ABCMdb

ABCG2 p.Arg482Thr

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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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
89 Early steps of the MCF-7 AdVp selection were found to contain the mutant allele (R482T). 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
88 Early steps of the MCF-7 AdVp selection were found to contain the mutant allele (R482T). 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
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
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
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

[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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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
160 However, the two BCRP-transfected clones used in the previous study (represented by the open diamonds in Fig. 1, B and C) were obtained using cDNA derived from the MCF7/ AdVp cells, before it was known that these cells contain the R482T mutation. 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
159 However, the two BCRP-transfected clones used in the previous study (represented by the open diamonds in Fig. 1, B and C) were obtained using cDNA derived from the MCF7/ AdVp cells, before it was known that these cells contain the R482T mutation. 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
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
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
61 Two internal complementary primer pairs were used with each containing the specific mutation as follows: the Arg- primer pairs were 5Ј-TTATTACCAATGCGCATGTTACC-3Ј and 5Ј-GG- TAACATGCGCATTGGTAATAA-3Ј; the R482T primer pairs were 5Ј-T- TATTACCTATGACGATGTTACC-3Ј and 5Ј-GGTAACATCGTCATAG- GTAATAA-3Ј; and the K86M primer pairs were 5Ј-TGGAGGCATGTC- TTCGTTATT-3Ј and 5Ј-TAATAACGAAGACATGCCTCCA-3Ј, respectively. 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
109 Site-directed mutagenesis was performed on a human ABCG2 cDNA, which possesses Gly at position 482 (4), to create the wild-type (Arg-482) and the R482T and K86M variants of the ABCG2 half-transporter. 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
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
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
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
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
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
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
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
280 In the present experiments a relatively high basal ATPase activity was found in case of the wild-type ABCG2 and the R482T variant, too. 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
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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No. Sentence Comment
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] Single-nucleotide polymorphism (SNP) analysis in t... Cancer Biol Ther. 2002 Nov-Dec;1(6):696-702. Honjo Y, Morisaki K, Huff LM, Robey RW, Hung J, Dean M, Bates SE
Single-nucleotide polymorphism (SNP) analysis in the ABC half-transporter ABCG2 (MXR/BCRP/ABCP1).
Cancer Biol Ther. 2002 Nov-Dec;1(6):696-702., [PMID:12642696]

Abstract [show]
Variations in the amino acid sequence of ABC transporters have been shown to impact substrate specificity. We identified two acquired mutations in ABCG2, the ABC half-transporter overexpressed in mitoxantrone-resistant cell lines. These mutations confer differences in substrate specificity and suggest that naturally occurring variants could also affect substrate specificity. To search for the existence of single nucleotide polymorphisms (SNPs) in ABCG2, we sequenced 90 ethnically diverse DNAs from the Single Nucleotide Polymorphism Discovery Resource representing the spectrum of human genotypes. We identified 3 noncoding SNPs in the untranslated regions, 3 nonsynonymous and 2 synonymous SNPs in the coding region and 7 SNPs in the intron sequences adjacent to the sixteen ABCG2 exons. Nonsynonymous SNPs at nucleotide 238 (V12M; exon 2) and nucleotide 625 (Q141K; exon 5) showed a greater frequency of heterozygosity (22.2% and 10%) than the SNP at 2062 (D620N; exon 16). Heterozygous changes at nucleotide 238 are in linkage disequilibrium with an SNP observed 36 bases downstream from the end of exon 2. No polymorphism at amino acid 482 was identified to correspond to the R to G or R to T mutations previously found in two drug resistant cell lines. Among 23 drug resistant sublines for which sequence at position 482 was determined, no additional mutations were found. Heterozygosity at amino acid 12 allowed us to identify overexpression of a single allele in a subset of drug resistant cell lines, a feature that could be exploited clinically in evaluating the significance of ABCG2 expression in malignancy. We conclude that ABCG2 is well conserved and that described amino acid polymorphisms seem unlikely to alter transporter stability or function.

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103 The doxorubicin-selected subline was cultured in the presence of verapamil to prevent emergence of P-glycoprotein and has been previously described.14 ABCG2 in these cells acquired a mutation at amino acid 482 (R482T) that conferred the ability to transport doxorubicin.9 In contrast, cells selected in epirubicin, which like doxorubicin is a poor ABCG2 substrate, do not overexpress ABCG2. 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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147 It has been reported that the R482G and R482T mutant variants of ABCG2 have altered substrate specificity. Login to comment

[hide] Differential effects of the breast cancer resistan... Cancer Res. 2003 Jun 15;63(12):3228-33. Rajendra R, Gounder MK, Saleem A, Schellens JH, Ross DD, Bates SE, Sinko P, Rubin EH
Differential effects of the breast cancer resistance protein on the cellular accumulation and cytotoxicity of 9-aminocamptothecin and 9-nitrocamptothecin.
Cancer Res. 2003 Jun 15;63(12):3228-33., 2003-06-15 [PMID:12810652]

Abstract [show]
Breast cancer resistance protein (BCRP)/MXR/ABCG2 is a new member of the family of ATP-dependent drug efflux proteins. Whereas overexpression of another member of this family, P-glycoprotein, minimally affects the cytotoxicity of camptothecins (CPTs), overexpression of wild-type as well as certain mutant BCRPs confers resistance to CPT analogues that are used clinically, including topotecan and irinotecan. Relatively little is known regarding the effects of BCRP on other CPT analogues. We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). The results indicate that overexpression of either P-glycoprotein, multidrug resistance protein type 1, or multidrug resistance protein type 2 has little effect on the cytotoxicity of 9-NC or 9-AC. By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC. Furthermore, overexpression of wild-type or mutant BCRP is associated with reduced intracellular accumulation of 9-AC, but not 9-NC. In addition, immunoblotting studies indicate that whereas increased BCRP expression is evident in cells selected for resistance to irinotecan, BCRP expression is not detectable in two different cell lines selected for resistance to 9-NC. Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Furthermore, the results suggest that polar groups at the 9 or 10 position of the CPT A ring facilitate interaction with BCRP and have implications for the clinical development of new CPT analogues.

