ABCMdb

ABCG2 p.Arg482Gly

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PMID: 11559526 [PubMed] Honjo Y et al: "Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells."

No. Sentence Comment

7 A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/ BCRP/ABCP forms transported mitoxantrone. Login to comment
8 Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. Login to comment
78 C, electropherograms for MCF-7 MX100 (R482, top), S1-M1-80 (R482G, middle), and MCF-7 AdVp3000 (R482T, bottom) MXR/BCRP/ABCP forms. Login to comment
103 However, the MCF-7 AdVp3000 cells with the R482T mutation were more resistant to adriamycin than the R482 wild type (P ϭ 0.001), whereas S1-M180 cells with the R482G mutation were also more resistant to adriamycin (P ϭ 0.004) but appeared to be less resistant to topotecan and SN-38. Login to comment
112 The absence of rhodamine and doxorubicin efflux is a sharp contrast from the phenotype observed in S1-M1-80 cells, with an R482G mutation, and in MCF-7 AdVp cells with an R482T mutation. Login to comment
116 When transmembrane domains for MXR/BCRP/ABCP were identified using the TMpred program, the third transmembrane domain was predicted to span amino acids 483-499 for wild-type MXR/ BCRP/ABCP (R482) but was predicted to span amino acids 478-497 for the R482G mutation and 478-499 for the R482T mutation. Login to comment
132 Four-day cytotoxicity assays were performed on the MCF-7 MX100 (R482), MCF-7 AdVp3000 (R482T), and S1-M1-80 (R482G) cells with topotecan, mitoxantrone, adriamycin, and SN-38. Login to comment
6 A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/ BCRP/ABCP forms transported mitoxantrone. Login to comment
77 C, electropherograms for MCF-7 MX100 (R482, top), S1-M1-80 (R482G, middle), and MCF-7 AdVp3000 (R482T, bottom) MXR/BCRP/ABCP forms. Login to comment
102 However, the MCF-7 AdVp3000 cells with the R482T mutation were more resistant to adriamycin than the R482 wild type (P ϭ 0.001), whereas S1-M180 cells with the R482G mutation were also more resistant to adriamycin (P ϭ 0.004) but appeared to be less resistant to topotecan and SN-38. Login to comment
111 The absence of rhodamine and doxorubicin efflux is a sharp contrast from the phenotype observed in S1-M1-80 cells, with an R482G mutation, and in MCF-7 AdVp cells with an R482T mutation. Login to comment
115 When transmembrane domains for MXR/BCRP/ABCP were identified using the TMpred program, the third transmembrane domain was predicted to span amino acids 483-499 for wild-type MXR/ BCRP/ABCP (R482) but was predicted to span amino acids 478-497 for the R482G mutation and 478-499 for the R482T mutation. Login to comment
131 Four-day cytotoxicity assays were performed on the MCF-7 MX100 (R482), MCF-7 AdVp3000 (R482T), and S1-M1-80 (R482G) cells with topotecan, mitoxantrone, adriamycin, and SN-38. Login to comment

PMID: 11804192 [PubMed] Bates SE et al: "The role of half-transporters in multidrug resistance."

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

PMID: 11956086 [PubMed] Allen JD et al: "A mutation hot spot in the Bcrp1 (Abcg2) multidrug transporter in mouse cell lines selected for Doxorubicin resistance."

No. Sentence Comment

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

PMID: 12208758 [PubMed] Volk EL et al: "Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance."

No. Sentence Comment

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

PMID: 12374800 [PubMed] Ozvegy C et al: "Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation."

No. Sentence Comment

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

PMID: 12488537 [PubMed] Wang X et al: "Breast cancer resistance protein (BCRP/ABCG2) induces cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors."

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

PMID: 12535572 [PubMed] Schinkel AH et al: "Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview."

No. Sentence Comment

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

PMID: 12544509 [PubMed] Zamber CP et al: "Natural allelic variants of breast cancer resistance protein (BCRP) and their relationship to BCRP expression in human intestine."

No. Sentence Comment

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

PMID: 12659681 [PubMed] Ujhelly O et al: "Application of a human multidrug transporter (ABCG2) variant as selectable marker in gene transfer to progenitor cells."

No. Sentence Comment

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

PMID: 12874005 [PubMed] Chen ZS et al: "Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport."

