2014.11.006. Epub 2014 Nov 14., [PMID:25445676]

Abstract [show]
Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.

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54 In cholesterol depletion experiments we used the HEK 293 cell line stably expressing the R482G mutant established earlier [15]. Login to comment
268 Alternatively, if we consider the possibility that cholesterol is transported by ABCG2, the results could be explained by a higher affinity to cholesterol by the ABCG2-Y413S mutant, also observed in the case of the R482G mutant [15]. Login to comment

[hide] Identification of residues in ABCG2 affecting prot... Biosci Rep. 2015 Jul 17;35(4). pii: e00241. doi: 10.1042/BSR20150150. Haider AJ, Cox MH, Jones N, Goode AJ, Bridge KS, Wong K, Briggs D, Kerr ID
Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.
Biosci Rep. 2015 Jul 17;35(4). pii: e00241. doi: 10.1042/BSR20150150., [PMID:26294421]

Abstract [show]
ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be 'rescued' by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2.

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32 Residue 482 in transmembrane helix (TM) 3 of the TMD is an arginine in the wild-type (WT) sequence, but a drug selected cell line expressing a mutant version (where the arginine is replaced by glycine; R482G) shows a broader resistance profile including doxorubicin and rhodamine 123 in its substrates [16-18]. Login to comment

[hide] Insights into Chemoresistance of Prostate Cancer. Int J Biol Sci. 2015 Aug 1;11(10):1160-70. doi: 10.7150/ijbs.11439. eCollection 2015. Zhang W, Meng Y, Liu N, Wen XF, Yang T
Insights into Chemoresistance of Prostate Cancer.
Int J Biol Sci. 2015 Aug 1;11(10):1160-70. doi: 10.7150/ijbs.11439. eCollection 2015., [PMID:26327810]

Abstract [show]
Prostate cancer (PCa) remains the most prevalent malignancy among males in the western world. Though hormonal therapies through chemical or surgical castration have been proposed many years ago, heretofore, such mainstay for the treatment on advanced PCa has not fundamentally changed. These therapeutic responses are temporary and most cases will eventually undergo PCa recurrence and metastasis, or even progress to castration-resistant prostate cancer (CRPC) due to persistent development of drug resistance. Prostate cancer stem cells (PCSCs) are a small population of cells, which possess unlimited self-renewal capacities, and can regenerate tumorigenic progenies, and play an essential role in PCa therapy resistance, metastasis and recurrence. Nowadays advanced progresses have been made in understanding of PCSC properties, roles of androgen receptor signaling and ATP-binding cassette sub-family G member 2 (ABCG2), as well as roles of genomic non-coding microRNAs and key signaling pathways, which have led to the development of novel therapies which are active against chemoresistant PCa and CRPC. Based on these progresses, this review is dedicated to address mechanisms underlying PCa chemoresistance, unveil crosstalks among pivotal signaling pathways, explore novel biotherapeutic agents, and elaborate functional properties and specific roles of chemoresistant PCSCs, which may act as a promising target for novel therapies against chemoresistant PCa.

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80 MCF7/AdVp3000 cells, highly resistant to both mitoxantrone and doxorubicin, have been demonstrated to express R482T and R482G variants respectively. Login to comment

[hide] Overcoming ABCG2-mediated drug resistance with imi... Cancer Chemother Pharmacol. 2015 Oct;76(4):853-64. doi: 10.1007/s00280-015-2858-9. Epub 2015 Sep 9. Darby RA, Unsworth A, Knapp S, Kerr ID, Callaghan R
Overcoming ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors.
Cancer Chemother Pharmacol. 2015 Oct;76(4):853-64. doi: 10.1007/s00280-015-2858-9. Epub 2015 Sep 9., [PMID:26351135]

Abstract [show]
PURPOSE: Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. METHODS: In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. RESULTS: The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [(3)H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. CONCLUSION: Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps.

