ABCMdb

ABCG2 p.Arg482Thr

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

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
150 In this context it is also noteworthy that cell lines carrying either the R482M or R482S Bcrp1 mutants showed greatly reduced (and Ko143-reversible) accumulation of the dye rhodamine 123 (Fig. 3C), as was observed previously for the R482G and R482T mutants of human BCRP (13). Login to comment
102 The identification of R482T and R482G mutations in human BCRP in highly drug-selected human cell lines (12, 13), which increase resistance to anthracyclines, suggested that the doxorubicin-selected mouse lines might harbor similar mutations. Login to comment
148 In this context it is also noteworthy that cell lines carrying either the R482M or R482S Bcrp1 mutants showed greatly reduced (and Ko143-reversible) accumulation of the dye rhodamine 123 (Fig. 3C), as was observed previously for the R482G and R482T mutants of human BCRP (13). Login to comment

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
160 However, the two BCRP-transfected clones used in the previous study (represented by the open diamonds in Fig. 1, B and C) were obtained using cDNA derived from the MCF7/ AdVp cells, before it was known that these cells contain the R482T mutation. Login to comment
183 Additionally, we found that the R482T and R482G mutations in BCRP abolish MTX resistance. Login to comment
6 In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Login to comment
159 However, the two BCRP-transfected clones used in the previous study (represented by the open diamonds in Fig. 1, B and C) were obtained using cDNA derived from the MCF7/ AdVp cells, before it was known that these cells contain the R482T mutation. Login to comment
182 Additionally, we found that the R482T and R482G mutations in BCRP abolish MTX resistance. Login to comment

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
43 We generated and expressed the human wild-type ABCG2 and its variants R482G and R482T in Sf9 cells, and we characterized their transport and ATP hydrolytic activity. Login to comment
60 Wild-type ABCG2 (Arg-482) and its variants (R482T and K86M) were created using ABCG2-R482G cDNA as a template (4) by overlap extension PCR (30). Login to comment
61 Two internal complementary primer pairs were used with each containing the specific mutation as follows: the Arg- primer pairs were 5Ј-TTATTACCAATGCGCATGTTACC-3Ј and 5Ј-GG- TAACATGCGCATTGGTAATAA-3Ј; the R482T primer pairs were 5Ј-T- TATTACCTATGACGATGTTACC-3Ј and 5Ј-GGTAACATCGTCATAG- GTAATAA-3Ј; and the K86M primer pairs were 5Ј-TGGAGGCATGTC- TTCGTTATT-3Ј and 5Ј-TAATAACGAAGACATGCCTCCA-3Ј, respectively. Login to comment
64 The PCR products containing the Arg-482 or R482T coding sequence were digested with PstI and MscI enzymes and ligated between the corresponding sites of the pAcUW21-L/ABCG2 vector. The PCR product coding for the K86M variant was digested with NotI and SpeI enzymes and ligated to the NotI and SpeI sites of the pAcUW21-L/ABCG2 (R482G) vector. The mutations were confirmed by sequencing the PstI-MscI or the NotI-SpeI fragments of the constructs, respectively. Login to comment
108 In the present study we utilized this expression system to produce the wild-type human ABCG2 protein and its variants, R482G and R482T, as well as a catalytic center mutant, K86M. Login to comment
109 Site-directed mutagenesis was performed on a human ABCG2 cDNA, which possesses Gly at position 482 (4), to create the wild-type (Arg-482) and the R482T and K86M variants of the ABCG2 half-transporter. Login to comment
125 We found equal expression levels for the wild-type, R482G, R482T, and K86M/ R482G mutant variants (data not shown). Login to comment
132 Lane 1, R482T/Sf9; lane 2, wild-type ABCG2/Sf9; lane 3, R482G/Sf9; lane 4, K86M-R482G/Sf9; lane 5, wild-type ABCG2/ HL60; lane 6, wild-type ABCG2/HL60 grown in the presence of 2.5 ␮g/ml tunicamycin; and lane 7, beta-galactosidase/Sf9. Login to comment
140 These experiments indicate that wtABCG2 and the R482G or R482T variants actively extrude MX in the intact Sf9 cells, whereas the K86M mutant is inactive in this respect. Login to comment
144 As documented, at increasing MX concentrations, the wild-type (R482) ABCG2 was found to be somewhat less effective in protecting Sf9 cells against MX accumulation than the other two amino acid 482 variants; the accumulation of MX was about 15 Ϯ 4% more in wtABCG2- expressing cells than in cells containing the R482G or R482T variants. Login to comment
145 In addition, the MX transport capacities of the amino acid 482 variants R482G and R482T were found to be similar in these experiments. Login to comment
146 Next we performed similar transport studies for the fluorescent dye rhodamine 123, which was indicated to be a transported substrate of the ABCG2 variants R482G and R482T (20). Login to comment
147 As shown in Fig. 3, we found that insect cells expressing the wild-type ABCG2 or the K86M mutant accumulated significantly more rhodamine 123 than cells expressing the R482G or R482T variants. Login to comment
148 In contrast to that found for the wild-type ABCG2, in the R482G or R482T variants the addition of FTC greatly increased rhodamine 123 accumulation indicating an ABCG2-dependent extrusion of this compound. Login to comment
149 These data support the fact that MX is a substrate for wtABCG2 and its amino acid 482 variants, although MX transport may be less efficient by the wild-type protein than by the R482G or R482T variants. Login to comment
150 In contrast, the wild-type ABCG2 is practically inactive for rhodamine 123 transport, whereas the R482G and R482T mutants actively extrude this fluorescent dye. Login to comment
167 Fig. 4, B and C, shows that the rate of Hst influx was low and was greatly increased by the inhibitor FTC in the wild-type ABCG2 (Fig. 4B) and also in the R482G and R482T variants (Fig. 4, B and C). Login to comment
173 In the present study we have compared the ATPase activity of the wild-type human ABCG2 and its variants (R482G, R482T, and K86M) in the presence and absence of a variety of potential ABCG2 substrates or inhibitors. Login to comment