No. Sentence Comment

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

PMID: 12960118 [PubMed] Nakanishi T et al: "Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients."

No. Sentence Comment

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

PMID: 14500392 [PubMed] Volk EL et al: "Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter."

No. Sentence Comment

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

PMID: 14566825 [PubMed] Miwa M et al: "Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants."

No. Sentence Comment

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

PMID: 14576842 [PubMed] Doyle LA et al: "Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)."

No. Sentence Comment

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

PMID: 14612912 [PubMed] Robey RW et al: "Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity."

No. Sentence Comment

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

PMID: 14633964 [PubMed] Dietrich CG et al: "ABC of oral bioavailability: transporters as gatekeepers in the gut."

No. Sentence Comment

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

PMID: 14645676 [PubMed] Nakanishi T et al: "Functional characterization of human breast cancer resistance protein (BCRP, ABCG2) expressed in the oocytes of Xenopus laevis."

No. Sentence 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

PMID: 14750175 [PubMed] Mizuarai S et al: "Single nucleotide polymorphisms result in impaired membrane localization and reduced atpase activity in multidrug transporter ABCG2."

No. Sentence Comment

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

PMID: 14754410 [PubMed] Han B et al: "Multidrug resistance in cancer chemotherapy and xenobiotic protection mediated by the half ATP-binding cassette transporter ABCG2."

No. Sentence Comment

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

PMID: 14973080 [PubMed] Robey RW et al: "Pheophorbide a is a specific probe for ABCG2 function and inhibition."

No. Sentence Comment

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

PMID: 14973083 [PubMed] Ee PL et al: "Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene."

No. Sentence Comment

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

PMID: 15075385 [PubMed] Bates SE et al: "ABCG2 mediates differential resistance to SN-38 (7-ethyl-10-hydroxycamptothecin) and homocamptothecins."

No. Sentence Comment

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

PMID: 15165903 [PubMed] Sarkadi B et al: "ABCG2 -- a transporter for all seasons."

No. Sentence Comment

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

PMID: 15251980 [PubMed] Burger H et al: "Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump."

No. Sentence Comment

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

PMID: 15260487 [PubMed] Polgar O et al: "Mutational analysis of ABCG2: role of the GXXXG motif."

No. Sentence Comment

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

PMID: 15362954 [PubMed] Janvilisri T et al: "Arginine-482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2)."

No. Sentence Comment

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

PMID: 15385942 [PubMed] Walter RB et al: "Breast cancer resistance protein (BCRP/ABCG2) does not confer resistance to gemtuzumab ozogamicin and calicheamicin-gamma1 in acute myeloid leukemia cells."

No. Sentence Comment

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

PMID: 15475413 [PubMed] Kobayashi D et al: "Functional assessment of ABCG2 (BCRP) gene polymorphisms to protein expression in human placenta."

No. Sentence Comment

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

PMID: 15486199 [PubMed] Loganzo F et al: "Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin."

No. Sentence Comment

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

PMID: 15516692 [PubMed] Ueda T et al: "Cloning and functional analysis of the rhesus macaque ABCG2 gene. Forced expression confers an SP phenotype among hematopoietic stem cell progeny in vivo."

No. Sentence Comment

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

PMID: 15517563 [PubMed] Garcia-Escarp M et al: "Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs."

No. Sentence Comment

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

PMID: 15557326 [PubMed] Ozvegy-Laczka C et al: "Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface."

No. Sentence Comment

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

PMID: 15588721 [PubMed] Rhee MS et al: "Lack of an effect of breast cancer resistance protein (BCRP/ABCG2) overexpression on methotrexate polyglutamate export and folate accumulation in a human breast cancer cell line."

No. Sentence Comment

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

PMID: 15598974 [PubMed] Ejendal KF et al: "Differential sensitivities of the human ATP-binding cassette transporters ABCG2 and P-glycoprotein to cyclosporin A."

No. Sentence Comment

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

PMID: 15618737 [PubMed] Itoda M et al: "Eight novel single nucleotide polymorphisms in ABCG2/BCRP in Japanese cancer patients administered irinotacan."

No. Sentence Comment

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

PMID: 15630450 [PubMed] Kager L et al: "Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics."