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152 Statistical comparisons were made using ANOVA, with the Bartlett`s post hoc test Cytotoxic drug Cell line Chemosensitisation agent Control FTC 0.3 bc;M K00135 0.1 bc;M K00135 0.3 bc;M K00486 0.3 bc;M K00486 1.0 bc;M Flavopiridol MCF7 52.6 &#b1; 7.3 42.7 &#b1; 5.9 57.7 &#b1; 4.6 52.7 &#b1; 5.9 44.7 &#b1; 6.6 40.4 &#b1; 5.3 MCF7FLV1000 2017 &#b1; 78 193 &#b1; 30* 728 &#b1; 87* 201 &#b1; 20* 347 &#b1; 24* 153 &#b1; 11* Resistance 38.3 4.5 12.6 3.8 7.8 3.8 Mitoxantrone MCF7 12.9 &#b1; 3.1 5.9 &#b1; 1.9 8.7 &#b1; 1.8 6.2 &#b1; 0.9 6.3 &#b1; 2.9 6.4 &#b1; 1.7 MCF7FLV1000 525 &#b1; 69 86 &#b1; 4* 209 &#b1; 51* 81 &#b1; 21* 174 &#b1; 49* 35 &#b1; 12* Resistance 40.7 15.5 24.1 13.1 27.4 5.4 Topotecan MCF7 8.1 &#b1; 1.3 4.5 &#b1; 0.4 6.3 &#b1; 0.7 5.2 &#b1; 0.5 6.8 &#b1; 1.1 4.9 &#b1; 0.8 MCF7FLV1000 2736 &#b1; 127 163 &#b1; 27* 1315 &#b1; 86* 353 &#b1; 32* 571 &#b1; 15* 177 &#b1; 13* Resistance 336 35.9 210 68.1 83.6 35.8 Doxorubicin MCF7 8.5 &#b1; 0.8 9.0 &#b1; 1.2 11.1 &#b1; 0.52 6.6 &#b1; 0.6 12.8 &#b1; 2.1 9.7 &#b1; 1.1 MCF7FLV1000 69.9 &#b1; 6.9 29.8 &#b1; 3.7 62.6 &#b1; 3.3* 34.7 &#b1; 4.8* 34 &#b1; 4.2* 24.5 &#b1; 3.6* Resistance 8.2 3.3 5.6 5.2 2.7 2.5 The well-characterised R482G mutant isoform of ABCG2 imparts a number of changes in the cross-resistance pattern compared to the wild-type isoform. Login to comment

[hide] A-803467, a tetrodotoxin-resistant sodium channel ... Oncotarget. 2015 Nov 17;6(36):39276-91. doi: 10.18632/oncotarget.5747. Anreddy N, Patel A, Zhang YK, Wang YJ, Shukla S, Kathawala RJ, Kumar P, Gupta P, Ambudkar SV, Wurpel JN, Chen ZS, Guo H
A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo.
Oncotarget. 2015 Nov 17;6(36):39276-91. doi: 10.18632/oncotarget.5747., [PMID:26515463]

Abstract [show]
ATP-binding cassette subfamily G member 2 (ABCG2) is a member of the ABC transporter superfamily proteins, which has been implicated in the development of multidrug resistance (MDR) in cancer, apart from its physiological role to remove toxic substances out of the cells. The diverse range of substrates of ABCG2 includes many antineoplastic agents such as topotecan, doxorubicin and mitoxantrone. ABCG2 expression has been reported to be significantly increased in some solid tumors and hematologic malignancies, correlated to poor clinical outcomes. In addition, ABCG2 expression is a distinguishing feature of cancer stem cells, whereby this membrane transporter facilitates resistance to the chemotherapeutic drugs. To enhance the chemosensitivity of cancer cells, attention has been focused on MDR modulators. In this study, we investigated the effect of a tetrodotoxin-resistant sodium channel blocker, A-803467 on ABCG2-overexpressing drug selected and transfected cell lines. We found that at non-toxic concentrations, A-803467 could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies demonstrated that A-803467 (7.5 muM) significantly increased the intracellular accumulation of [3H]-mitoxantrone by inhibiting the transport activity of ABCG2, without altering its expression levels. In addition, A-803467 stimulated the ATPase activity in membranes overexpressed with ABCG2. In a murine model system, combination treatment of A-803467 (35 mg/kg) and topotecan (3 mg/kg) significantly inhibited the tumor growth in mice xenografted with ABCG2-overexpressing cancer cells. Our findings indicate that a combination of A-803467 and ABCG2 substrates may potentially be a novel therapeutic treatment in ABCG2-positive drug resistant cancers.