175 Despite the similar ABCG2 expression levels, this basal ATPase activity was ϳ1.5-fold higher in case of the R482G variant (71 Ϯ 10.8 nmol of Pi/mg of membrane protein/ min) than in case of the wild-type ABCG2 or the R482T variant (45 Ϯ 8.85 and 46 Ϯ 9.04 nmol of Pi/mg of membrane protein/ min, respectively) and negligible in the ABCG2-K86M mutant (5 Ϯ 0.5 nmol of Pi/mg of membrane protein/min). Login to comment
177 In wtABCG2 and in R482G and R482T proteins FTC (or Ko 143) powerfully inhibited the vanadate-sensitive ATPase activity. Login to comment
179 In case of the R482G and R482T mutants, vanadate-sensitive ATPase activity could be greatly stimulated by several potential ABCG2 substrates (e.g. prazosin, mitoxantrone, adriamycin, rhodamine 123, benzamil, and camptothecin), corresponding to the transport activity of these proteins. Login to comment
183 We found that the effect of different ABCG2 substrates on the ATPase activity of variants R482G and R482T was almost similar. Login to comment
186 Rhodamine 123, a specific substrate of the amino acid 482 mutants, gave a maximum of 30% stimulation, and the Kact values were 4.5 and 4 ␮M, respectively, for the R482G and R482T mutants. Login to comment
187 The maximum extent of stimulation of the R482G and R482T-ATPases by prazosin was 100 and 70%, and the Kact values were 7 and 3 ␮M. Login to comment
188 Mitoxantrone showed a maximum of 50% stimulation of the ATPase activity of the R482G and R482T variants, with Kact values of 1 and 0.8 ␮M, respectively. Login to comment
189 FTC was found to be an effective inhibitor for the vanadate-sensitive ABCG2-ATPase activity both for the wild-type enzyme (76% inhibition at 10 ␮M) and the R482G variant (74% inhibition at 10 ␮M); however, its inhibitory effect was smaller and required higher FTC concentrations in the case of the R482T variant (37% inhibition at 10 ␮M) (see Fig. 5). Login to comment
190 The concentration of FTC causing half-maximal inhibition of the ABCG2-ATPases was 0.4 ␮M for the wild-type enzyme, 0.5 ␮M for the R482G variant, and 0.7 ␮M for the R482T mutant. Login to comment
192 A, MX accumulation in Sf9 cells expressing beta-galactosidase (beta-gal), wild-type ABCG2 (Arg-482), and the R482G, R482T, or K86M mutants. Login to comment
214 We found that 8-azido-ATP binding was similar in the wild-type and in the R482G, R482T, and K86M mutant variants under these conditions (Fig. 6B). Login to comment
224 Rhodamine 123 accumulation in Sf9 cells expressing the wild-type (Arg-482), R482G, R482T, or K86M/R482G variants of ABCG2. Login to comment
228 As shown in Fig. 8, we found significant differences in the adenine nucleotide trapping of the wild-type, R482G, and R482T ABCG2 transporters. Login to comment
229 When we analyzed these data with a quantitative PhosphorImager, in the absence of added drugs the wild-type ABCG2 (Fig. 8, lane 4) showed a more pronounced nucleotide trapping activity, 2.6 Ϯ 0.03- and 2.05 Ϯ 0.5-fold of the R482G and R482T variants (lanes 7 and 10), respectively. Login to comment
230 The addition of prazosin, an activator of the ABCG2-ATPase of the R482G and R482T variants, significantly stimulated nucleotide trapping in the same variants (see Fig. 8, lanes 6 and 9, the stimulations were 2.0 Ϯ 0.22- and 1.7 Ϯ 0.04-fold, respectively). Login to comment
237 DISCUSSION In this paper we describe the expression and detailed functional analysis of the wild-type human ABCG2 multidrug transporter and its mutant variants R482G, R482T, and K86M. Login to comment
241 According to the established sequence in the human genome data base (GenBankTM accession number AF103796), the wild-type ABCG2 contains arginine at this position, whereas the other two variants R482G and R482T most probably were generated during drug selection in tumor cell lines (20, 22). Login to comment
245 The aim of the present study was to provide a quantitative characterization both for the transport and the ATP hydrolytic activity of the wtABCG2 and its variants R482G and R482T. Login to comment
255 B and C, the rate of Hst influx (⌬ fluorescence/⌬ time) into Sf9 cells expressing the wtABCG2 or the R482T mutant (B) and R482G or K86M/R482G (C) was determined at different Hst concentrations with or without the ABCG2 inhibitor FTC. Login to comment
258 ATPase activity measured in membranes of Sf9 cells expressing the wild-type, R482G, R482T, or K86M/R482G variants of human ABCG2. Login to comment
263 ATPase activity determined in membranes of wtABCG2- (B), R482G- (C), or R482T (D)-expressing Sf9 cells. Login to comment
273 Comparing the transport activity of the wtABCG2 and its mutant variants, we found that the wild-type and the two amino acid 482 variants actively exported mitoxantrone, whereas rhodamine 123 extrusion could only be observed in cells expressing the R482G and R482T proteins. Login to comment
280 In the present experiments a relatively high basal ATPase activity was found in case of the wild-type ABCG2 and the R482T variant, too. Login to comment
281 However, stimulation of the ABCG2-ATPase activity was observed only in Sf9 membranes containing the R482G or R482T variants and not the wild-type ABCG2. Login to comment
283 [␣-32 P]8-azido-ATP binding of the wild-type and R482G, R482T, or K86M/R482G mutant ABCG2 proteins. Login to comment
296 Comparison of the effect of different compounds on the [␣-32 P]8-azido-ATP trapping of the wild-type, and R482G or R482T variants of the ABCG2 protein. Login to comment
297 Sf9 membranes (150 ␮g) containing wtABCG2 (lanes 2-4), ABCG2-R482G (lanes 5-7), ABCG2-R482T (lanes 8-10), or beta-galactosidase (lane 1) were incubated for 2 min at 37 °C with 2 ␮M 8-azido-[␣-32 P]ATP, 1 mM sodium orthovanadate, and 2 mM Co2ϩ as described under "Experimental Procedures." Login to comment
309 In case of the R482G and R482T variants, this endogenous stimulation (caused by endogenous activators, producing what we observe as a high base-line ATPase) is only partial, and further stimulation by the transported compounds is observed. Login to comment
325 We found that the nucleotide trapping characteristics of the R482G or R482T variants were different from the wild-type ABCG2. Login to comment
326 Whereas this transition state formation in variants R482G and R482T could be significantly stimulated by various ABCG2 substrates (prazosin and mitoxantrone), the transition state formation of the wild-type ABCG2 could not be stimulated but rather was inhibited by these compounds. Login to comment