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5 We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). Login to comment
7 By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC. Login to comment
10 Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Login to comment
24 In this report, we demonstrate that whereas overexpression of Pgp, MRP1, or MRP2 has little effect on the cytotoxicity of either 9-NC or 9-AC, overexpression of either wild-type or mutant R482T BCRP confers resistance to 9-AC, but not to 9-NC. Login to comment
72 HEK293 pcDNA3-10 Human embryonic kidney cells, stably transfected with pcDNA3 control vector 27 HEK293- ABCG2R482-2 HEK293 cells, stably transfected with pcDNA3-BCRP, expressing wild-type BCRP under control of a CMVa promoter 27 HEK293- ABCG2T482-10 HEK293 cells, stably transfected with pcDNA3- BCRPR482T, expressing a mutant form of BCRP with a threonine for arginine substitution at codon 482 27 MDA-MB-231- pcDNA3 Human breast carcinoma cells, stably transfected with pcDNA3 control vector 24 MDA-MB-231- pcDNA3- BCRP(R482T) MDA-MB-231 cells, stably transfected with pcDNA3-BCRP expressing R482T mutant BCRP under control of a CMV promoter 24 and 35 MDCKII MDCK epithelial cells 48 MDCKII-Pgp MDCKII cells, stably transfected with a vector expressing human MDR1 under control of a CMV promoter 49 MDCKII-MRP1 MDCKII cells, stably expressing human MRP1 under control of a CMV promoter 50 MDCKII-MRP2 MDCKII cells, stably expressing human MRP2 under control of a CMV promoter 51 U-937 Human monoblastic leukemia cells 31 U-937-CR U-937 cells selected for resistance to 9-NC 31 U-937-RERC U-937 cells selected for resistance to 9-NC and etoposide 28 RPMI-8402 Human T-cell lymphoblastic leukemia cells 36 CPT-K5 RPMI-8402 cells selected for resistance to irinotecan 36 a CMV, cytomegalovirus. Login to comment
78 Similar studies were done using HEK293 cells overexpressing wild-type BCRP or R482T BCRP and with MDA-MB-231 cells overexpressing R482T BCRP (Table 1). Login to comment
80 As observed previously (27), expression of the R482T protein was slightly lower than that of the wild-type protein in the stable HEK293 transfectants (Fig. 2). Login to comment
81 Similar to previous results (27, 34, 35), overexpression of wild-type or R482T BCRP conferred resistance to both TPT and SN-38 (Fig. 3; Table 3). Login to comment
83 In HEK293 cells expressing wild-type or mutant BCRP, relative resistances to 9-AC were similar to those observed for TPT and SN-38, whereas in MDA-MB-231 cells, R482T BCRP conferred slightly less resistance to 9-AC compared with TPT or SN-38 (Table 3). Login to comment
84 Furthermore, in HEK293 cells, overexpression of the R482T BCRP mutant was associated with levels of resistance that were similar to those observed with the wild-type protein for TPT, SN-38, and 9-AC (Fig. 3; Table 3). Login to comment
85 By contrast, overexpression of either wild-type or R482T BCRP had no effect on cellular sensitivity to 9-NC (Fig. 3; Table 3). Login to comment
91 Cells consisting of vector only transfectants (pcDNA) or stably expressing wild-type (WT) or R482T mutant BCRP were subjected to sequential immunoblotting using BCRP and beta-actin antibodies as described in "Materials and Methods." Login to comment
97 Effects of overexpression of wild-type or R482T mutant BCRP on the cytotoxicity of 9-NC and 9-AC. HEK293 cells consisting of stable transfectants of vector alone (ࡗ) or vectors expressing wild-type (Œ) or mutant (X) BCRP were incubated for 72 h with various concentrations of the indicated compounds. Login to comment
100 3230 DIFFERENTIAL EFFECTS OF BCRP ON 9-AC AND 9-NC American Association for Cancer ResearchCopyright (c) 2003 on May 3, exposure to 10 ␮M 9-AC, intracellular drug levels were about -fold higher in vector controls compared with wild-type or R482T BCRP transfectants (Fig. 4). Login to comment
102 These results are consistent with those obtained from antiproliferative studies and suggest that wild-type and mutant R482T BCRP mediate cellular efflux of 9-AC, but not 9-NC. Login to comment
117 By contrast, overexpression of wild-type or R482T BCRP confers resistance to 9-AC, associated with reduced intracellular accumulation of this drug. Login to comment
119 Similar to results reported for TPT (26, 27), we found that overexpression of the R482T mutant yielded resistance to 9-AC that was similar to that observed with overexpression of the wild-type protein. Login to comment
122 Effects of overexpression of wild-type or R482T mutant BCRP on the intracellular accumulation of 9-NC and 9-AC. HEK293 cells stably transfected with vector alone (pcDNA) or vectors expressing wild-type (WT) or R482T mutant BCRP were incubated with 10 ␮M 9-NC or 9-AC at 37°C for 30 min. Login to comment
131 Table 3 Antiproliferative effects of camptothecin analogues in the presence and absence of forced expression of wild-type or mutant BCRP Drug HEK293 HEK293 BCRP RRa HEK293 BCRP R482T RRa MDA-MB-231 MDA-MB-231 BCRP R482T RR TPT 0.034 Ϯ 0.01b 0.39 Ϯ 0.19c 11.5 0.40 Ϯ 0.16c 11.8 0.04 Ϯ 0.01 1.13 Ϯ 0.09c 28 SN-38 0.022 Ϯ 0.00 0.17 Ϯ 0.08c 7.7 0.14 Ϯ 0.02c 6.4 0.10 Ϯ 0.03 3.17 Ϯ 0.15c 32 9-AC 0.033 Ϯ 0.01 0.24 Ϯ 0.13c 7.3 0.40 Ϯ 0.13c 12 0.22 Ϯ 0.07 1.56 Ϯ 0.51c 7.1 9-NC 0.030 Ϯ 0.01 0.03 Ϯ 0.01 1 0.03 Ϯ 0.01 1 0.24 Ϯ 0.02 0.32 Ϯ 0.17 1.4 a RR, relative resistance of BCRP-expressing cell line compared with control cell line. b Results are expressed as mean-SDs (␮M) for calculated IC50s for six replicate experiments. Login to comment
8 We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptoth- ecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T). Login to comment
13 Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC. Login to comment
27 In this report, we demonstrate that whereas overexpression of Pgp, MRP1, or MRP2 has little effect on the cytotoxicity of either 9-NC or 9-AC, overexpression of either wild-type or mutant R482T BCRP confers resistance to 9-AC, but not to 9-NC. Login to comment
75 HEK293 pcDNA3-10 Human embryonic kidney cells, stably transfected with pcDNA3 control vector 27 HEK293- ABCG2R482-2 HEK293 cells, stably transfected with pcDNA3-BCRP, expressing wild-type BCRP under control of a CMVa promoter 27 HEK293- ABCG2T482-10 HEK293 cells, stably transfected with pcDNA3- BCRPR482T, expressing a mutant form of BCRP with a threonine for arginine substitution at codon 482 27 MDA-MB-231- pcDNA3 Human breast carcinoma cells, stably transfected with pcDNA3 control vector 24 MDA-MB-231- pcDNA3- BCRP(R482T) MDA-MB-231 cells, stably transfected with pcDNA3-BCRP expressing R482T mutant BCRP under control of a CMV promoter 24 and 35 MDCKII MDCK epithelial cells 48 MDCKII-Pgp MDCKII cells, stably transfected with a vector expressing human MDR1 under control of a CMV promoter 49 MDCKII-MRP1 MDCKII cells, stably expressing human MRP1 under control of a CMV promoter 50 MDCKII-MRP2 MDCKII cells, stably expressing human MRP2 under control of a CMV promoter 51 U-937 Human monoblastic leukemia cells 31 U-937-CR U-937 cells selected for resistance to 9-NC 31 U-937-RERC U-937 cells selected for resistance to 9-NC and etoposide 28 RPMI-8402 Human T-cell lymphoblastic leukemia cells 36 CPT-K5 RPMI-8402 cells selected for resistance to irinotecan 36 a CMV, cytomegalovirus. Login to comment
86 In HEK293 cells expressing wild-type or mutant BCRP, relative resistances to 9-AC were similar to those observed for TPT and SN-38, whereas in MDA-MB-231 cells, R482T BCRP conferred slightly less resistance to 9-AC compared with TPT or SN-38 (Table 3). Login to comment
87 Furthermore, in HEK293 cells, overexpression of the R482T BCRP mutant was associated with levels of resistance that were similar to those observed with the wild-type protein for TPT, SN-38, and 9-AC (Fig. 3; Table 3). Login to comment
88 By contrast, overexpression of either wild-type or R482T BCRP had no effect on cellular sensitivity to 9-NC (Fig. 3; Table 3). Login to comment
94 Cells consisting of vector only transfectants (pcDNA) or stably expressing wild-type (WT) or R482T mutant BCRP were subjected to sequential immunoblotting using BCRP and beta-actin antibodies as described in "Materials and Methods." Login to comment
103 3230 DIFFERENTIAL EFFECTS OF BCRP ON 9-AC AND 9-NC exposure to 10 ␮M 9-AC, intracellular drug levels were about 2-fold higher in vector controls compared with wild-type or R482T BCRP transfectants (Fig. 4). Login to comment
105 These results are consistent with those obtained from antiproliferative studies and suggest that wild-type and mutant R482T BCRP mediate cellular efflux of 9-AC, but not 9-NC. Login to comment
120 By contrast, overexpression of wild-type or R482T BCRP confers resistance to 9-AC, associated with reduced intracellular accumulation of this drug. Login to comment
125 Effects of overexpression of wild-type or R482T mutant BCRP on the intracellular accumulation of 9-NC and 9-AC. HEK293 cells stably transfected with vector alone (pcDNA) or vectors expressing wild-type (WT) or R482T mutant BCRP were incubated with 10 ␮M 9-NC or 9-AC at 37°C for 30 min. Login to comment
134 Table 3 Antiproliferative effects of camptothecin analogues in the presence and absence of forced expression of wild-type or mutant BCRP Drug HEK293 HEK293 BCRP RRa HEK293 BCRP R482T RRa MDA-MB-231 MDA-MB-231 BCRP R482T RR TPT 0.034 Ϯ 0.01b 0.39 Ϯ 0.19c 11.5 0.40 Ϯ 0.16c 11.8 0.04 Ϯ 0.01 1.13 Ϯ 0.09c 28 SN-38 0.022 Ϯ 0.00 0.17 Ϯ 0.08c 7.7 0.14 Ϯ 0.02c 6.4 0.10 Ϯ 0.03 3.17 Ϯ 0.15c 32 9-AC 0.033 Ϯ 0.01 0.24 Ϯ 0.13c 7.3 0.40 Ϯ 0.13c 12 0.22 Ϯ 0.07 1.56 Ϯ 0.51c 7.1 9-NC 0.030 Ϯ 0.01 0.03 Ϯ 0.01 1 0.03 Ϯ 0.01 1 0.24 Ϯ 0.02 0.32 Ϯ 0.17 1.4 a RR, relative resistance of BCRP-expressing cell line compared with control cell line. b Results are expressed as mean-SDs (␮M) for calculated IC50s for six replicate experiments. 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
25 However, a puzzling feature of this study was that MCF7 cells stably transfected with an ABCG2 cDNA, which has since been determined to have an R482T mutation, did not recapitulate MTX resistance. 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
91 For authentic BCRP in vitro-transcribed RNA (cRNA) R482 and R482T standards, the Tms of the hybridization probes are 62°C and 57.2°C, respectively. 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