No. Sentence Comment

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

PMID: 15670731 [PubMed] Ozvegy-Laczka C et al: "Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition."

No. Sentence Comment

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

PMID: 15684613 [PubMed] Robey RW et al: "ABCG2-mediated transport of photosensitizers: potential impact on photodynamic therapy."

No. Sentence Comment

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

PMID: 15694832 [PubMed] Staud F et al: "Breast cancer resistance protein (BCRP/ABCG2)."

No. Sentence Comment

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

PMID: 15716365 [PubMed] Xia CQ et al: "Expression, localization, and functional characteristics of breast cancer resistance protein in Caco-2 cells."

No. Sentence Comment

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

PMID: 15769853 [PubMed] Henriksen U et al: "Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2."

No. Sentence Comment

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

PMID: 15807535 [PubMed] Diop NK et al: "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."

No. Sentence Comment

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

PMID: 15838659 [PubMed] Morisaki K et al: "Single nucleotide polymorphisms modify the transporter activity of ABCG2."

No. Sentence Comment

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

PMID: 15882131 [PubMed] Lepper ER et al: "Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2."

No. Sentence Comment

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

PMID: 15930306 [PubMed] Ahmed-Belkacem A et al: "Flavonoid structure-activity studies identify 6-prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein ABCG2."

No. Sentence 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

PMID: 16086592 [PubMed] Bhatia A et al: "Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers."

No. Sentence Comment

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

PMID: 16107343 [PubMed] Henriksen U et al: "Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2."

No. Sentence Comment

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

PMID: 16108826 [PubMed] Sugimoto Y et al: "Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/pharmacodynamics."

No. Sentence Comment

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

PMID: 16146333 [PubMed] Mao Q et al: "Role of the breast cancer resistance protein (ABCG2) in drug transport."

No. Sentence Comment

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

PMID: 16158227 [PubMed] Krishnamurthy P et al: "The ABC transporter Abcg2/Bcrp: role in hypoxia mediated survival."

No. Sentence Comment

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

PMID: 16160819 [PubMed] Ishikawa T et al: "Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design."

No. Sentence Comment

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

PMID: 16169662 [PubMed] Huang Y et al: "Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells."

No. Sentence Comment

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

PMID: 16259577 [PubMed] Sakurai A et al: "Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications."

No. Sentence Comment

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

PMID: 16337012 [PubMed] Breedveld P et al: "Use of P-glycoprotein and BCRP inhibitors to improve oral bioavailability and CNS penetration of anticancer drugs."

No. Sentence Comment

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

PMID: 16337740 [PubMed] Cervenak J et al: "The role of the human ABCG2 multidrug transporter and its variants in cancer therapy and toxicology."

No. Sentence 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

PMID: 16399366 [PubMed] Ishikawa T et al: "High-speed screening of human ATP-binding cassette transporter function and genetic polymorphisms: new strategies in pharmacogenomics."

No. Sentence Comment

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

PMID: 16404634 [PubMed] Gupta A et al: "Cyclosporin A, tacrolimus and sirolimus are potent inhibitors of the human breast cancer resistance protein (ABCG2) and reverse resistance to mitoxantrone and topotecan."

No. Sentence Comment

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

PMID: 16442095 [PubMed] Cooray HC et al: "Modulation of p-glycoprotein and breast cancer resistance protein by some prescribed corticosteroids."

No. Sentence Comment

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

PMID: 16489126 [PubMed] Saito H et al: "A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions."

No. Sentence Comment

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

PMID: 16520651 [PubMed] Ahmed-Belkacem A et al: "Inhibitors of cancer cell multidrug resistance mediated by breast cancer resistance protein (BCRP/ABCG2)."

No. Sentence Comment

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

PMID: 16608919 [PubMed] Tamura A et al: "Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport."

No. Sentence Comment

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

PMID: 16702730 [PubMed] Maekawa K et al: "Genetic variation and haplotype structure of the ABC transporter gene ABCG2 in a Japanese population."

No. Sentence Comment

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

PMID: 16715370 [PubMed] Asashima T et al: "ATP-binding cassette transporter G2 mediates the efflux of phototoxins on the luminal membrane of retinal capillary endothelial cells."

No. Sentence Comment

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

PMID: 16815914 [PubMed] Ejendal KF et al: "The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding."