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: 12642696 [PubMed] Honjo Y et al: "Single-nucleotide polymorphism (SNP) analysis in the ABC half-transporter ABCG2 (MXR/BCRP/ABCP1)."

No. Sentence Comment

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

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

147 It has been reported that the R482G and R482T mutant variants of ABCG2 have altered substrate specificity. Login to comment

PMID: 12810652 [PubMed] Rajendra R et al: "Differential effects of the breast cancer resistance protein on the cellular accumulation and cytotoxicity of 9-aminocamptothecin and 9-nitrocamptothecin."

No. Sentence Comment

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

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
25 However, a puzzling feature of this study was that MCF7 cells stably transfected with an ABCG2 cDNA, which has since been determined to have an R482T mutation, did not recapitulate MTX resistance. Login to comment
29 The results of these experiments show that wild-type ABCG2 is indeed capable of mediating the transport of MTX, but that neither the R482G nor the R482T mutants are able to transport this agent to any extent. Login to comment
49 HEK293 cells transfected with ABCG2-R482, ABCG2-R482G, and ABCG2-R482T were generated using the respective cDNAs cloned into pcDNA3.4 LLC/PK1 cells transfected with MRP2 expression vector, and HEK293 cells transfected with MRP3 expression vector were described previously (19, 20). Login to comment
61 RESULTS Analysis of MTX Transport by Wild-Type ABCG2, ABCG2R482G, and ABCG2-R482T. Login to comment
68 Membrane vesicles were prepared from HEK293 cells transfected with ABCG2-R482G and ABCG2-R482T, so as to analyze both types of R482 mutations that have been identified in drug-resistant cell lines (4). Login to comment
70 However, by contrast with ABCG2-R482, neither of the mutants were able to transport [3 H]MTX to any extent, in that MgATP-dependent uptake of this agent by ABCG2-R482G and ABCG2-R482T-enriched vesicles was indistinguishable from uptake by the same vesicles in the presence of MgAMP, or uptake by negative control vesicles in the presence of either MgATP or MgAMP (Fig. 2, B and C). Login to comment
82 Membrane vesicles were prepared from HEK293 cells transfected with parental vector (Lane 1), ABCG2-R482 (Lane 2), ABCG2-R482G (Lane 3), and ABCG2-R482T (Lane 4). Login to comment
99 We reported previously that several members of the MRP family that are capable of transporting MTX are also competent in mediating the Fig. 2. Time course of ATP-dependent uptake of [3 H]MTX by ABCG2-R482, ABCG2-R482G, and ABCG2-R482T. Login to comment
100 Membrane vesicles (10 ␮g) prepared from HEK293 cells transfected with ABCG2-R482 (A), ABCG2-R482G (B), and ABCG2-R482T (C), and from parental plasmid-transfected control cells, were incubated at 37°C in uptake medium containing 100 ␮M [3 H]MTX and 4 mM MgATP (solid symbols) or 4 mM MgAMP (open symbols). Login to comment
107 The results of experiments in which uptake of FA was examined were in complete accord with the properties of ABCG2 as determined from the MTX transport experiments, in that whereas membrane vesicles prepared from wild-type ABCG2-transfected cells were able to catalyze the uptake of 100 ␮M [3 H]FA, uptake was not detected for R482G or R482T (Fig. 6, A-C). Login to comment
143 Membrane vesicles (10 ␮g) prepared from HEK293 cells transfected with ABCG2-R482 (A and D), ABCG2-R482G (B), ABCG2-R482T (C), and MRP3 (E), from LLC/PK1 cells transfected with MRP2 (F), and from the respective parental plasmid-transfected counterparts, were incubated at 37°C in uptake medium containing 100 ␮M [3 H]FA (A-C) or 100 ␮M [3 H]leucovorin (D-F), and 4 mM MgATP (solid symbols) or 4 mM MgAMP (open symbols). Login to comment

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
91 For authentic BCRP in vitro-transcribed RNA (cRNA) R482 and R482T standards, the Tms of the hybridization probes are 62°C and 57.2°C, respectively. Login to comment
218 Recently, the R482T and R482G mutations of BCRP have been described. Login to comment
220 Additionally, a recent study (31) indicates that the R482T and R482G mutations result in loss of the ability to transport methotrexate. Login to comment

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

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

13 A doxorubicin-resistant human breast cancer cell line MCF-7 AdVp3000 and a mitoxantrone-resistant human colon carcinoma cell line S1-M1-80 expressed R482T- and R482G-BCRP, respectively and showed high resistance to mitoxantrone and doxorubicin.5,6,13 Doxorubicin-resistant murine fibroblast cell lines also expressed R482M- or R482S-BCRP and showed high resistance to mitoxantrone and doxorubicin.14 We recently identified the substitution R482M in a doxorubicin-resistant human T cell line MT-4/DOX500.23 We made 32 mutant BCRP cDNAs with an amino acid substitution in the TMs and examined the effect of the substitutions on cellular drug resistance. Login to comment
64 PA/R482N, PA/R482C, PA/R482M, PA/R482S, PA/R482T, PA/R482V, PA/R482A, PA/R482G, PA/R482E PA/R482W and PA/R482D (Group 2) showed higher degrees of resistance to mitoxantrone than to SN-38. Login to comment
135 MCF-7 AdVp3000 established by treating MCF-7 cells with 3 ␮g/ml of doxorubicin and 5 ␮g/ml of verapamil overexpressed R482T-BCRP.5,13 S1-M1-80 established by treating human colon carcinoma S1 cells with 80 ␮M mitoxantrone overexpressed R482G-BCRP.6,13 These resistant cells exhibited high resistance to doxorubicin and mitoxantrone.5,6,13 We found recently that MT-4/DOX500 cells established by treating human T cell MT-4 cells with 500 ng/ml of doxorubicin overexpressed R482M-BCRP.23 Two doxorubicin-resistant murine fibroblast lines 88.6/D800-A and 88.6/D800-B overexpressed R482M-BCRP and R482S-BCRP, respectively.14 Another doxorubicin-resistant cell line KOT52/D800 from mouse fibroblasts co-expressed wild-type BCRP and R482M-BCRP.14 In addition to anthracyclines and mitoxantrone, cells transfected with R482-mutant BCRP cDNAs also showed high resistance to methotrexate.28 Theoretically, 9 FIGURE 6 - Accumulation of [3 H]mitoxantrone in R482-mutant BCRP transfectants. PA/WT2, PA/R482G and PA/R482S were mixed populations of the transfected cells established after the 2-step selection with 120 ng/ml methotrexate and 1 ng/ml mitoxantrone. Login to comment
147 Cells transfected with R482G- (GGG), R482M- (ATG), R482T- (ACG), or R482S- (AGT and AGC) BCRP cDNA showed greater resistance to mitoxantrone and doxorubicin than PA/WT2 (Fig. 2). Login to comment
148 As described above, human drug-resistant cell lines MCF-7 AdVp3000, S1-M1-80, MT-4/DOX500 and a mouse drug-resistant line 88.6/D800-B overexpressed R482T- (ACG), R482G- (GGG), R482M- (ATG) and R482S- (AGT) BCRP, respectively.5,6,13,14,23 The other possible mutations are R482W (TGG) and R482K (AAG). Login to comment
149 PA317 cells expressing R482W- BCRP (PA/R482W) showed somewhat higher levels of resistance to mitoxantrone and doxorubicin than PA/WT2, but the resistance levels were lower than those in PA/ R482G, PA/R482M, PA/R482T and PA/R482S. Login to comment
163 Group 2 members (PA/R482N, PA/R482C, PA/R482M, PA/R482S, PA/R482T, PA/R482V, PA/ R482A, PA/R482G, PA/R482E PA/R482W and PA/R482D) showed higher degrees of resistance to mitoxantrone than to SN-38. Login to comment

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
160 A Japanese study also suggested that crossresistance to mitoxantrone was relatively high in cell lines transfected with BCRP R482T as compared with those transfected with BCRP R482 (Komatani et al., 2001). Login to comment
161 This conclusion was refuted by another study demonstrating high levels of mitoxantrone resistance in cell lines expressing wild-type R482 as well as mutated R482G and R482T (Volk et al., 2002a). Login to comment
163 However, MCF-7/AdrVp cells, which overexpress the R482T mutation of BCRP and display considerable resistance to daunorubicin (25-40-fold) (Ross et al., 1995; Doyle et al., 1998), accumulate and retain idarubicin well, and are only minimally resistant to idarubicin (1.8-fold) (Ross et al., 1995). Login to comment
166 However, in this same work, investigations of the BCRP-transfected MCF-7 or MDAMB-231 cells developed in our laboratory did not find resistance or low accumulation of methotrexate in the transfected cells. Since these MCF-7 or MDAMB231 cells were transfected with BCRP cDNA derived from MCF-7/AdrVp cells, the transfected cells consequently overexpressed the R482T mutation of BCRP. Login to comment
167 This realization led to further studies of methotrexate transport, which revealed that methotrexate resistance, reversible with GF120918, correlated with BCRP expression in cell lines that overexpressed wild-type BCRP, but not the mutant forms (R482T or R482G) (Volk et al., 2002b). Login to comment

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: 14617793 [PubMed] Brooks TA et al: "Taxane-based reversal agents modulate drug resistance mediated by P-glycoprotein, multidrug resistance protein, and breast cancer resistance protein."