[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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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
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
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
160 A Japanese study also suggested that crossresistance to mitoxantrone was relatively high in cell lines transfected with BCRP R482T as compared with those transfected with BCRP R482 (Komatani et al., 2001). 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
163 However, MCF-7/AdrVp cells, which overexpress the R482T mutation of BCRP and display considerable resistance to daunorubicin (25-40-fold) (Ross et al., 1995; Doyle et al., 1998), accumulate and retain idarubicin well, and are only minimally resistant to idarubicin (1.8-fold) (Ross et al., 1995). Login to comment
166 However, in this same work, investigations of the BCRP-transfected MCF-7 or MDAMB-231 cells developed in our laboratory did not find resistance or low accumulation of methotrexate in the transfected cells. Since these MCF-7 or MDAMB231 cells were transfected with BCRP cDNA derived from MCF-7/AdrVp cells, the transfected cells consequently overexpressed the R482T mutation of BCRP. 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] Taxane-based reversal agents modulate drug resista... Mol Cancer Ther. 2003 Nov;2(11):1195-205. Brooks TA, Minderman H, O'Loughlin KL, Pera P, Ojima I, Baer MR, Bernacki RJ
Taxane-based reversal agents modulate drug resistance mediated by P-glycoprotein, multidrug resistance protein, and breast cancer resistance protein.
Mol Cancer Ther. 2003 Nov;2(11):1195-205., [PMID:14617793]

Abstract [show]
Overexpression of ATP-binding cassette transport proteins, including P-glycoprotein (Pgp), multidrug resistance (MDR) protein (MRP-1), and breast cancer resistance protein (BCRP), is a well-characterized mechanism of MDR in tumor cells. Although the cytotoxic taxanes paclitaxel and docetaxel are substrates for Pgp-mediated efflux, the semisynthetic taxane analogue ortataxel inhibits drug efflux mediated by Pgp as well as, as we recently demonstrated, MRP-1 and BCRP. Nevertheless, ortataxel is not optimal for development as a clinical MDR modulator because of its cytotoxicity [corrected]. We sought to identify noncytotoxic taxane-based broad-spectrum modulators from a library of noncytotoxic taxane-based reversal agents (tRAs) designed by eliminating the C-13 side chain of the taxane molecule, which inhibits microtubule depolymerization. Twenty tRAs, selected based on modulation of paclitaxel cytotoxicity in Pgp-overexpressing MDA435/LCC6(mdr1) cells, were studied for modulation of retention and cytotoxicity of substrates of MRP-1 and BCRP as well as Pgp in established cell lines overexpressing each of these transporters. Four tRAs modulated MRP-1 and 17 modulated BCRP in addition to Pgp. The four broad-spectrum tRAs strongly modulated daunorubicin and mitoxantrone efflux and enhanced their cytotoxicity in cell lines overexpressing the three MDRs, decreasing IC(50) values by as much as 97% [corrected]. These tRAs, especially tRA 98006, have promise for development as clinical broad-spectrum MDR modulators and warrant more preclinical analysis to determine pharmacokinetic interactions and efficacy.