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41 HEK293 cells transfected with wild-type (HEK293/R482) and mutant (HEK293/ R482G and HEK293/R482T) ABCG2 (Supplementary Figure S2) showed significant resistance to MX and topotecan compared to HEK293/pcDNA3.1 (Table 1). Login to comment
60 Table 1: A-803467 enhances the cytotoxicity of mitoxantrone and topotecan in HEK293/pcDNA3.1 cells overexpressing the wild-type as well as mutant ABCG2 Treatments IC50 &#b1; SD (nM) HEK293/ pcDNA3.1 FR HEK293/ R482 FR HEK293/ R482G FR HEK293/ R482T FR Mitoxantrone 24.8 &#b1; 0.9 1.0 258.5 &#b1; 12.8 10.4# 723.8 &#b1; 12.5 29.1# 808.0 &#b1; 38.2 32.4# +A-803467 (2.5 bc;M) 21.5 &#b1; 0.8 0.8 57.5 &#b1; 0.9 2.3* 66.2 &#b1; 1.2 2.6* 74.0 &#b1; 18.9 3.0* +A-803467 (7.5 bc;M) 20.4 &#b1; 2.0 0.9 19.5 &#b1; 0.2 0.8* 24.2 &#b1; 1.5 0.9* 34.5 &#b1; 16.7 1.3* +FTC (5 bc;M) 21.5 &#b1; 0.8 0.8 17.7 &#b1; 0.1 0.7* 22.4 &#b1; 1.2 0.9 36.5 &#b1; 18.7 1.4* Topotecan 10.2 &#b1; 2.5 1.0 280.9 &#b1; 30.6 27.5 224.2 &#b1; 12.6 22.0 187.2 &#b1; 19.6 18.4 +A-803467 (2.5 bc;M) 10.5 &#b1; 3.6 0.9 182.3 &#b1; 23.8 17.9 131.4 &#b1; 21.6 12.9 137.7 &#b1; 15.6 13.5 +A-803467 (7.5 bc;M) 9.4 &#b1; 3.7 0.9 18.6 &#b1; 4.6 1.8* 15.3 &#b1; 2.8 1.5* 17.4 &#b1; 3.8 1.7* +FTC (5 bc;M) 9.8 &#b1; 2.8 0.9 19.8 &#b1; 2.5 1.9* 16.4 &#b1; 2.4 1.6* 16.9 &#b1; 1.4 1.6* Cisplatin 2945.8 &#b1; 55.9 1.0 2636.0 &#b1; 94.1 0.9 2566.4 &#b1; 88.2 0.8 2745.6 &#b1; 141.8 0.9 +A-803467 (2.5 bc;M) 2486.7 &#b1; 90.1 0.8 2486.5 &#b1; 125.5 0.8 2478.8 &#b1; 70.6 0.8 2399.4 &#b1; 106.4 0.8 +A-803467 (7.5 bc;M) 2507.6 &#b1; 186.1 0.8 2377.7 &#b1; 125.3 0.8 2378.2 &#b1; 55.5 0.8 2377.7 &#b1; 125.3 0.8 +FTC (5 bc;M) 2641.4 &#b1; 84.2 0.8 2396.2 &#b1; 127.02 0.8 2367.5 &#b1; 27.6 0.9 2347.7 &#b1; 43.5 0.8 Data represents the mean IC50 values for each cell line &#b1; SD obtained from three independent sets of experiments. Login to comment
65 The fold resistance (FR) was determined by dividing the IC50 value of anticancer drug for HEK293/pcDNA3.1, HEK293/R482, HEK293/R482G and HEK293/R482T, in the absence or presence of reversal agents, by the IC50 value of respective anticancer drug for HEK293/pcDNA3.1 in the absence of reversal agent. FTC was used as a positive control of ABCG2 inhibitor Table 3: A-803467 cannot enhance the cytotoxicity of ABCB1 and ABCC10 substrate anticancer agents in HEK293/PCDNA3.1 cells overexpressing ABCB1 and ABCC10 Treatments IC50 &#b1; SD (nM) HEK293/pc DNA3.1 FR HEK293/ ABCB1 RF HEK293/ ABCC10 FR Paclitaxel 8.3 &#b1; 0.2 1.0 525.2 &#b1; 20.1 63.2# 95.2 &#b1; 6.1 11.4# +A-803467 (7.5 bc;M) 7.9 &#b1; 0.4 0.9 453 &#b1; 18.9 54.5 77.4 &#b1; 5.6 9.3 +Verapamil (5 bc;M) 8.2 &#b1; 0.6 1.0 9.5 &#b1; 1.5 1.0 * - - +Cepharanthine (2.5 bc;M) 7.2 &#b1; 0.3 0.8 - - 12.3 &#b1; 2.5 1.4* Data represents the mean IC50 values for each cell line &#b1; SD obtained from three independent sets of experiments. Login to comment
94 A. A-803467 at 7.5 bc;M significantly increased intracellular accumulation of [3 H]-MX in ABCG2-expressing cells HEK293/R482, HEK293/R482G and HEK293/R482T cells. Login to comment
164 Further functional analysis was performed by measuring the intracellular accumulation of [3 H]-MX in wild-type HEK293/R482, mutant HEK293/R482T, and mutant HEK293/R482G cells (Fig. 1A). Login to comment
238 Cell lines and cell culture HEK293/pcDNA3.1, wild-type HEK293/R482, mutant HEK293/R482T and mutant HEK293/R482G cells were established by transfecting HEK293 cell with either the empty pcDNA3.1 vector or pcDNA3.1 vector containing a full-length ABCG2, with coding arginine (R), threonine (T), or glycine (G) at amino acid position 482, respectively, after selection with G418 and maintained in medium with 2 mg/ml of G418 [26]. Login to comment
243 Cells were harvested and resuspended in a final concentration of 6 &#d7; 103 cells/well for HEK293/ pcDNA3.1, HEK/ABCB1, HEK/ABCC10, HEK293/R482, HEK293/R482G and HEK293/R482T cells, and 4 &#d7; 103 cells/well for H460 and H460/MX20 cells. Login to comment
295 Susan E. Bates and Robert W. Robey (NCI, NIH) for providing us HEK293/pcDNA3.1 (parental), HEK293/R482, HEK293/ R482G and HEK293/R482T, H460 and H460/MX20 cell lines. We thank Anna Maria Barbuti (St. John`s University, NY) for her critical reading and editing of the article. Login to comment