No. Sentence Comment

60 MRP-1 expression in cell lines Cell line Pgp MRP-1 BCRP HL60-wt À À À HL60-ADR À + À 8226-wt À À + 8226-Dox6 + À + 8226-MR20 À À + MCF7/S À À + MCF7/R + À + MCF7/MRP1-10 À + + MCF7/AdrVp À À +a a R482T mutation in BCRP. Login to comment

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

0 Functional Characterization of Human Breast Cancer Resistance Protein (BCRP, ABCG2) Expressed in the Oocytes of Xenopus laevis TAKEO NAKANISHI, L. AUSTIN DOYLE, BRET HASSEL, YUETONG WEI, KENNETH S. BAUER, SUHLAN WU, DAVID W. PUMPLIN, HONG-BIN FANG, and DOUGLAS D. ROSS The Program in Experimental Therapeutics (T.N., L.A.D., B.H., Y.W., K.S.B., S.W.) and Division of Biostatistics (H.-B.F.), University of Maryland Greenebaum Cancer Center, the Division of Hematology and Oncology, Departments of Medicine (T.N., L.A.D., D.D.R.), Anatomy and Neurobiology (D.W.P.), Microbiology (B.H.), and Epidemiology and Preventative Medicine (H.-B.F.), University of Maryland School of Medicine, Baltimore Maryland; School of Pharmacy, University of Maryland, Baltimore, Baltimore, Maryland (K.S.B.); and the Baltimore Veterans Administration Medical Center, Baltimore Maryland (D.D.R.) Received May 16, 2003; accepted August 26, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT To evaluate the function and substrate specificity of human breast cancer resistance protein (BCRP, ABCG2) in the absence of cofactors or heterologous partner proteins, Xenopus laevis oocytes were injected with cRNA of wild-type or mutant (R482T) BCRP. Login to comment
2 Accumulation and efflux assays revealed that oocytes expressing R482T transported daunorubicin (DNR), mitoxantrone (MX), rhodamine 123, and flavopiridol (FLV), whereas wild-type BCRP transported only MX and FLV, in agreement with observations in mammalian and other systems. Login to comment
5 When oocytes were coinjected with R482T and R482T/S187T, DNR transport was inhibited in a manner dependent on the amount of R482T/S187T cRNA added, consistent with the idea that the active form of BCRP is a homodimer or homomultimer. Login to comment
28 Mutant forms of BCRP with threonine or glycine in place of arginine at codon 482 (R482T or R482G) have been described in drug-selected cell lines (Honjo et al., 2001; Komatani et al., 2001), including the original isolate of BCRP from MCF-7/ AdrVp cells (Doyle et al., 1998), which express the R482T mutation. Login to comment
29 Compared with the wild-type form, the R482T or R482G mutations are able to transport anthracyclines and Rho123 (Honjo et al., 2001; Ozvegy et al., 2002) but not methotrexate (Volk et al., 2002). Login to comment
57 The PCR product was ligated into the pCR Blunt TOPO II TABLE 1 Summary of cDNA constructs made as templates for producing BCRP cRNA Construct Plasmid Features of the BCRP cRNA Produced RNA Polymerase Consensus Sequence Upstream from BCRP cDNA I pSP64poly(A) R482T-poly(A) SP6 II pCR Blunt TOPO II Kozak-R482T-poly(A) T7 III pSD64TR 5ЈUTR-Kozak-R482T-3ЈUTR-poly(A) other BCRP forms: 5ЈUTR-Kozak-R482-3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/S187T- 3ЈUTR-poly(A) 5ЈUTR-Kozak-R482T/ S187A-3ЈUTR-poly(A) SP6 III- pSD64TR 5ЈUTR-R482T-3ЈUTR-poly(A) SP6 5ЈUTR, 3ЈUTR, portions of the 5Ј- and 3Ј-UTR of the Xenopus laevis beta-globin gene; Kozak, modified sequences proximate to the start codon, as described under Materials and Methods; Poly(A), addition of a poly(A) tail, as described under Materials and Methods. Login to comment
105 Oocytes injected with water or BCRP cRNA (wild type, R482T) were fixed in 4% paraformaldehyde in PBS, then immersed overnight in 30% sucrose in PBS. Login to comment
116 However, injection of oocytes with BCRP cRNA directly transcribed from BCRP (R482T) cDNA (Doyle et al., 1998) failed to produce BCRP protein, as detected by Western blot examination or by functional assays. Login to comment
128 Oocytes were injected with cRNA coding for the wild-type (R482) or mutant R482T form of BCRP, then fixed and sectioned as described under Materials and Methods. Login to comment
129 Exposure of these sections to the BXP-34 monoclonal antibody to BCRP resulted in immunoreactivity only in the plasma membranes of oocytes expressing wild-type (Fig. 2A) or the mutant R482T form of BCRP (Fig. 2B), as detected by immunofluorescence microscopic analysis. Login to comment
132 Functional expression of BCRP (R482T) was examined by 10 ␮M DNR accumulation (A) or Western blot (B) in oocytes injected with 50 nl of water (control) or cRNA (1 ␮g/␮l) transcribed from construct I, II, or III. Login to comment
133 For convenience, the R482T form of BCRP was used to enable monitoring of function by DNR accumulation. Login to comment
140 Western blot examination of oocytes injected with wild-type or BCRP (R482T) cRNA at the time of the immunofluorescence experiments confirmed robust expression of BCRP, with a molecular mass of 70 kDa (Fig. 2D). Login to comment
141 Accumulation and Efflux of MX or DNR in Oocytes Expressing BCRP (R482T). Login to comment
142 To determine whether functional BCRP was expressed in X. laevis oocytes, we monitored the accumulation of DNR and [3 H]MX in control or BCRP(R482T) injected oocytes. Login to comment
144 Oocytes expressing BCRP (R482T) showed a remarkable reduction in the accumulation of DNR or MX over the 120-min period. Login to comment
147 To examine whether the diminished accumulation of DNR or MX in the BCRP (R482T)-expressing oocytes was caused by enhanced drug efflux, the efflux of these compounds in the presence or absence of 5 ␮M FTC was compared with that of control oocytes. Login to comment
148 Before the efflux studies, the drug of interest was preloaded into the control or BCRP (R482T)-expressing oocytes by incubating with drug for 90 min. Login to comment
149 In the case of BCRP (R482T)-expressing oocytes, 5 ␮M FTC was added, which resulted in intracellular drug accumulation comparable with levels attained in the control (water injected) oocytes; after preloading and subtraction of background, the accumulation of DNR in BCRP (R482T)-injected oocytes was 3.79 Ϯ 0.15 pmol/oocyte, whereas that in control was 4.55 Ϯ 0.31 pmol/oocyte. Login to comment
150 [3 H]MX accumulation in control and preloaded R482T-expressing oocytes was 0.87 Ϯ 0.16 and 0.96 Ϯ 0.91 pmol/oocyte, respectively. Login to comment
151 Both DNR and MX were effluxed more rapidly from the BCRP (R482T)-expressing oocytes, compared with control (Fig. 3, D and E). Login to comment
152 This rapid efflux was not observed in R482T-expressing oocytes in the presence of FTC. Login to comment
156 Substrate Specificity of Wild-Type and Mutant (R482T) BCRP Expressed in X. laevis Oocytes. Login to comment
157 Accumulation assays using DNR, MX, FLV, and Rho123 were performed to study the effect of the Arg-to-Thr mutation at codon 482 on the substrate specificity of BCRP in the X. laevis oocyte system. Login to comment
158 Figure 4 shows the intracellular accumulation of the four compounds in oocytes injected with R482 or R482T BCRP in the absence or presence of FTC. Login to comment
159 Accumulation of all four compounds in the BCRP (R482T)-expressing oocytes was markedly reduced. Login to comment