No. Sentence Comment

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

PMID: 16842198 [PubMed] Perez-Tomas R et al: "Multidrug resistance: retrospect and prospects in anti-cancer drug treatment."

No. Sentence Comment

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

PMID: 16863427 [PubMed] Xia CQ et al: "Breast cancer resistance protein in pharmacokinetics and drug-drug interactions."

No. Sentence Comment

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

PMID: 16877258 [PubMed] Wakabayashi K et al: "Human ABC transporter ABCG2 in xenobiotic protection and redox biology."

No. Sentence Comment

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

PMID: 16981002 [PubMed] Clark R et al: "Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter."

No. Sentence Comment

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

PMID: 16983557 [PubMed] Kusuhara H et al: "ATP-binding cassette, subfamily G (ABCG family)."

No. Sentence Comment

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

PMID: 17015488 [PubMed] Sarkadi B et al: "Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system."

No. Sentence Comment

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

PMID: 17027309 [PubMed] Li YF et al: "Towards understanding the mechanism of action of the multidrug resistance-linked half-ABC transporter ABCG2: a molecular modeling study."

No. Sentence Comment

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

PMID: 17032904 [PubMed] Breedveld P et al: "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."

No. Sentence Comment

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

PMID: 17098188 [PubMed] McDevitt CA et al: "Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2."

No. Sentence Comment

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

PMID: 17228519 [PubMed] Tamura A et al: "Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system."

No. Sentence Comment

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

PMID: 17297656 [PubMed] Tamura A et al: "Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2."

No. Sentence Comment

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

PMID: 17323126 [PubMed] Huang Y et al: "Pharmacogenetics/genomics of membrane transporters in cancer chemotherapy."

No. Sentence Comment

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

PMID: 17347325 [PubMed] Pal A et al: "Cholesterol potentiates ABCG2 activity in a heterologous expression system: improved in vitro model to study function of human ABCG2."

No. Sentence Comment

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

PMID: 17375082 [PubMed] Hardwick LJ et al: "The emerging pharmacotherapeutic significance of the breast cancer resistance protein (ABCG2)."

No. Sentence Comment

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

PMID: 17380269 [PubMed] Lee G et al: "Expression of the ATP-binding cassette membrane transporter, ABCG2, in human and rodent brain microvessel endothelial and glial cell culture systems."

No. Sentence Comment

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

PMID: 17487233 [PubMed] Yeboah D et al: "Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor."

No. Sentence Comment

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

PMID: 17504223 [PubMed] Xia CQ et al: "Evaluation of drug-transporter interactions using in vitro and in vivo models."

No. Sentence Comment

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

PMID: 17537873 [PubMed] Glavinas H et al: "ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions."

No. Sentence Comment

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

PMID: 17616561 [PubMed] Matsson P et al: "A global drug inhibition pattern for the human ATP-binding cassette transporter breast cancer resistance protein (ABCG2)."

No. Sentence Comment

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

PMID: 17662239 [PubMed] Telbisz A et al: "Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter."

No. Sentence Comment

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

PMID: 17904109 [PubMed] Korynevska A et al: "Mechanisms underlying the anticancer activities of the angucycline landomycin E."

No. Sentence Comment

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

PMID: 17909048 [PubMed] Zhang Y et al: "ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging."

No. Sentence Comment

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

PMID: 17923155 [PubMed] Gedeon C et al: "Breast cancer resistance protein: mediating the trans-placental transfer of glyburide across the human placenta."

No. Sentence Comment

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

PMID: 17943230 [PubMed] Gounder MK et al: "Effects of drug efflux proteins and topoisomerase I mutations on the camptothecin analogue gimatecan."

No. Sentence Comment

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

PMID: 17979803 [PubMed] Saito H et al: "High-speed screening and quantitative SAR analysis of human ABC transporter ABCG2 for molecular modeling of anticancer drugs to circumvent multidrug resistance."

No. Sentence Comment

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

PMID: 18006847 [PubMed] Shi Z et al: "Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance."

No. Sentence Comment

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

PMID: 18089722 [PubMed] Wu CP et al: "Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance linked ABCG2 transporter."

No. Sentence Comment

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

PMID: 18154452 [PubMed] Sharom FJ et al: "ABC multidrug transporters: structure, function and role in chemoresistance."