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60 MRP-1 expression in cell lines Cell line Pgp MRP-1 BCRP HL60-wt À À À HL60-ADR À + À 8226-wt À À + 8226-Dox6 + À + 8226-MR20 À À + MCF7/S À À + MCF7/R + À + MCF7/MRP1-10 À + + MCF7/AdrVp À À +a a R482T mutation in BCRP. 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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0 Functional Characterization of Human Breast Cancer Resistance Protein (BCRP, ABCG2) Expressed in the Oocytes of Xenopus laevis TAKEO NAKANISHI, L. AUSTIN DOYLE, BRET HASSEL, YUETONG WEI, KENNETH S. BAUER, SUHLAN WU, DAVID W. PUMPLIN, HONG-BIN FANG, and DOUGLAS D. ROSS The Program in Experimental Therapeutics (T.N., L.A.D., B.H., Y.W., K.S.B., S.W.) and Division of Biostatistics (H.-B.F.), University of Maryland Greenebaum Cancer Center, the Division of Hematology and Oncology, Departments of Medicine (T.N., L.A.D., D.D.R.), Anatomy and Neurobiology (D.W.P.), Microbiology (B.H.), and Epidemiology and Preventative Medicine (H.-B.F.), University of Maryland School of Medicine, Baltimore Maryland; School of Pharmacy, University of Maryland, Baltimore, Baltimore, Maryland (K.S.B.); and the Baltimore Veterans Administration Medical Center, Baltimore Maryland (D.D.R.) Received May 16, 2003; accepted August 26, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT 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. Login to comment
2 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. Login to comment
5 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. Login to comment
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
57 The PCR product was ligated into the pCR Blunt TOPO II TABLE 1 Summary of cDNA constructs made as templates for producing BCRP cRNA Construct Plasmid Features of the BCRP cRNA Produced RNA Polymerase Consensus Sequence Upstream from BCRP cDNA I pSP64poly(A) R482T-poly(A) SP6 II pCR Blunt TOPO II Kozak-R482T-poly(A) T7 III pSD64TR 5ЈUTR-Kozak-R482T-3ЈUTR-poly(A) other BCRP forms: 5ЈUTR-Kozak-R482-3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/S187T- 3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/ S187A-3ЈUTR-poly(A) SP6 III- pSD64TR 5ЈUTR-R482T-3ЈUTR-poly(A) SP6 5ЈUTR, 3ЈUTR, portions of the 5Ј- and 3Ј-UTR of the Xenopus laevis beta-globin gene; Kozak, modified sequences proximate to the start codon, as described under Materials and Methods; Poly(A), addition of a poly(A) tail, as described under Materials and Methods. Login to comment
105 Oocytes injected with water or BCRP cRNA (wild type, R482T) were fixed in 4% paraformaldehyde in PBS, then immersed overnight in 30% sucrose in PBS. Login to comment
116 However, injection of oocytes with BCRP cRNA directly transcribed from BCRP (R482T) cDNA (Doyle et al., 1998) failed to produce BCRP protein, as detected by Western blot examination or by functional assays. Login to comment
128 Oocytes were injected with cRNA coding for the wild-type (R482) or mutant R482T form of BCRP, then fixed and sectioned as described under Materials and Methods. Login to comment
129 Exposure of these sections to the BXP-34 monoclonal antibody to BCRP resulted in immunoreactivity only in the plasma membranes of oocytes expressing wild-type (Fig. 2A) or the mutant R482T form of BCRP (Fig. 2B), as detected by immunofluorescence microscopic analysis. Login to comment
132 Functional expression of BCRP (R482T) was examined by 10 ␮M DNR accumulation (A) or Western blot (B) in oocytes injected with 50 nl of water (control) or cRNA (1 ␮g/␮l) transcribed from construct I, II, or III. Login to comment
133 For convenience, the R482T form of BCRP was used to enable monitoring of function by DNR accumulation. Login to comment
140 Western blot examination of oocytes injected with wild-type or BCRP (R482T) cRNA at the time of the immunofluorescence experiments confirmed robust expression of BCRP, with a molecular mass of 70 kDa (Fig. 2D). Login to comment
141 Accumulation and Efflux of MX or DNR in Oocytes Expressing BCRP (R482T). Login to comment
142 To determine whether functional BCRP was expressed in X. laevis oocytes, we monitored the accumulation of DNR and [3 H]MX in control or BCRP(R482T) injected oocytes. Login to comment
144 Oocytes expressing BCRP (R482T) showed a remarkable reduction in the accumulation of DNR or MX over the 120-min period. Login to comment
147 To examine whether the diminished accumulation of DNR or MX in the BCRP (R482T)-expressing oocytes was caused by enhanced drug efflux, the efflux of these compounds in the presence or absence of 5 ␮M FTC was compared with that of control oocytes. Login to comment
148 Before the efflux studies, the drug of interest was preloaded into the control or BCRP (R482T)-expressing oocytes by incubating with drug for 90 min. Login to comment
149 In the case of BCRP (R482T)-expressing oocytes, 5 ␮M FTC was added, which resulted in intracellular drug accumulation comparable with levels attained in the control (water injected) oocytes; after preloading and subtraction of background, the accumulation of DNR in BCRP (R482T)-injected oocytes was 3.79 Ϯ 0.15 pmol/oocyte, whereas that in control was 4.55 Ϯ 0.31 pmol/oocyte. Login to comment
150 [3 H]MX accumulation in control and preloaded R482T-expressing oocytes was 0.87 Ϯ 0.16 and 0.96 Ϯ 0.91 pmol/oocyte, respectively. Login to comment
151 Both DNR and MX were effluxed more rapidly from the BCRP (R482T)-expressing oocytes, compared with control (Fig. 3, D and E). Login to comment
152 This rapid efflux was not observed in R482T-expressing oocytes in the presence of FTC. Login to comment
156 Substrate Specificity of Wild-Type and Mutant (R482T) BCRP Expressed in X. laevis Oocytes. Login to comment
157 Accumulation assays using DNR, MX, FLV, and Rho123 were performed to study the effect of the Arg-to-Thr mutation at codon 482 on the substrate specificity of BCRP in the X. laevis oocyte system. Login to comment
158 Figure 4 shows the intracellular accumulation of the four compounds in oocytes injected with R482 or R482T BCRP in the absence or presence of FTC. Login to comment
159 Accumulation of all four compounds in the BCRP (R482T)-expressing oocytes was markedly reduced. Login to comment
162 A Mutation in the ABC Signature Motif Inactivates BCRP (R482T) Transport of DNR; BCRP Expressed in X. laevis Oocytes Functions as a Homodimer or Homomultimer. Login to comment
163 To determine whether dimerization is required for BCRP activity, a mutant construct was created by substituting the highly conserved serine at residue 187 in the ABC signature motif with threonine (R482T/S187T) or alanine (R482T/S187A). Login to comment
165 As shown in Fig. 5A, no difference was observed in expression levels of BCRP protein among oocytes injected with cRNA encoding BCRP (R482T) or these codon 187 mutant BCRP constructs. Login to comment
167 Confocal immunofluorescence microscopic analysis was performed in oocytes injected with 50 nl of cRNA of BCRP (wild-type, R482) (A), BCRP (R482T) (B), or water as control (C), using the BXP-34 antibody. Login to comment
168 A and B show the distribution of BCRP (R482 or R482T) at the oocyte surface (C). Login to comment
170 Cell lysate (25 ␮g) from the oocytes injected with water (control), BCRP (R482), or BCRP (R482T) cRNA was loaded per lane of the gel. Login to comment
173 Accumulation of 25 ␮M DNR (A), 10.8 ␮M MX (B) or varying concentrations of DNR (C, DNR: 10 to 250 ␮M) in control (E) or BCRP (R482T)-expressing oocytes (F). Login to comment
174 Efflux of DNR (D) or MX (E) from control (E) or BCRP (R482T)-expressing oocytes (F) afterpreloading with DNR or MX in the presence of 5 ␮M FTC was monitored at room temperature, as described under Materials and Methods. Login to comment
175 Œ, drug efflux in BCRP (R482T)-expressing oocytes in the presence of 5 ␮M FTC. Login to comment
178 *, statistically significant difference (p Ͻ 0.05, student`s t test) between control and R482T-expressing oocytes. Login to comment
180 a manner analogous to a dominant-negative mutation, if coinjected with the active BCRP (R482T) cRNA. Login to comment
181 Indeed, when BCRP (R482T) was coinjected with varying amounts of the "dominant-negative" construct S187T/R482T, the transport of DNR was significantly inhibited in a manner dependent on the amount of the S187T mutant construct added (Fig. 5C), strongly suggesting that homodimerization or multimerization is essential for BCRP activity. Login to comment
185 First, DNR accumulation in BCRP (R482T)-expressing oocytes was studied (Fig. 6A). Login to comment
188 In contrast, MX and Rho123 only partially inhibited DNR transport by BCRP (R482T), and 10-fold molar excess of TPT was not inhibitory at all. Login to comment
189 When BCRP substrate inhibition of MX accumulation was studied (Fig. 6B) using a 23-fold molar excess of Rho123, TPT, DNR, or FLV as competitors, only FLV caused partial but statistically significant reversal of BCRP R482 or R482T transport of MX. Login to comment
190 Interestingly, when the competition studies were done with respect to FLV accumulation (Fig. 6C), none of the competing BCRP substrates was able to inhibit FLV efflux by wild-type or R482T BCRP, despite their presence in a 10-fold or, in the case of DNR, a 100-fold molar excess relative to FLV. Login to comment
193 Accumulation of BCRP substrates DNR (25 ␮M, A), MX (0.5 ␮M, B), FLV (25 ␮M, C), and Rho123 (5 ␮M, D) was measured in control (Ⅺ), BCRP (R482T)-expressing oocytes (f) or BCRP (R482, wild-type)-expressing oocytes (u). Login to comment
199 Expression of dominant-negative construct was confirmed by Western blot (A) and by DNR accumulation (25 ␮M, B) in control or R482T-expressing oocytes (f), R482T/S187T- expressing oocytes (o), or R482T/S187A-expressing oocytes (gray o). Login to comment
201 DNR accumulation (25 ␮M, C) was determined in oocytes coinjected with 24 ng of R482T cRNA and different amounts (0 to 72 ng) of the R482T/S187T construct cRNA (f). Login to comment
202 As control, the accumulation of 25 ␮M DNR was also determined in control (Ⅺ) or oocytes injected with 24 ng of R482T/S187T cRNA only (o). Login to comment
204 *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001 represent statistically significant differences (Student`s t test) in coinjected oocytes compared with accumulation in oocytes injected with R482T cRNA only. Login to comment
207 Our results further demonstrate that oocytes injected with mutant R482T or wild-type BCRP cRNA express BCRP in the oocyte plasma membrane, as evidenced by confocal immunofluorescence microscopic analysis. Login to comment
212 Validation of the assay is based our observation that the function of BCRP expressed in the oocytes parallels that observed in mammalian systems (Doyle et al., 1998; Honjo et al., 2001; Robey et al., 2001; Minderman et al., 2002); the R482T mutant form of BCRP expressed in the oocytes efficiently transported DNR, Rho123, MX, and FLV, whereas the oocyte-expressed wild-type form transported only MX and FLV. Login to comment
213 Furthermore, FTC, in concentrations known to inhibit BCRP function in mammalian systems (Rabindran et al., 2000) also caused complete inhibition of both mutant R482T and wild-type BCRP forms expressed in the oocytes. Login to comment
216 In our studies, the diminished accumulation of DNR or MX displayed by BCRP R482T-expressing oocytes correlated with greater initial efflux rates of these drugs compared with control oocytes. Login to comment
224 Ⅺ, accumulation of these drugs in control oocytes for each condition (100%); f, accumulation in BCRP (R482T)-expressing oocytes; u, accumulation in BCRP (R482, wild-type)-expressing oocytes. Login to comment
233 Hence, we made two mutations of serine 187 in this signature motif of BCRP R482T, one to an amino acid that, like serine, has a polar side chain (threonine, S187T) and the other to a nonpolar side chain amino acid (alanine, S187A). Login to comment
234 The R482T mutant form of BCRP was used because it allowed us to monitor BCRP function by DNR accumulation. Login to comment
239 When the S187T mutation was coexpressed with BCRP R482T in the oocytes, the S187T mutant protein acted as would a dominant-negative inhibitor of BCRP transport of DNR, with the degree of inhibition increasing in proportion to the amount of S187T cRNA injected. Login to comment
249 In the substrate interaction studies in BCRP expressing X. laevis oocytes presented here (Fig. 6 and Table 2), we found a significant inhibitory effect of FLV on BCRP-mediated MX transport, in good agreement with Robey et al. (2001); furthermore, FLV and MX significantly inhibited DNR accumulation in R482T-expressing oocytes. Login to comment
255 It is possible that BCRP also has multiple substrate interaction sites; however, because we did TABLE 2 Summary of substrate interaction studies Substrate Competitor Inhibitor Substrate (BCRP form) DNR MX FLV TPT Rho123 FTC DNR (R482T) Partial Yes No Ϯ Yes MX (W or R482T) No Partial No No Yes FLV (W or R482T) No No No N.D. Yes W, wild type; R482T, BCRP R482T; No, no inhibition by competitor; Yes, inhibition by competitor, P Ͻ 0.01; Partial, partial (approximately 50%) inhibition by competitor, P Ͻ 0.01; Ϯ, slight (approximately 20%) inhibition by competitor, P Ͻ 0.05; N.D., no data. 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 overexpression in colon cancer cells resista... Int J Cancer. 2004 May 10;109(6):848-54. Candeil L, Gourdier I, Peyron D, Vezzio N, Copois V, Bibeau F, Orsetti B, Scheffer GL, Ychou M, Khan QA, Pommier Y, Pau B, Martineau P, Del Rio M
ABCG2 overexpression in colon cancer cells resistant to SN38 and in irinotecan-treated metastases.
Int J Cancer. 2004 May 10;109(6):848-54., 2004-05-10 [PMID:15027118]

Abstract [show]
Overcoming drug resistance has become an important issue in cancer chemotherapy. Among all known mechanisms that confer resistance, active efflux of chemotherapeutic agents by proteins from the ATP-binding cassette family has been extensively reported. The aim of the present study was to determine the involvement of ABCG2 in resistance to SN38 (the active metabolite of irinotecan) in colorectal cancer. By progressive exposure to increasing concentrations of SN38, we isolated 2 resistant clones from the human colon carcinoma cell line HCT116. These clones were 6- and 53-fold more resistant to SN38 than the HCT116-derived sensitive clone. Topoisomerase I expression was unchanged in our resistant variants. The highest resistance level correlated with an ABCG2 amplification. This overexpression was associated with a marked decrease in the intracellular accumulation of SN38. The inhibition of ABCG2 function by Ko143 demonstrated that enhanced drug efflux from resistant cells was mediated by the activity of ABCG2 protein and confirmed that ABCG2 is directly involved in acquired resistance to SN38. Furthermore, we show, for the first time in clinical samples, that the ABCG2 mRNA content in hepatic metastases is higher after an irinotecan-based chemotherapy than in irinotecan-naive metastases. In conclusion, this study supports the potential involvement of ABCG2 in the development of irinotecan resistance in vivo.

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127 The lack of cross-resistance to doxorubicin has been explained by a single mutation at codon 482 (doxorubicin is poorly transported by the wild-type variant of ABCG2 in which the amino acid at position 482 is an arginine but can be transported by the R482T mutant32). 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] Arginine482 to threonine mutation in the breast ca... Biochem J. 2004 Sep 1;382(Pt 2):711-6. Alqawi O, Bates S, Georges E
Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding.
Biochem J. 2004 Sep 1;382(Pt 2):711-6., 2004-09-01 [PMID:15139851]

Abstract [show]
ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells. This mutation was shown to modulate the transport of Rh123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482. In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.