162 A Mutation in the ABC Signature Motif Inactivates BCRP (R482T) Transport of DNR; BCRP Expressed in X. laevis Oocytes Functions as a Homodimer or Homomultimer. Login to comment
163 To determine whether dimerization is required for BCRP activity, a mutant construct was created by substituting the highly conserved serine at residue 187 in the ABC signature motif with threonine (R482T/S187T) or alanine (R482T/S187A). Login to comment
165 As shown in Fig. 5A, no difference was observed in expression levels of BCRP protein among oocytes injected with cRNA encoding BCRP (R482T) or these codon 187 mutant BCRP constructs. Login to comment
167 Confocal immunofluorescence microscopic analysis was performed in oocytes injected with 50 nl of cRNA of BCRP (wild-type, R482) (A), BCRP (R482T) (B), or water as control (C), using the BXP-34 antibody. Login to comment
168 A and B show the distribution of BCRP (R482 or R482T) at the oocyte surface (C). Login to comment
170 Cell lysate (25 ␮g) from the oocytes injected with water (control), BCRP (R482), or BCRP (R482T) cRNA was loaded per lane of the gel. Login to comment
173 Accumulation of 25 ␮M DNR (A), 10.8 ␮M MX (B) or varying concentrations of DNR (C, DNR: 10 to 250 ␮M) in control (E) or BCRP (R482T)-expressing oocytes (F). Login to comment
174 Efflux of DNR (D) or MX (E) from control (E) or BCRP (R482T)-expressing oocytes (F) afterpreloading with DNR or MX in the presence of 5 ␮M FTC was monitored at room temperature, as described under Materials and Methods. Login to comment
175 Œ, drug efflux in BCRP (R482T)-expressing oocytes in the presence of 5 ␮M FTC. Login to comment
178 *, statistically significant difference (p Ͻ 0.05, student`s t test) between control and R482T-expressing oocytes. Login to comment
180 a manner analogous to a dominant-negative mutation, if coinjected with the active BCRP (R482T) cRNA. Login to comment
181 Indeed, when BCRP (R482T) was coinjected with varying amounts of the "dominant-negative" construct S187T/R482T, the transport of DNR was significantly inhibited in a manner dependent on the amount of the S187T mutant construct added (Fig. 5C), strongly suggesting that homodimerization or multimerization is essential for BCRP activity. Login to comment
185 First, DNR accumulation in BCRP (R482T)-expressing oocytes was studied (Fig. 6A). Login to comment
188 In contrast, MX and Rho123 only partially inhibited DNR transport by BCRP (R482T), and 10-fold molar excess of TPT was not inhibitory at all. Login to comment
189 When BCRP substrate inhibition of MX accumulation was studied (Fig. 6B) using a 23-fold molar excess of Rho123, TPT, DNR, or FLV as competitors, only FLV caused partial but statistically significant reversal of BCRP R482 or R482T transport of MX. Login to comment
190 Interestingly, when the competition studies were done with respect to FLV accumulation (Fig. 6C), none of the competing BCRP substrates was able to inhibit FLV efflux by wild-type or R482T BCRP, despite their presence in a 10-fold or, in the case of DNR, a 100-fold molar excess relative to FLV. Login to comment
193 Accumulation of BCRP substrates DNR (25 ␮M, A), MX (0.5 ␮M, B), FLV (25 ␮M, C), and Rho123 (5 ␮M, D) was measured in control (Ⅺ), BCRP (R482T)-expressing oocytes (f) or BCRP (R482, wild-type)-expressing oocytes (u). Login to comment
199 Expression of dominant-negative construct was confirmed by Western blot (A) and by DNR accumulation (25 ␮M, B) in control or R482T-expressing oocytes (f), R482T/S187T- expressing oocytes (o), or R482T/S187A-expressing oocytes (gray o). Login to comment
201 DNR accumulation (25 ␮M, C) was determined in oocytes coinjected with 24 ng of R482T cRNA and different amounts (0 to 72 ng) of the R482T/S187T construct cRNA (f). Login to comment
202 As control, the accumulation of 25 ␮M DNR was also determined in control (Ⅺ) or oocytes injected with 24 ng of R482T/S187T cRNA only (o). Login to comment
204 *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001 represent statistically significant differences (Student`s t test) in coinjected oocytes compared with accumulation in oocytes injected with R482T cRNA only. Login to comment
207 Our results further demonstrate that oocytes injected with mutant R482T or wild-type BCRP cRNA express BCRP in the oocyte plasma membrane, as evidenced by confocal immunofluorescence microscopic analysis. Login to comment
212 Validation of the assay is based our observation that the function of BCRP expressed in the oocytes parallels that observed in mammalian systems (Doyle et al., 1998; Honjo et al., 2001; Robey et al., 2001; Minderman et al., 2002); the R482T mutant form of BCRP expressed in the oocytes efficiently transported DNR, Rho123, MX, and FLV, whereas the oocyte-expressed wild-type form transported only MX and FLV. Login to comment
213 Furthermore, FTC, in concentrations known to inhibit BCRP function in mammalian systems (Rabindran et al., 2000) also caused complete inhibition of both mutant R482T and wild-type BCRP forms expressed in the oocytes. Login to comment
216 In our studies, the diminished accumulation of DNR or MX displayed by BCRP R482T-expressing oocytes correlated with greater initial efflux rates of these drugs compared with control oocytes. Login to comment
224 Ⅺ, accumulation of these drugs in control oocytes for each condition (100%); f, accumulation in BCRP (R482T)-expressing oocytes; u, accumulation in BCRP (R482, wild-type)-expressing oocytes. Login to comment
233 Hence, we made two mutations of serine 187 in this signature motif of BCRP R482T, one to an amino acid that, like serine, has a polar side chain (threonine, S187T) and the other to a nonpolar side chain amino acid (alanine, S187A). Login to comment
234 The R482T mutant form of BCRP was used because it allowed us to monitor BCRP function by DNR accumulation. Login to comment
239 When the S187T mutation was coexpressed with BCRP R482T in the oocytes, the S187T mutant protein acted as would a dominant-negative inhibitor of BCRP transport of DNR, with the degree of inhibition increasing in proportion to the amount of S187T cRNA injected. Login to comment
249 In the substrate interaction studies in BCRP expressing X. laevis oocytes presented here (Fig. 6 and Table 2), we found a significant inhibitory effect of FLV on BCRP-mediated MX transport, in good agreement with Robey et al. (2001); furthermore, FLV and MX significantly inhibited DNR accumulation in R482T-expressing oocytes. Login to comment
255 It is possible that BCRP also has multiple substrate interaction sites; however, because we did TABLE 2 Summary of substrate interaction studies Substrate Competitor Inhibitor Substrate (BCRP form) DNR MX FLV TPT Rho123 FTC DNR (R482T) Partial Yes No Ϯ Yes MX (W or R482T) No Partial No No Yes FLV (W or R482T) No No No N.D. Yes W, wild type; R482T, BCRP R482T; No, no inhibition by competitor; Yes, inhibition by competitor, P Ͻ 0.01; Partial, partial (approximately 50%) inhibition by competitor, P Ͻ 0.01; Ϯ, slight (approximately 20%) inhibition by competitor, P Ͻ 0.05; N.D., no data. Login to comment

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: 15027118 [PubMed] Candeil L et al: "ABCG2 overexpression in colon cancer cells resistant to SN38 and in irinotecan-treated metastases."