No. Sentence Comment

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

PMID: 18237272 [PubMed] Nakagawa H et al: "Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2."

No. Sentence Comment

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

PMID: 18249138 [PubMed] Hazai E et al: "Homology modeling of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

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

PMID: 18253130 [PubMed] Lemos C et al: "Drug transporters: recent advances concerning BCRP and tyrosine kinase inhibitors."

No. Sentence Comment

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

PMID: 18363541 [PubMed] Tamura A et al: "Drug-induced phototoxicity evoked by inhibition of human ABC transporter ABCG2: development of in vitro high-speed screening systems."

No. Sentence Comment

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

PMID: 18430864 [PubMed] Liu Y et al: "Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2."

No. Sentence Comment

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

PMID: 18523872 [PubMed] Muenster U et al: "Characterization of substrates and inhibitors for the in vitro assessment of Bcrp mediated drug-drug interactions."

No. Sentence Comment

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

PMID: 18644784 [PubMed] Ozvegy-Laczka C et al: "Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter."

No. Sentence Comment

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

PMID: 18657189 [PubMed] McDevitt CA et al: "Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2?"

No. Sentence Comment

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

PMID: 18668433 [PubMed] Koshiba S et al: "Human ABC transporters ABCG2 (BCRP) and ABCG4."

No. Sentence Comment

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

PMID: 18673259 [PubMed] Nakamura T et al: "Pharmacogenetics of intestinal absorption."

No. Sentence Comment

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

PMID: 18824523 [PubMed] Merino G et al: "Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors."

No. Sentence Comment

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

PMID: 18829547 [PubMed] Dai CL et al: "Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2."

No. Sentence Comment

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

PMID: 18855611 [PubMed] Zhou SF et al: "Clinical pharmacogenetics and potential application in personalized medicine."

No. Sentence Comment

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

PMID: 18958403 [PubMed] Furukawa T et al: "Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations."

No. Sentence Comment

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

PMID: 19002564 [PubMed] Polgar O et al: "The 315-316 deletion determines the BXP-21 antibody epitope but has no effect on the function of wild type ABCG2 or the Q141K variant."

No. Sentence Comment

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

PMID: 19056916 [PubMed] Giri N et al: "Substrate-dependent breast cancer resistance protein (Bcrp1/Abcg2)-mediated interactions: consideration of multiple binding sites in in vitro assay design."

No. Sentence Comment

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

PMID: 19059384 [PubMed] Shi Z et al: "Inhibiting the function of ABCB1 and ABCG2 by the EGFR tyrosine kinase inhibitor AG1478."

No. Sentence Comment

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

PMID: 19111841 [PubMed] Noguchi K et al: "Functions of the breast cancer resistance protein (BCRP/ABCG2) in chemotherapy."

No. Sentence Comment

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

PMID: 19111842 [PubMed] Wakabayashi-Nakao K et al: "Quality control of human ABCG2 protein in the endoplasmic reticulum: ubiquitination and proteasomal degradation."

No. Sentence Comment

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

PMID: 19124053 [PubMed] McDevitt CA et al: "Purification and structural analyses of ABCG2."

No. Sentence Comment

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

PMID: 19135105 [PubMed] Hegedus C et al: "Ins and outs of the ABCG2 multidrug transporter: an update on in vitro functional assays."

No. Sentence Comment

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

PMID: 19135106 [PubMed] Nicolle E et al: "QSAR analysis and molecular modeling of ABCG2-specific inhibitors."

No. Sentence Comment

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

PMID: 19156141 [PubMed] Day JM et al: "BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure."

No. Sentence Comment

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

PMID: 19232821 [PubMed] Dai CL et al: "Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2."

No. Sentence Comment

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

PMID: 19251825 [PubMed] Bram EE et al: "Structural determinants of imidazoacridinones facilitating antitumor activity are crucial for substrate recognition by ABCG2."

No. Sentence Comment

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

PMID: 19270533 [PubMed] Mathew G et al: "ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate."

No. Sentence Comment

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

PMID: 19406100 [PubMed] Polgar O et al: "Arginine 383 is a crucial residue in ABCG2 biogenesis."

No. Sentence Comment

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

PMID: 19427995 [PubMed] Tiwari AK et al: "Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters."