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1 J. (2004) 382, 711-716 (Printed in Great Britain) 711 Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding Omar ALQAWI*, Susan BATES† and Elias GEORGES*1 *Institute of Parasitology, McGill University, Macdonald Campus, Ste-Anne de Bellevue, Quebec, Canada H9 X3V9, and †Cancer Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, U.S.A. ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. Login to comment
3 Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/ AdrVp1000 cells. Login to comment
26 Analysis of ABCG2 cDNA sequences from these cell lines revealed mutations at position 482: Arg482 to Thr in MCF-7/ AdrVp1000 cells, and to Gly in S1-M1-81 cells. Login to comment
139 For example, it is plausible that the Arg482 to Thr change in the third transmembrane domain affects ABCG2 protein interactions (either homo- or hetero-protein interactions), which in turn leads to a gain in transport function. 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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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

[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] 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
36 Given that BCRP-mediated drug resistance profile partially overlaps those associated with Pgp or MRP, and that additional unidentified mechanisms likely play a role in resistance to GO,3,8 we investigated whether BCRP (wild type or R482T) could confer resistance to GO or to the unconjugated calicheamicin-g1 derivative. Login to comment
37 For this purpose, lentiviral pRRLsin.cPPT.MSCV.BCRP-IRES-EGFP vectors were generated by cloning BCRP cDNA (wild type or R482T; provided by DD Ross, University of Maryland, Baltimore, MD, USA) into pRRLsin.cPPT.MSCV as a BCRP-IRES-EGFP cassette. Login to comment
43 In comparison, lentivirus-mediated transduction of wild-type or R482T BCRP at an MOI of up to 100 yielded stable subpopulations of cells with significant BCRP protein expression and activity (Figure 1). Login to comment
49 As expected,4,5 cells overexpressing either wild-type or R482T BCRP were resistant to mitoxantrone, and coincubation with Ko143 restored sensitivity (Tables 1 and 2). Login to comment
50 In contrast, overexpression of either wild-type or R482T BCRP did not alter GOor calicheamicin-g1-induced cytotoxicities, and Ko143 had no effect on drug susceptibilities (Tables 1 and 2; note that R482T-transduced HL-60 cells were not established). Login to comment
53 They further suggest that calicheamicin-g1 is not a substrate for either wild-type or R482T BCRP. Login to comment
56 Figure 1 BCRP wild-type and R482T protein expression and activity in parental and transduced CD33þ hematopoietic cell lines. Login to comment
59 For this assay, cells were incubated for 30 min at 371C in the dark in 20 mM mitoxantrone in the presence or absence of 200 nM Ko143 (for R482T transduced NB4 and ML-1 cells) or 600 nM Ko143 (for all other transduced cells). Login to comment
61 HL-60 cells were not infected with R482T BCRP (ND). Login to comment
90 Table 2 Effect of BCRP R482T on cytotoxity induced by mitoxantrone, calicheamicin-g1, and GO MSCV-EGFP BCRP/R482T +DMSO +Ko143 +DMSO +Ko143 U937 Mitoxantrone 25 nM 37.2724.1 (5) 51.7724.7 (5) 3.571.8 (5) 33.7719.3 (5) 100 nM 49.6724.0 (5) 61.0722.7 (5) 8.176.1 (5) 41.4722.0 (5) Calicheamicin 2.5 ng/ml 65.5711.2 (3) 70.578.3 (3) 60.577.4 (3) 62.675.2 (3) 10 ng/ml 87.770.8 (3) 89.270.6 (3) 86.872.2 (3) 86.473.3 (3) GO 2.5 ng/ml 13.672.7 (2) 15.073.4 (2) 21.573.5 (2) 20.076.0 (2) 40 ng/ml 27.772.8 (2) 26.070.6 (2) 33.476.1 (2) 35.7710.9 (2) ML-1 Mitoxantrone 2.5 nM 16.078.4 (3) 16.478.6 (3) 1.670.9 (3) 12.577.8 (3) 10 nM 25.275.5 (3) 26.073.9 (3) 6.574.0 (3) 28.272.0 (3) Calicheamicin 1 ng/ml 27.875.4 (2) 18.6715.6 (2) 38.073.6 (2) 34.973.3 (2) 10 ng/ml 69.173.5 (2) 61.377.3 (2) 74.673.0 (2) 73.873.1 (2) GO 0.1 ng/ml 4.672.4 (3) 4.170.8 (2) 12.877.0 (3) 6.871.9 (2) 0.5 ng/ml 33.275.4 (3) 31.273.0 (2) 31.077.7 (3) 27.675.6 (2) NB4 Mitoxantrone 10 nM 27.9713.3 (5) 29.6714.1 (5) 3.271.2 (5) 27.2715.8 (5) 25 nM 52.9716.2 (5) 65.2720.0 (5) 9.473.0 (5) 52.6730.1 (5) Calicheamicin 1 ng/ml 57.2711.5 (3) 54.176.1 (3) 60.077.1 (3) 61.176.9 (3) 10 ng/ml 90.670.6 (3) 91.370.6 (3) 92.770.8 (3) 91.471.5 (3) GO 2.5 ng/ml 9.673.2 (2) 8.971.7 (2) 10.070.7 (2) 9.870.9 (2) 40 ng/ml 29.974.2 (2) 30.373.1 (2) 31.873.8 (2) 31.976.8 (2) Empty-vector-transduced cells (MSCV-EGFP) and BCRP R482T vector-transduced cells (at MOI 10: NB4 cells, or MOI 100: U937 and ML-1 cells) were treated with various concentrations of mitoxantrone, calicheamicin-g1, or GO in the presence or absence of Ko143 at the predetermined optimal concentration (200 nM for NB4 and ML-1 cells; 600 nM for U937 cells) or vehicle (DMSO) for 72 h prior to the determination of cytotoxicity by flow cytometry using PI with duplicate or triplicate measurements. Values represent percentages of PI+ cells compared to corresponding cells treated with DMSO vehicle or Ko143 only. Results for relevant concentrations of the cytotoxic drugs are shown as mean7s.e.m. with the number of independent experiments denoted within parentheses. Login to comment
91 R482T BCRP-transduced HL-60 cells were not established. 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] 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
183 In contrast, the R482T mutation may play a critical role in this decreased MXR retention, rather than in the increased velocity of the transporter. 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
85 Microsomal membranes were isolated from transiently cotransfected-infected HeLa cells, drug-sensitive MCF-7 cells, drug-resistant MCF-7/AdVp3000, which overexpress the R482T variant of human ABCG2, and drug resistant MCF-7/DX1 cells, which overexpress human P-gp. 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
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
201 A, 50 ␮g of membrane protein from MCF-7 or MCF-7/AdVp3000 cells expressing the R482T variant of ABCG2. 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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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
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

[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] 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] Gefitinib ("Iressa", ZD1839), an epidermal growth ... Cancer Res. 2005 Feb 15;65(4):1541-6. Nakamura Y, Oka M, Soda H, Shiozawa K, Yoshikawa M, Itoh A, Ikegami Y, Tsurutani J, Nakatomi K, Kitazaki T, Doi S, Yoshida H, Kohno S
Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance.
Cancer Res. 2005 Feb 15;65(4):1541-6., 2005-02-15 [PMID:15735043]

Abstract [show]
Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.

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42 The transfectants have a mutant form of BCRP (R482T; ref. 25). 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
120 Similar migration patterns are observed for wild-type ABCG2 and the R482T variant (data not shown). Login to comment
158 In the stable drug-selected cell line MCF-7/AdVp3000 that overexpresses ABCG2 (R482T), the protein also migrates as a single species but at a higher molecular mass closer to the 75 kDa marker (Figure 4B, lane 1). Login to comment
183 (B) Crude membranes derived from MCF-7/AdVp3000 cells (10 µg) overexpressing ABCG2 (R482T) were treated with (+) or without (-) PNGase F. Login to comment
214 DISCUSSION Our studies demonstrate that ABCG2 transiently expressed in HeLa cells and ABCG2 (R482T) overexpressed in the drug-selected cell line, MCF-7/AdVp3000, are N-linked glycosylated. 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
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
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
174 Outside of cell Cell membrane C terminus ATP-binding site N terminus Inside of cellGln141Lys Walker A Walker B Signature motif Arg482Thr Val12Met Table 5. 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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10 The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Login to comment
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
40 Interestingly, the R482T mutation limited this prenylation-dependent effect, whereas the inhibitory potency of tectochrysin was even increased, especially towards rhodamine transport. Login to comment

[hide] Chronic imatinib mesylate exposure leads to reduce... Cancer Biol Ther. 2005 Jul;4(7):747-52. Epub 2005 Jul 9. Burger H, van Tol H, Brok M, Wiemer EA, de Bruijn EA, Guetens G, de Boeck G, Sparreboom A, Verweij J, Nooter K
Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps.
Cancer Biol Ther. 2005 Jul;4(7):747-52. Epub 2005 Jul 9., [PMID:15970668]