No. Sentence Comment

127 The lack of cross-resistance to doxorubicin has been explained by a single mutation at codon 482 (doxorubicin is poorly transported by the wild-type variant of ABCG2 in which the amino acid at position 482 is an arginine but can be transported by the R482T mutant32). Login to comment

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: 15139851 [PubMed] Alqawi O et al: "Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding."

No. Sentence Comment

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

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

No. Sentence 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

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

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: 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
183 In contrast, the R482T mutation may play a critical role in this decreased MXR retention, rather than in the increased velocity of the transporter. Login to comment

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
85 Microsomal membranes were isolated from transiently cotransfected-infected HeLa cells, drug-sensitive MCF-7 cells, drug-resistant MCF-7/AdVp3000, which overexpress the R482T variant of human ABCG2, and drug resistant MCF-7/DX1 cells, which overexpress human P-gp. Login to comment
97 Our data show that ABCG2 is localized at the surface in both HeLa cells transfected with any of the three ABCG2 variants (R482G, R482, or R482T) and in MCF-7/AdVp3000 cells (Fig. 2, A and B). Login to comment
98 The R482 (wild type), R482G, and R482T ABCG2 variants are expressed equivalently in HeLa cells (Fig. 2A). Login to comment
113 Rhodamine 123 is a well established substrate of the R482G and R482T variants of ABCG2 (Honjo et al., 2001) and of P-gp (Hrycyna et al., 1998), but it is not a substrate of wild-type R482 variant of ABCG2 (Honjo et al., 2001). Login to comment
117 As can be seen in Fig. 3A, cells expressing ABCG2 (R482G or R482T) or P-gp exclude rhodamine 123 equivalently in the absence of inhibitor. Login to comment
123 Transport of mitoxantrone by the R482T ABCG2 variant seems to be the least sensitive to addition of 5 ␮M cyclosporin A, followed by the R482G and the wild-type R482 ABCG2 proteins. Login to comment
130 A, transiently transfected HeLa cells expressing ABCG2 labeled with 5D3; R482G (-), R482 (⅐ ⅐ ⅐), R482T (-), R482G cells labeled with IgG2b (- ⅐ - ⅐ -). Login to comment
156 A to F, histograms show HeLa cells transfected with the empty pTM1 vector (shaded peak), R482G (-), R482 (- ⅐ - ⅐ -), R482T (- - -), or P-gp (-). Login to comment
168 These experiments show that cells expressing ABCG2 (R482T or R482G) or P-gp extrude daunomycin in the absence of the inhibitor and that this transport is inhibited in the presence of GF120918. Login to comment
170 Together, our data demonstrate that cyclosporin A is only transported by P-gp, whereas daunomycin is transported by P-gp and the R482G and R482T variants of ABCG2 but not the wild-type ABCG2 (R482). Login to comment
175 To assess whether different variants of ABCG2 can be labeled with [125 I]IAAP, we performed the experiment with membrane isolates from S1-M1-80, MCF-7/FLV1000, and MCF-7/AdVp3000 cells that express the R482G, R482, and R482T variants of ABCG2, respectively. Login to comment
197 The ATPase activity of the R482G and R482T variants of ABCG2 are stimulated 2.2- and 1.4-fold, respectively, by the addition of 40 ␮M prazosin (Fig. 6, A and C). Login to comment
201 A, 50 ␮g of membrane protein from MCF-7 or MCF-7/AdVp3000 cells expressing the R482T variant of ABCG2. Login to comment
224 The assays were performed both in the absence (᭜) and presence (E) of 40 ␮M prazosin (for ABCG2) or 30 ␮M verapamil (for P-gp), at increasing concentrations of cyclosporin A. ATPase activity of ABCG2 proteins transiently expressed in HeLa cells R482G (A), R482 (B), and R482T (C), and in MCF-7/AdVp3000 cells (D). Login to comment
228 In this study, we used both the drug-resistant cell lines MCF-7/AdVp3000 that overexpress ABCG2 with the R482T gain of function mutation (Miyake et al., 1999) and MCF-7/ DX1 that overexpress P-gp, as well as HeLa cells transiently expressing ABCG2 (R482G, R482, or R482T variants) or P-gp to explore, in detail, the molecular effects that the immunomodulator cyclosporin A has on these transporters. Login to comment
233 In addition, we observe subtle differences in the transport of mitoxantrone in the absence and presence of cyclosporin A between the three ABCG2 variants (R482G, Arg482, and R482T) (Fig. 3, D-F), corroborating previous data suggesting amino acid 482 is an important determinant of substrate specificity. Login to comment
254 In conclusion, our findings demonstrate that the immunomodulator cyclosporin A is a substrate and an inhibitor of human P-glycoprotein, but that under the conditions examined, it is neither a substrate nor an inhibitor of any of the variants (R482G, R482, and R482T) of the related ABC transporter ABCG2. Login to comment

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

116 Prazosin is a substrate of the wtABCG2, R482G and R482T [34], and it stimulates the ATPase activity of R482G and T mutants, but it has no major effect on the ATPase activity of wtABCG2 [25]. Login to comment
149 Flow cytometry assay of mitoxantrone and rhodamine 123 extrusion from intact Sf9 cells expressing wtABCG2 and its Arg-482 mutants It has been documented earlier that mitoxantrone (MX) is a transported substrate both of the wtABCG2 and its R482G or T mutants, while rhodamine 123 (R123) is transported only by the R482G and R482T variants [22,25]. Login to comment

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: 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: 15735043 [PubMed] Nakamura Y et al: "Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance."

No. Sentence Comment

42 The transfectants have a mutant form of BCRP (R482T; ref. 25). 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
120 Similar migration patterns are observed for wild-type ABCG2 and the R482T variant (data not shown). Login to comment
158 In the stable drug-selected cell line MCF-7/AdVp3000 that overexpresses ABCG2 (R482T), the protein also migrates as a single species but at a higher molecular mass closer to the 75 kDa marker (Figure 4B, lane 1). Login to comment
183 (B) Crude membranes derived from MCF-7/AdVp3000 cells (10 µg) overexpressing ABCG2 (R482T) were treated with (+) or without (-) PNGase F. Login to comment
214 DISCUSSION Our studies demonstrate that ABCG2 transiently expressed in HeLa cells and ABCG2 (R482T) overexpressed in the drug-selected cell line, MCF-7/AdVp3000, are N-linked glycosylated. Login to comment

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
95 Results Establishment of stable transfectants We had previously generated stable transfectants containing empty vector, wild-type (R482) or mutant (R482G, R482T) ABCG2 [38]. Login to comment
122 Representative results are shown clones each of ABCG2-transfected cells expressing R482, R482T, R482G, V12M, Q141K or D620N ABCG2. Login to comment
132 Nearly all the values for the efflux and expression for cells transfected with mutant R482G and R482T ABCG2 fell above the regression line for wild-type ABCG2, confirming a gain-of-function in the transport of mitoxantrone in these cells. Login to comment
138 Efflux per unit expression values in cells transfected with mutant 482G and 482T ABCG2 were significantly higher than for wild-type ABCG2 (P=0.0003 R482G; P=0.0013 R482T). Login to comment
189 Values from the experiment in a were obtained for ABCG2-transfected HEK-293 clones expressing varying levels of 482R, R482G, R482T, V12M, Q141K, and D620N ABCG2 and a box plot was generated. Login to comment

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
174 Outside of cell Cell membrane C terminus ATP-binding site N terminus Inside of cellGln141Lys Walker A Walker B Signature motif Arg482Thr Val12Met Table 5. Login to comment

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

10 The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Login to comment
24 The precise substrate profile depends on a hotspot mutation at position 482 such that methotrexate is only transported by wild-type ABCG2 (R482) and anthracyclines and rhodamine 123 only by mutant ABCG2 (R482T or R482G), whereas Hoechst33342 and mitoxantrone are transported by all variants (9). Login to comment
40 Interestingly, the R482T mutation limited this prenylation-dependent effect, whereas the inhibitory potency of tectochrysin was even increased, especially towards rhodamine transport. Login to comment

PMID: 15970668 [PubMed] Burger H et al: "Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps."