No. Sentence Comment

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

PMID: 19827267 [PubMed] Ishikawa T et al: "Human ABC transporter ABCG2 in cancer chemotherapy and pharmacogenomics."

No. Sentence Comment

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

PMID: 19949921 [PubMed] Calcagno AM et al: "Molecular mechanisms of drug resistance in single-step and multi-step drug-selected cancer cells."

No. Sentence Comment

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

PMID: 19949928 [PubMed] Ross DD et al: "Impact of breast cancer resistance protein on cancer treatment outcomes."

No. Sentence Comment

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

PMID: 20088606 [PubMed] Polgar O et al: "Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2."

No. Sentence Comment

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

PMID: 20203106 [PubMed] Cai X et al: "Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport."

No. Sentence Comment

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

PMID: 20345483 [PubMed] Kawahara H et al: "Pharmacological interaction with sunitinib is abolished by a germ-line mutation (1291T>C) of BCRP/ABCG2 gene."

No. Sentence Comment

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

PMID: 20812902 [PubMed] Ni Z et al: "Structure and function of the human breast cancer resistance protein (BCRP/ABCG2)."

No. Sentence Comment

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

PMID: 20876799 [PubMed] Mi YJ et al: "Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters."

No. Sentence Comment

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

PMID: 21103975 [PubMed] Meyer zu Schwabedissen HE et al: "In vitro and in vivo evidence for the importance of breast cancer resistance protein transporters (BCRP/MXR/ABCP/ABCG2)."

No. Sentence Comment

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

PMID: 21175590 [PubMed] Kerr ID et al: "The ABCG family of membrane-associated transporters: you don't have to be big to be mighty."

No. Sentence Comment

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

PMID: 21188243 [PubMed] Ishikawa T et al: "Key Role of Human ABC Transporter ABCG2 in Photodynamic Therapy and Photodynamic Diagnosis."

No. Sentence Comment

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

PMID: 21402712 [PubMed] Shi Z et al: "Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance."

No. Sentence Comment

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

PMID: 21424391 [PubMed] Jacobs A et al: "Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study."

No. Sentence Comment

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

PMID: 21605668 [PubMed] Munic V et al: "Characterization of rhodamine-123, calcein and 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) export via MRP2 (ABCC2) in MES-SA and A549 cells."

No. Sentence Comment

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

PMID: 21930826 [PubMed] Haslam IS et al: "Intestinal ciprofloxacin efflux: the role of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

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

PMID: 22614107 [PubMed] Sun YL et al: "Zafirlukast antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance."

No. Sentence Comment

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

PMID: 22548830 [PubMed] Hegedus C et al: "Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance."

No. Sentence Comment

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

PMID: 22593228 [PubMed] Wei Y et al: "New use for an old drug: inhibiting ABCG2 with sorafenib."

No. Sentence Comment

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

PMID: 22549112 [PubMed] Wang F et al: "Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2."

No. Sentence Comment

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

PMID: 22497316 [PubMed] Mo W et al: "Different roles of TM5, TM6, and ECL3 in the oligomerization and function of human ABCG2."

No. Sentence Comment

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

PMID: 22285911 [PubMed] Fukuda Y et al: "ABC transporters and their role in nucleoside and nucleotide drug resistance."

No. Sentence Comment

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

PMID: 22115866 [PubMed] Telbisz A et al: "Antibody binding shift assay for rapid screening of drug interactions with the human ABCG2 multidrug transporter."

No. Sentence Comment

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

PMID: 22509477 [PubMed] Mo W et al: "Human ABCG2: structure, function, and its role in multidrug resistance."

No. Sentence Comment

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

PMID: 21625222 [PubMed] Singh RR et al: "ABCG2 is a direct transcriptional target of hedgehog signaling and involved in stroma-induced drug tolerance in diffuse large B-cell lymphoma."

No. Sentence Comment

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

PMID: 21628496 [PubMed] Li L et al: "pH-Dependent transport of pemetrexed by breast cancer resistance protein."

No. Sentence Comment

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

PMID: 21531129 [PubMed] Wang F et al: "Prognostic value of the multidrug resistance transporter ABCG2 gene polymorphisms in Chinese patients with de novo acute leukaemia."