Abstract [show]
Imatinib mesylate is a selective tyrosine kinase inhibitor that is successfully used in the treatment of Philadelphia-positive chronic and acute leukaemia's, and gastrointestinal stromal tumors. We investigated whether the intended chronic oral administration of imatinib might lead to the induction of the intestinal ABC transport proteins ABCB1, ABCC1 (MRP1), ABCC2 (MRP2) and ABCG2. Using Caco2 cells as an in vitro model for intestinal drug transport, we found that continuous exposure (up to 100 days) with imatinib (10 microM) specifically upregulates the expression of ABCG2 (maximal approximately 17-fold) and ABCB1 (maximal approximately 5-fold). The induction of gene expression appeared to be biphasic in time, with a significant increase in ABCG2 and ABCB1 at day 3 and day 25, respectively, and was not mediated through activation of the human orphan nuclear receptor SXR/NR1I2. Importantly, chronic imatinib exposure of Caco2 cells resulted in a approximately 50% decrease in intracellular accumulation of imatinib, probably by enhanced ABCG2- and ABCB1-mediated efflux, as a result of upregulated expression of these drug pumps. Both ABCG2 and ABCB1 are normally expressed in the gastrointestinal tract and it might be anticipated that drug-induced upregulation of these intestinal pumps could reduce the oral bioavailability of imatinib, representing a novel mechanism of acquired pharmacokinetic drug resistance in cancer patients that are chronically treated with imatinib.

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25 Cell lines used: Caco2 parental, a human colon carcinoma cell line (ATCC, HTB-37); MCF-7 parental, a human breast carcinoma cell line (ATCC, HTB-22); MCF-7/MR, a mitoxantrone resistant counterpart of MCF-7 overexpressing wild-type ABCG2;11 MCF-7/AdVp3000, an MCF-7 derivative overexpressing the R482T variant of BCRP; KB-3-1, a human epidermoid carcinoma cell line (ATCC CCL-17) and the colchicine-selected derivative KB-8-5 overexpressing ABCB1;12 COS-1, an African green monkey cell line (ATCC, CRL-1650), and HEP3B, a hepatocarcinoma cell line (ATCC HB-8064). Login to comment
26 Cell lines used: Caco2 parental, a human colon carcinoma cell line (ATCC, HTB-37); MCF-7 parental, a human breast carcinoma cell line (ATCC, HTB-22); MCF-7/MR, a mitoxantrone resistant counterpart of MCF-7 overexpressing wild-type ABCG2;11 MCF-7/AdVp3000, an MCF-7 derivative overexpressing the R482T variant of BCRP; KB-3-1, a human epidermoid carcinoma cell line (ATCC CCL-17) and the colchicine-selected derivative KB-8-5 overexpressing ABCB1;12 COS-1, an African green monkey cell line (ATCC, CRL-1650), and HEP3B, a hepatocarcinoma cell line (ATCC HB-8064). 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
52 MCF-7 cells (breast adenocarcinoma) selected and maintained in 3 µg/ mL doxorubicin and 5 µg/mL verapamil (MCF-7/AdVp3000) overexpress ABCG2 (R482T) at the plasma membrane. Login to comment
173 Because the cross-linker is not cell permeable, these experiments were performed in crude membrane preparations of drug-selected MCF-7/AdVp3000 cells that overexpress a homogeneously glycosylated population of ABCG2 (R482T) (Figure 3A, lane 1). Login to comment
174 Membrane preparations from these cells were used because of their uniform banding pattern on SDS-PAGE and their high level of ABCG2 (R482T) expression. Login to comment
175 In the absence of the cross-linker, ABCG2 (R482T) migrates as a monomer by SDS-PAGE analysis (Figure 3A, lane 1). Login to comment
195 In six-well plates, MCF-7/ AdVp3000 cells overexpressing ABCG2 (R482T) were incubated with a 1 mM solution of DPDPB for 2 h at 4 °C. Login to comment
201 When ABCG2 (R482T) was cross-linked with DPDPB, a higher molecular mass species at a molecular mass consistent with an ABCG2 (R482T) dimer (Figure 4, lane 2) was apparent, as compared to the monomer species observed in the absence of the cross-linker (Figure 4, lane 1). Login to comment
211 To determine if the extracellular cysteine residues are crucial for activity, the ABCG2∆EC-C variant was expressed in HeLa cells, and drug transport activity was measured as described in Experimental Procedures. We observed that the ABCG2∆EC-C variant had comparable levels of transport FIGURE 3: Nucleotide cross-linking of ABCG2 (R482T) in cell membranes. Login to comment
214 Proteins (5 µg in panel B and 25 µg in panel C) were separated by SDS-PAGE (7.5% gel) and detected by immunoblot analysis using the monoclonal antibody BXP-21 (1:1000) as described in Experimental Procedures. FIGURE 4: Sulfhydryl-reactive chemical cross-linking of ABCG2 (R482T) in whole 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
134 Other BCRP inhibitors include reserpine (a rauwolfia alkaloid),84 the pipecolinate derivatives VX-710 (Biricodar or Incel),85 and tryprostatin A (an Aspergillus fumigatus second metabolite).86 VX-710 increased uptake, retention, and cytotoxicity of mitoxantrone in cells overexpressing wild-type BCRP but had little effect on uptake, retention, and cytotoxicity of mitoxantrone and topotecan or SN-38 in cells expressing the BCRP mutant R482T.85 These results suggest that VX-710 is an inhibitor of wild-type BCRP only. 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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5 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. Login to comment
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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168 Subsequently, these authors showed that the MCF-7/AdrVp subline, which does not express P-gp, expresses high levels of a BCRP mutant (R482T) which can transport daunorubicin [10]. 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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47 Interestingly, however, they were able to inhibit only wild-type (R482) ABCG2, but not the mutant (R482T) transporter [23], as this was also the case with the third-generation P-gp inhibitor VX-710 (biricodar, Incel) [24]. Login to comment
59 Cyclosporin A was considered, however, as a broad-spectrum inhibitor on the basis of its 3-fold induced increase in mitoxantrone accumulation and cell growth sensitization [29], and was reported as a potent ATPase inhibitor of recombinant R482T mutant ABCG2 in insect cell membranes [8]. Login to comment
69 Interestingly, the R482T hotspot mutation altered the impact of prenylation on the inhibitory potency; tectochrysin being the best compound with an IC50 of 1.9 mmol/l. Login to comment
88 The interaction was altered 2-fold by the R482T/G hotspot mutations. 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] Modulation of multidrug resistance efflux pump act... Curr Opin Pharmacol. 2006 Aug;6(4):350-4. Epub 2006 May 11. Modok S, Mellor HR, Callaghan R
Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer.
Curr Opin Pharmacol. 2006 Aug;6(4):350-4. Epub 2006 May 11., [PMID:16690355]

Abstract [show]
Early publications using cultured cancer cells immediately recognized the phenomenon of resistance to anticancer agents. However, it was not until 1973 that it was first demonstrated that a major factor in the resistance of cancer cells was that of reduced drug accumulation. This year marks the 30th anniversary of the discovery by Juliano and Ling that P-glycoprotein mediates this active efflux of chemotherapeutic drugs from cancer cells. Since this seminal finding, the investigation of P-glycoprotein (MDR1, ATP binding cassette [ABC]B1) has proceeded with great vigour. However, it soon became apparent that P-glycoprotein was not expressed in all drug-resistant cells that displayed an accumulation deficiency, which led to the discovery of other ABC transporters involved in drug efflux. In 1992, the multidrug resistance-associated protein (MRP1, ABCC1) was identified in small cell lung cancer followed by breast cancer resistance protein (mitoxantrone resistance protein, ABCG2) in 1999. After three decades of research, can we confidently define the contribution of multidrug resistance transporters to chemoresistance and do we have clinically useful drugs to sensitise cancers?

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46 Ortataxel also inhibits MRP1, whereas both compounds are ineffective against the R482T BCRP isoform [15]. 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
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
125 Alqawi and colleagues have also shown that ABCG2, overexpressed in MCF-7/AdVp1000 cells (R482T) and MCF-7/ Mito (R482wt), binds directly and specifically to a photoactive drug analog of rhodamine 123, [125 I]Iodoarylazi- dorhodamine123 ([125 I]IAARh123) (Alqawi et al. 2004). 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
211 Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. Login to comment

[hide] Purification of breast cancer resistance protein A... Cell Mol Life Sci. 2006 Aug;63(16):1912-22. Pozza A, Perez-Victoria JM, Sardo A, Ahmed-Belkacem A, Di Pietro A
Purification of breast cancer resistance protein ABCG2 and role of arginine-482.
Cell Mol Life Sci. 2006 Aug;63(16):1912-22., [PMID:16847575]

Abstract [show]
Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding.