No. Sentence Comment

25 Cell lines used: Caco2 parental, a human colon carcinoma cell line (ATCC, HTB-37); MCF-7 parental, a human breast carcinoma cell line (ATCC, HTB-22); MCF-7/MR, a mitoxantrone resistant counterpart of MCF-7 overexpressing wild-type ABCG2;11 MCF-7/AdVp3000, an MCF-7 derivative overexpressing the R482T variant of BCRP; KB-3-1, a human epidermoid carcinoma cell line (ATCC CCL-17) and the colchicine-selected derivative KB-8-5 overexpressing ABCB1;12 COS-1, an African green monkey cell line (ATCC, CRL-1650), and HEP3B, a hepatocarcinoma cell line (ATCC HB-8064). Login to comment
26 Cell lines used: Caco2 parental, a human colon carcinoma cell line (ATCC, HTB-37); MCF-7 parental, a human breast carcinoma cell line (ATCC, HTB-22); MCF-7/MR, a mitoxantrone resistant counterpart of MCF-7 overexpressing wild-type ABCG2;11 MCF-7/AdVp3000, an MCF-7 derivative overexpressing the R482T variant of BCRP; KB-3-1, a human epidermoid carcinoma cell line (ATCC CCL-17) and the colchicine-selected derivative KB-8-5 overexpressing ABCB1;12 COS-1, an African green monkey cell line (ATCC, CRL-1650), and HEP3B, a hepatocarcinoma cell line (ATCC HB-8064). Login to comment

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
52 MCF-7 cells (breast adenocarcinoma) selected and maintained in 3 µg/ mL doxorubicin and 5 µg/mL verapamil (MCF-7/AdVp3000) overexpress ABCG2 (R482T) at the plasma membrane. Login to comment
173 Because the cross-linker is not cell permeable, these experiments were performed in crude membrane preparations of drug-selected MCF-7/AdVp3000 cells that overexpress a homogeneously glycosylated population of ABCG2 (R482T) (Figure 3A, lane 1). Login to comment
174 Membrane preparations from these cells were used because of their uniform banding pattern on SDS-PAGE and their high level of ABCG2 (R482T) expression. Login to comment
175 In the absence of the cross-linker, ABCG2 (R482T) migrates as a monomer by SDS-PAGE analysis (Figure 3A, lane 1). Login to comment
195 In six-well plates, MCF-7/ AdVp3000 cells overexpressing ABCG2 (R482T) were incubated with a 1 mM solution of DPDPB for 2 h at 4 °C. Login to comment
201 When ABCG2 (R482T) was cross-linked with DPDPB, a higher molecular mass species at a molecular mass consistent with an ABCG2 (R482T) dimer (Figure 4, lane 2) was apparent, as compared to the monomer species observed in the absence of the cross-linker (Figure 4, lane 1). Login to comment
211 To determine if the extracellular cysteine residues are crucial for activity, the ABCG2∆EC-C variant was expressed in HeLa cells, and drug transport activity was measured as described in Experimental Procedures. We observed that the ABCG2∆EC-C variant had comparable levels of transport FIGURE 3: Nucleotide cross-linking of ABCG2 (R482T) in cell membranes. Login to comment
214 Proteins (5 µg in panel B and 25 µg in panel C) were separated by SDS-PAGE (7.5% gel) and detected by immunoblot analysis using the monoclonal antibody BXP-21 (1:1000) as described in Experimental Procedures. FIGURE 4: Sulfhydryl-reactive chemical cross-linking of ABCG2 (R482T) in whole cells. Login to comment

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
134 Other BCRP inhibitors include reserpine (a rauwolfia alkaloid),84 the pipecolinate derivatives VX-710 (Biricodar or Incel),85 and tryprostatin A (an Aspergillus fumigatus second metabolite).86 VX-710 increased uptake, retention, and cytotoxicity of mitoxantrone in cells overexpressing wild-type BCRP but had little effect on uptake, retention, and cytotoxicity of mitoxantrone and topotecan or SN-38 in cells expressing the BCRP mutant R482T.85 These results suggest that VX-710 is an inhibitor of wild-type BCRP only. Login to comment

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

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

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

168 Subsequently, these authors showed that the MCF-7/AdrVp subline, which does not express P-gp, expresses high levels of a BCRP mutant (R482T) which can transport daunorubicin [10]. Login to comment

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

47 Interestingly, however, they were able to inhibit only wild-type (R482) ABCG2, but not the mutant (R482T) transporter [23], as this was also the case with the third-generation P-gp inhibitor VX-710 (biricodar, Incel) [24]. Login to comment
59 Cyclosporin A was considered, however, as a broad-spectrum inhibitor on the basis of its 3-fold induced increase in mitoxantrone accumulation and cell growth sensitization [29], and was reported as a potent ATPase inhibitor of recombinant R482T mutant ABCG2 in insect cell membranes [8]. Login to comment
69 Interestingly, the R482T hotspot mutation altered the impact of prenylation on the inhibitory potency; tectochrysin being the best compound with an IC50 of 1.9 mmol/l. Login to comment
88 The interaction was altered 2-fold by the R482T/G hotspot mutations. Login to comment

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: 16690355 [PubMed] Modok S et al: "Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer."

No. Sentence Comment

46 Ortataxel also inhibits MRP1, whereas both compounds are ineffective against the R482T BCRP isoform [15]. 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
71 Analysis of the substrate binding properties of wild-type and six mutant ABCG2 proteins In order to further investigate the effects of the R482X mutations, we studied the drug-binding ability of a selection of ABCG2 mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type ABCG2 (R482wt). Login to comment
74 R482K was chosen because it is completely devoid of any transport, and the R482G and R482T mutants were chosen because these two mutants have been used in many different studies and would therefore be of broader interest. Login to comment
86 We analyzed expression of ABCG2 in the membranes using the monoclonal antibody BXP-21 (Fig. 5A), which shows that the R482G, R482wt, and R482T membranes used here express less ABCG2, compared with the membranes expressing the R482H, R482K, R482P, and R482Y variants. Login to comment
89 The R482G, R482P, and R482T variants are stimulated 1.9-, 2.1-, and 1.8-fold, respectively, by the addition of 20 mM prazosin. Login to comment
96 Specific [125 I]IAAP photoaffinity labeling of crude membranes derived from HeLa cells expressing wild-type ABCG2 (R482wt) and the ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y. Login to comment
106 Basal and drug-stimulated ATPase activity of wild-type ABCG2 (R482wt) and ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y. Login to comment
124 We have previously shown that the prazosin substrate analog [125 I]iodoarylazidoprazosin ([125 I]IAAP) can be photoaffinity cross-linked to R482wt, R482G, and R482T variants of ABCG2 when overexpressed in MCF-7/FLV1000, S1-M1-80, and MCF-7/AdVp3000 cells, respectively (Ejendal and Hrycyna 2005). Login to comment
125 Alqawi and colleagues have also shown that ABCG2, overexpressed in MCF-7/AdVp1000 cells (R482T) and MCF-7/ Mito (R482wt), binds directly and specifically to a photoactive drug analog of rhodamine 123, [125 I]Iodoarylazi- dorhodamine123 ([125 I]IAARh123) (Alqawi et al. 2004). Login to comment
131 Previously, we have shown that, out of seven agents tested, only prazosin and GF120918 can compete for [125 I]IAAP labeling of R482G, R482wt, and R482T and that known substrates such as rhodamine 123 and mitoxantrone do not (Ejendal and Hrycyna 2005). Login to comment
211 Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. Login to comment

PMID: 16847575 [PubMed] Pozza A et al: "Purification of breast cancer resistance protein ABCG2 and role of arginine-482."