No. Sentence Comment

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

PMID: 20873966 [PubMed] Poguntke M et al: "Drug transport by breast cancer resistance protein."

No. Sentence Comment

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

PMID: 16815813 [PubMed] Choudhuri S et al: "Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters."

No. Sentence Comment

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

PMID: 18271955 [PubMed] Tang R et al: "Zosuquidar restores drug sensitivity in P-glycoprotein expressing acute myeloid leukemia (AML)."

No. Sentence Comment

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

PMID: 18094074 [PubMed] Velamakanni S et al: "A functional steroid-binding element in an ATP-binding cassette multidrug transporter."

No. Sentence Comment

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

PMID: 19148526 [PubMed] Shi Z et al: "The epidermal growth factor tyrosine kinase inhibitor AG1478 and erlotinib reverse ABCG2-mediated drug resistance."

No. Sentence Comment

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

PMID: 21821808 [PubMed] Real R et al: "Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models."

No. Sentence Comment

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

PMID: 21935580 [PubMed] Eddabra L et al: "Arginine 482 to glycine mutation in ABCG2/BCRP increases etoposide transport and resistance to the drug in HEK-293 cells."

No. Sentence Comment

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

PMID: 23084250 [PubMed] Zhang Y et al: "Influence of bioluminescence imaging dynamics by D-luciferin uptake and efflux mechanisms."

No. Sentence Comment

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

PMID: 23153455 [PubMed] Wu CP et al: "Overexpression of ATP-binding cassette transporter ABCG2 as a potential mechanism of acquired resistance to vemurafenib in BRAF(V600E) mutant cancer cells."

No. Sentence Comment

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

PMID: 23205634 [PubMed] Telbisz A et al: "Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein."

No. Sentence Comment

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

PMID: 23467750 [PubMed] Zoernig I et al: "Sequence mutations of the substrate binding pocket of stem cell factor and multidrug resistance protein ABCG2 in renal cell cancer: a possible link to treatment resistance."

No. Sentence Comment

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

PMID: 23470221 [PubMed] Strouse JJ et al: "Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters."

No. Sentence Comment

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

PMID: 23586520 [PubMed] Hazai E et al: "Predicting substrates of the human breast cancer resistance protein using a support vector machine method."

No. Sentence Comment

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

PMID: 24021215 [PubMed] Stacy AE et al: "Molecular pharmacology of ABCG2 and its role in chemoresistance."

No. Sentence Comment

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

PMID: 24384916 [PubMed] Telbisz A et al: "Regulation of the function of the human ABCG2 multidrug transporter by cholesterol and bile acids: effects of mutations in potential substrate and steroid binding sites."

No. Sentence Comment

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

PMID: 24388985 [PubMed] Deppe S et al: "Impact of genetic variability in the ABCG2 gene on ABCG2 expression, function, and interaction with AT1 receptor antagonist telmisartan."

No. Sentence Comment

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

PMID: 24468191 [PubMed] Gu YY et al: "Baicalein decreases side population proportion via inhibition of ABCG2 in multiple myeloma cell line RPMI 8226 in vitro."

No. Sentence Comment

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

PMID: 24626598 [PubMed] Kathawala RJ et al: "Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance."

No. Sentence Comment

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

PMID: 24726739 [PubMed] Zhang H et al: "Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance."

No. Sentence Comment

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

PMID: 24747122 [PubMed] Zhang H et al: "AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance."

No. Sentence Comment

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

PMID: 24777822 [PubMed] Jani M et al: "Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics."

No. Sentence Comment

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

PMID: 25036722 [PubMed] Szafraniec MJ et al: "Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2)."

No. Sentence Comment

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

PMID: 25236865 [PubMed] Mao Q et al: "Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update."

No. Sentence Comment

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

PMID: 25445676 [PubMed] Gal Z et al: "Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter."

No. Sentence Comment

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

PMID: 26294421 [PubMed] Haider AJ et al: "Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences."

No. Sentence Comment

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

PMID: 26327810 [PubMed] Zhang W et al: "Insights into Chemoresistance of Prostate Cancer."

No. Sentence Comment

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

PMID: 26351135 [PubMed] Darby RA et al: "Overcoming ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors."

No. Sentence Comment

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

PMID: 26515463 [PubMed] Anreddy N et al: "A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo."

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