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3 Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Login to comment
5 The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. Login to comment
23 To decide between these alternatives and establish structure-function relationships, the aim of this study was to overexpress and purify both wild-type (R482) and mutant (R482T) ABCG2 to determine parameters ofATPase activity and measure direct binding of ligands, i.e. nucleotides, substrate drugs and inhibitors. Login to comment
26 The present results show that the R482T mutation represents a gain-of-function for ATPase activity by increasing both maximal rate of hydrolysis and affinity for MgATP. Login to comment
36 The pcDNA3 plasmid containing R482T-ABCG2 cDNA, provided by Dr. D. D. Ross, was used for subcloning into the pTriex-4-Neo plasmid (Novagen, VWR, Fontenay-sous- Bois, France). Login to comment
37 The R482T cDNA was mutated to obtain R482 cDNA by site-directed mutagenesis using a QuickChange Site-directed mutagenesis Kit (Stratagene, La Jolla). Login to comment
40 Recombinant baculoviruses carrying either R482T- or R482-ABCG2 human cDNA were generated with a BacVector transfection kit (Novagen), according to manufacturer`s instructions. Login to comment
52 Inverted membrane vesicles of High Five cells infected by baculovirus vector encoding the R482 or R482T transporter were treated with solubilization buffer {50 mM HEPES/NaOH, pH 8, 18 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.5 M NaCl, 10 mM imidazole, 20% glycerol} at a final protein concentration of 2 mg/ml, for 30 min with gently shaking at 4 °C. Login to comment
72 Lane 1: prestained molecular weight markers; lane 2: inverted membrane vesicles of High Five cells infected with baculovirus vector encoding R482 or R482T ABCG2; lane 3: supernatant after solubilization; lane 4: supernatant after centrifugation (15000 g, 30 min); lane 5: detergent-insoluble pellet after centrifugation; lane 6: supernatant after binding for 2 h; lane 7: Ni-NTA agarose gel after binding for 2 h; lane 8: washing; lane 9: Ni-NTA agarose gel after washing; lane 10: elution; lane 11: Ni-NTA agarose gel after elution; lane 12: after imidazole removal by gel filtration. Login to comment
74 Buffer-corrected fluorescence emission spectra for purified wild-type R482 (■) or mutant R482T (▲) transporter at (0.045 mg protein/ml); the fluorescence emission spectra were recorded at 25 °C upon excitation at 295 nm. Login to comment
89 The ATPase activity of purified R482 (■) or R482T (▲) ABCG2 was measured with 0.5 μg protein in the presence of 50 μg azolectin at 37 °C for 40 min. Login to comment
91 The inhibition of ATPase activity of R482 (■) and R482T transporter (▲) in the presence of 5 mM MgATP was measured at increasing orthovanadate concentrations (0.001-10 mM). Login to comment
92 (c, d) nucleotide binding as monitored by quenching of the intrinsic tryptophan fluorescence of purified R482 (■) and R482T (▲) transporter at 0.045 mg/ml: ATP (c) and ADP/Mg2+ (d). Login to comment
97 A similar procedure was used to overexpress, solubilize and purify mutant R482T ABCG2: qualitatively similar results were obtained as in Fig. 1a and b, but with a twofold lower yield (data not shown). Login to comment
99 It is worthwhile mentioning that the R482T point mutation significantly increased fluorescence intensity, by nearly 20%, giving rise to a more symmetrical emission spectrum. Login to comment
103 Interestingly, the R482T point mutation both increased Vmax, up to 1111 ± 79 nmol ATP hydrolyzed/min/mg, and lowered the Km(MgATP) to 1.0 ± 0.1 mM. Login to comment
107 Interestingly, the R482T mutation increased the binding affinity for ATP (in the absence of magnesium ions to avoid hydrolysis), with a shift in apparent KD from 81.3 ± 10.4 to 36.1 ± 7.4 μM, whereas it lowered the binding affinity for MgADP, the hydrolysis product, with a shift in KD from 82.0 ± 13.5 to 149 ± 22 μM (cf. Table 1). Login to comment
112 Interestingly, the R482T point mutation, reported to abolish methotrexate transport by whole cells and inverted membrane vesicles, was found here to only slightly alter KD1 (7.30 ± 0.57 μM), while KD2 remained unchanged (87.0 ± 29.4 μM), whereas maximal quenching was higher for mutant as compared with wild-type ABCG2. Login to comment
113 Conversely, although wild-type ABCG2 was known not to transport rhodamine 123, in contrast to R482T mutant [8], displaying here apparent KD1 and KD2 values of 0.52 ± 0.10 and 16.6 ± 6.6 μM, it in fact bound the fluo- Table 1. Login to comment
119 Wild-type R482 Mutant R482T KD1 (μM) ΔF1 (%) KD2 (μM) ΔFmax (%) KD1 (μM) ΔF1 (%) KD2 (μM) ΔFmax (%) Nucleotides ATP 81.3 ± 10.4 5.42 ± 0.19 36.1 ± 7.4 2.99 ± 0.14 MgADP 82.0 ± 13.5 6.42 ± 0.23 149 ± 22 11.1 ± 0.4 Substrate drugs Methotrexate 4.48 ± 0.40 13.7 ± 3.4 84.2 ± 25.6 66.3 ± 3.4 7.30 ± 0.57 15.7 ± 4.7 87.0 ± 29.4 84.3 ± 4.7 Rhodamine 123 0.52 ± 0.10 5.66 ± 1.21 16.6 ± 6.6 24.4 ± 1.2 0.42 ± 0.09 6.11 ± 1.23 15.4 ± 6.6 23.9 ± 1.2 Doxorubicin 1.63 ± 0.45 12.4 ± 3.4 22.2 ± 11.1 47.6 ± 3.4 2.91 ± 0.60 7.32 ± 2.36 25.9 ± 9.0 40.7 ± 2.4 Mitoxantrone 0.65 ± 0.06 4.03 ± 0.42 20.3 ± 4.3 16.0 ± 0.4 0.80 ± 0.10 4.53 ± 0.10 17.8 ± 4.6 25.5 ± 0.1 Pheophorbide a 0.94 ± 0.12 10.0 ± 1.3 17.7 ± 4.2 40.0 ± 1.3 1.50 ± 0.23 6.77 ± 1.75 21.7 ± 6.3 38.2 ± 1.8 Hoechst33342 2.04 ± 0.67 12.8 ± 2.3 28.7 ± 19.6 32.2 ± 2.3 1.69 ± 0.40 14.0 ± 4.1 23.1 ± 9.6 66.0 ± 4.1 Inhibitors GF120918 2.71 ± 0.66 12.6 ± 5.1 25.7 ± 10.9 72.4 ± 5.1 6.23 ± 1.46 9.6 ± 5.2 30.9 ± 11.7 70.9 ± 5.2 6-prenylchrysin 0.80 ± 0.11 8.22 ± 2.39 26.8 ± 9.2 51.8 ± 2.4 0.57 ± 0.07 11.0 ± 2.7 23.4 ± 6.1 69.0 ± 2.7 Ko143 0.012 ± 0.0016 13.2 ± 7.9 0.14 ± 0.11 66.8 ± 7.9 0.012 ± 0.0005 8.63 ± 3.63 0.096 ± 0.02 91.4 ± 3.6 Novobiocin 2.56 ± 0.56 4.45 ± 2.07 23.8 ± 10.8 27.6 ± 2.1 1.49 ± 0.33 8.75 ± 2.87 21.1 ± 10.0 41.3 ± 2.9 rescent dye similarly (0.42 ± 0.09 and 15.4 ± 6.6 μM, respectively) (Fig. 3b, and Table 1). Login to comment
128 Fluorescence emission was recorded, as in Fig. 2c, d for R482 (■) and R482T (▲) transporters, in the presence of increasing concentrations of either substrate drugs such as methotrexate (a) and rhodamine 123 (b), or known inhibitors such as GF120918 (c) and 6-prenylchrysin (d). Login to comment
145 The R482T mutant ABCG2 could be also prepared by the same procedure despite a twofold lower yield that might indicate some loss in protein stability. Login to comment
148 The functionality of both recombinant purified transporters was assessed by (i) the high ATPase activity, sensitive to vanadate and dependent on the R482T mutation, (ii) the direct binding of nucleotides, in the presence or absence of magnesium ions and their dependence on the mutation, (iii) the binding of most substrate drugs and inhibitors in the micromolar range, consistent with their known cellular effects, and (iv) the very high-affinity binding, in the nanomolar range, of Ko143 recognized as the most efficient ABCG2 inhibitor. Login to comment
149 The R482T hot-spot mutation as a gain-of-function for ATP hydrolysis. Login to comment
152 It is further shown here that the gain-of-function induced by the R482T mutation is also characterized by a twofold increase in affinity for MgATP, concomitant with a better binding of substrate ATP and a better release of product ADP. Login to comment
182 In conclusion, the R482T mutation, which occurs in vitro on cell cultures submitted to high drug pressure, induces a gain-of-function for ATPase activity and for substrate-drug transport, but not drug binding. Login to comment
313 39 Alqawi, O., Bates, S. and Georges, E. (2004) Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. 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
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] Modulation of the function of the multidrug resist... Mol Cancer Ther. 2006 Aug;5(8):1995-2006. Chearwae W, Shukla S, Limtrakul P, Ambudkar SV
Modulation of the function of the multidrug resistance-linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin.
Mol Cancer Ther. 2006 Aug;5(8):1995-2006., [PMID:16928820]

Abstract [show]
Curcumin (curcumin I), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the major forms of curcuminoids found in the turmeric powder, which exhibit anticancer, antioxidant, and anti-inflammatory activities. In this study, we evaluated the ability of purified curcuminoids to modulate the function of either the wild-type 482R or the mutant 482T ABCG2 transporter stably expressed in HEK293 cells and drug-selected MCF-7 FLV1000 and MCF-7 AdVp3000 cells. Curcuminoids inhibited the transport of mitoxantrone and pheophorbide a from ABCG2-expressing cells. However, both cytotoxicity and [(3)H]curcumin I accumulation assays showed that curcuminoids are not transported by ABCG2. Nontoxic concentration of curcumin I, II, and III sensitized the ABCG2-expressing cells to mitoxantrone, topotecan, SN-38, and doxorubicin. This reversal was not due to reduced expression because ABCG2 protein levels were unaltered by treatment with 10 mumol/L curcuminoids for 72 hours. Curcumin I, II, and III stimulated (2.4- to 3.3-fold) ABCG2-mediated ATP hydrolysis and the IC(50)s were in the range of 7.5 to 18 nmol/L, suggesting a high affinity of curcuminoids for ABCG2. Curcuminoids also inhibited the photolabeling of ABCG2 with [(125)I]iodoarylazidoprazosin and [(3)H]azidopine as well as the transport of these two substrates in ABCG2-expressing cells. Curcuminoids did not inhibit the binding of [alpha-(32)P]8-azidoATP to ABCG2, suggesting that they do not interact with the ATP-binding site of the transporter. Collectively, these data show that, among curcuminoids, curcumin I is the most potent modulator of ABCG2 and thus should be considered as a treatment to increase the efficacy of conventional chemotherapeutic drugs.

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117 Results Curcuminoids Inhibit the Efflux of Mitoxantrone and Pheophorbide a from Both Wild-type and R482T Mutant ABCG2-Expressing Cells To investigate the effect of curcuminoids on the accumulation of ABCG2 substrates, mitoxantrone and pheophorbide a accumulation assays (31) were done in wild-type 482R-HEK293 cells and mutant 482T ABCG2-expressing MCF-7 AdVp3000 cells. 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

[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] Interactions of cyclosporin a with breast cancer r... Drug Metab Dispos. 2007 Apr;35(4):576-82. Epub 2007 Jan 12. Xia CQ, Liu N, Miwa GT, Gan LS
Interactions of cyclosporin a with breast cancer resistance protein.
Drug Metab Dispos. 2007 Apr;35(4):576-82. Epub 2007 Jan 12., [PMID:17220244]

Abstract [show]
The objective of this study was to investigate whether cyclosporin A (CsA) is a modulator for breast cancer resistance protein (BCRP). The interactions between CsA and BCRP were evaluated by using both membrane- and cell-based assays. CsA inhibited BCRP or BCRP R482T mutant-associated ATPase with an IC(50) of 26.1 and 7.3 microM (31,388 and 8779 ng/ml), respectively, indicating that CsA is a modulator for BCRP and its R482T mutant. The apparent permeability (P(app)) of CsA was not affected by the BCRP-specific inhibitor Ko143 in both apical-to-basolateral (A-to-B) and basolateral-to-apical (B-to-A) directions in hBCRP- or mBcrp-transfected MDCKII cells, whereas CsA at 50 microM significantly increased the A-to-B transport and decreased B-to-A transport of BCRP substrates, [(3)H]estrone-3-sulfate ([(3)H]E3S) and [(3)H]methotrexate ([(3)H]MTX), in hBCRP- and mBcrp1-trasfected MDCKII cells. Similar to cellular transport studies, CsA did not exhibit ATP-dependent uptake in BCRP-expressed membrane vesicles but inhibited the ATP-mediated E3S and MTX uptake in the same vesicles. The inhibitory constant (K(i)) of CsA toward BCRP was 6.7 microM (8507 ng/ml) and 7.8 microM (9380 ng/ml) when using E3S or MTX, respectively, as a BCRP substrate. The inhibitory potency of CsA on BCRP wild type or its R482T mutant was lower than that on P-glycoprotein. The present studies demonstrate that CsA is an inhibitor but not a substrate for BCRP, and has low potential to cause drug-drug interactions with BCRP substrate drugs due to its weak inhibitory effect on BCRP and BCRP R482T mutant at its normal therapeutic blood concentrations (200-400 ng/ml) (Blood 91:362-363, 1998).

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2 CsA inhibited BCRP or BCRP R482T mutant-associated ATPase with an IC50 of 26.1 and 7.3 ␮M (31,388 and 8779 ng/ml), respectively, indicating that CsA is a modulator for BCRP and its R482T mutant. Login to comment
6 The inhibitory potency of CsA on BCRP wild type or its R482T mutant was lower than that on P-glycoprotein. Login to comment
7 The present studies demonstrate that CsA is an inhibitor but not a substrate for BCRP, and has low potential to cause drug-drug interactions with BCRP substrate drugs due to its weak inhibitory effect on BCRP and BCRP R482T mutant at its normal therapeutic blood concentrations (200-400 ng/ml) (Blood 91:362-363, 1998). Login to comment
43 We also demonstrated that CsA reduced the ATPase activities of both wild-type BCRP and BCRP R482T mutant with an IC50 of 26.1 and 7.3 ␮M (31,388 and 8779 ng/ml), respectively, and inhibited BCRP-mediated efflux of estrone-3-sulfate (E3S) and MTX with a Ki of 6.7 and 7.8 ␮M (8507 and 9380 ng/ml), respectively. Login to comment
45 Compared with the effect on daunorubicin-stimulated P-gp ATPase activity, CsA had less potency on the inhibition of daunorubicin-stimulated BCRP R482T mutant ATPase activity. Login to comment
47 Materials and Methods Human BCRP or BCPR R482T mutant-expressed cell membranes and human BCRP-expressing and control membrane vesicles were obtained from Solvo Biotechnology, Inc. (Budao¨rs, Hungary). Login to comment
53 BCRP, BCRP R482T mutant, or P-gp-associated ATPase activities were measured according to the method of Sarkadi et al. (1992). Login to comment
98 CsA reduced vanadate-sensitive ATPase activities of wild-type BCRP and BCRP R482T mutant with the IC50 of 26.1 and 7.3 ␮M (31,388 and 8779 ng/ml), respectively (Fig. 1). Login to comment
99 The inhibitory effects of CsA on BCRP and BCRP R482T mutant-associated ATPase activities indicate that CsA is a modulator for both BCRP and BCRP R482T mutant. Login to comment
100 Effects of CsA on Daunorubicin-Stimulated P-gp or BCRP R482T Mutant ATPase Activities. Login to comment
101 To compare the inhibitory potency of CsA on P-gp and mutant BCRP, the effects of CsA on daunorubicin-stimulated P-gp or BCRP R482T mutant ATPase activities were evaluated. Login to comment
106 Effects of CsA on BCRP (A)- or BCRP R482T mutant (B)-associated ATPase activities. Login to comment
112 The effect of CsA on the daunorubicin-stimulated P-gp and BCRP R482T mutant ATPase activity. Login to comment
168 Similar to the P-gp ATPase activity, both BCRP and BCRP R482T mutant ATPase activities were vanadate-sensitive and could be stimulated or inhibited by its substrates or inhibitors (Ozvegy et al., 2001, 2002; Xia et al., 2004). Login to comment
169 Although the BCRP R482T mutant was only found in the drug-resistant human tumor cell lines (Honjo et al., 2001) but not in human individuals (Honjo et al., 2002; Zamber et al., 2003), it is still of interest to know whether CsA can be a modulator of this mutant because CsA is a commonly used MDR-reversing reagent in anticancer therapy. Login to comment
170 CsA reduced vanadate-sensitive ATPase activities of wild-type BCRP and BCRP R482T mutant with an IC50 of 26.1 and 7.3 ␮M (31,388 and 8779 ng/ml), respectively (Fig. 1). Login to comment
171 Because the ATPase assay is not a transport functional assay and cannot distinguish substrates from inhibitors, the inhibitory effects of CsA on BCRP ATPase activities (Fig. 1) indicate that CsA is a modulator of BCRP and BCRP R482T mutant activity. Login to comment
172 The lower IC50 of CsA on the BCRP R482T mutant ATPase activity than that on the wild-type BCRP ATPase activity (Fig. 1) suggests that CsA binding to the BCRP R482T mutant is stronger than that to the wild-type BCRP. Login to comment
191 Daunorubicin is known to be a more selective substrate for BCRP R482T mutant (Honjo et al., 2001) than wild-type BCRP. Login to comment
192 The inhibitory effect of CsA on daunorubicin- simulated BCRP R482T mutant ATPase activity was 3.0-fold less than that on P-gp ATPase activity (Fig. 2B), indicating that CsA has less inhibitory potency on BCRP mutant than on P-gp. Login to comment
195 CsA can modulate both wild-type BCRP and BCRP R482T mutant activity, with Ki of 6.7 to ϳ7.8 ␮M (8507-9380 ng/ml) for the wild-type BCRP using E3S and MTX as substrates. 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.