No. Sentence Comment

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

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
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: 16928820 [PubMed] Chearwae W et al: "Modulation of the function of the multidrug resistance-linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin."

No. Sentence Comment

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

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

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: 17220244 [PubMed] Xia CQ et al: "Interactions of cyclosporin a with breast cancer resistance protein."

No. Sentence Comment

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

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: 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: 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
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
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
204 Similarly, the MTX transport rate by the ABCG2-R482T variant was below 50 pmol/mg membrane protein/min, irrespective of the membrane cholesterol content. 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
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
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
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
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
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

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
285 The cell line we used in this study was transfected with a BCRP plasmid, which has the R482T mutation [4,41] and has been shown to confer resistance against ADR and DNR [4]. Login to comment
387 [41] Alqawi O, Bates S, Georges E. Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. 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
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

132 December 2007 American Association for Cancer ResearchCopyright (c) 2007 on May 3, (Fig. 1, top), ABCG-expressing MCF-7 FLV1000 cells, and ABCG2 mutant R482T MCF-7 AdVp3000 cells (Fig. 1, bottom), the intensities of the accumulated fluorescent substrates were then analyzed as described in Materials and Methods. Login to comment
112 Using flow cytometry with wild type R482-HEK293 cells (Fig. 1, top panel), ABCG2-expressing MCF-7 FLV1000 cells and ABCG2 mutant R482T MCF-7 AdVp3000 cells (Fig. 1, bottom panel), the intensities of the accumulated fluorescent substrates were then analyzed as described in Materials and Methods. Login to comment

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

No. Sentence Comment

101 Anticancer drugs • Mitoxantrone • Bisantrene (R482T mutant form) • Epipodophyllotoxins (etoposide and teniposide) • Camptothecins (topotecan and irinotecan) • Flavopiridol • Anthracyclines (doxorubicin and daunorubicin; R482T mutant form) Antifolates • Methotrexate Porphyrins • Pheophorbide a • Protoporphyrin IX • Hematoporphyrin Tyrosine kinase inhibitors • Imatinib mesylate • Gefitinib Flavonoids • Genestein • Quercetin Carcinogens • Aflatoxin B • PhiP Fungal toxins • Fumitremorgin C • Ko143 Drug & metabolite conjugates • Acetominaphen sulfate • Estrone-3-sulfate • Dehydroepiandrosterone sulfate • Estradiol-17-β-D-glucuronide • Dinitrophenyl-S-glutathione HMG CoA reductase inhibitors • Rosuvastatin • Pravastatin • Cerivastatin Antihypertensives • Reserpine Antibiotics • Ciprofloxacin • Norfloxacin Fluorescent compounds • Hoechst 33342 • BODIPY-prazosin • Rhodamine 123 (R482T/G mutants) Antiviral drugs • Zidovudine • Lamivudine Natural products • Curcuminoids the Walker A and B motifs of one NBD subunit, and the LSGGQ signature C motif of the partner NBD subunit. Login to 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

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

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

142 Also Nakanishi et al. speculated a complex interaction of substrate with the BCRP transport protein (WT-BCRP and R482T variant), since in their substrate interaction studies using Rhodamin 123, topotecan, mitoxantrone, and flavopiridol, no two substrates reciprocally inhibited the efflux of the other (32). 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

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: 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

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: 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

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
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
795 This study reported a percentage yield of ABCG2 of ~1.3% (membrane protein) and an approximately two fold lower yield for the R482T isoform. 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

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
1061 Importantly, [(3)H]azidopine can covalently link to both wtABCG2 and to the ABCG2 R482T mutant variant. 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

1136 Interestingly, the R482T/G hot-spot mutation, frequently observed in cell cultures selected at high concentration of drugs [55], has been found to be a gain-of-function for drug efflux by extending the transport to anthracyclins and bisantrene [56] as well as to rhodamine 123 [54], an exception being methotrexate which is no longer transported. Login to comment
1137 Direct ligand interactions with purified ABCG2 have shown that the R482T mutation does not modify the binding affinity for a given substrate whether it is transported or not (methotrexate still binds), suggesting that arginine-482, assumed to be located at the inner edge of the third membrane span, is involved in substrate transport, not in binding [57]. Login to comment
1220 2.1.1. Cellular-based assays Whole cells, either transfected or drug-selected, are mainly used by flow cytometry for studying small series of inhibitors for their ability to induce intracellular accumulation of fluorescent drugs such as mitoxantrone, pheophorbide A, BODIPY-prazosin, LysoTracker, topotecan or Hoechst 33342, and also rhodamine 123 and doxorubicin with the R482T/G mutant [109]. Login to comment
1293 Interestingly, the R482T hotspot mutation altered the positive impact of prenylation on the inhibitory potency, tectochrysin being then the best compound with an IC50 of 1.9 μM. The relatively low toxicity of tectochrysin and 6-prenylchrysin and efficient sensitization of cell growth to mitoxantrone made these compounds promising for future potential use in clinical trials. 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

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

77 Unlike the MCF-7/FLV1000, the MCF-7 AdVp3000 expressed the mutant ABCG2 R482T. 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: 19949923 [PubMed] Aszalos A et al: "Flow cytometric evaluation of multidrug resistance proteins."

No. Sentence Comment

194 The BCRPT482 cell line (containing a mutated BCRP in which arginine 482 was replaced with threonine) was also studied. 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
138 BCRP R482T or G mutation: Altered Substrate Specificity (62). 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

32 Rhodamine123 [Rho123; 2Ј-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-5Ј-bi-1h-benzimidazole] and anthracyclines such as doxorubicin (Dox) and daunorubicin are not substrates of wild-type BCRP, but can be efficiently transported by the mutants with Gly or Thr substitution of Arg482 (Honjo et al., 2001; Miwa et al., 2003; Ozvegy-Laczka et al., 2005). Login to comment
326 R482T exhibited markedly increased resistance to MX (Miwa et al., 2003; Robey et al., 2003). Login to comment
367 More importantly, the Km and Vmax/Km values for basal ATPase activity of R482T with a similar gain of function have also been shown to be decreased and increased, respectively (Pozza et al., 2006). Login to comment
377 Given the nature of gain of function associated with K452A and H457A, a similar observation was reported in which R482T was shown to be much less intensively photolabeled by a photoactive analog of Rho123 than wild-type BCRP (Alqawi et al., 2004), indicating the binding affinity of the photoactive substrate to R482T was decreased even though R482T can effectively transport Rho123, but wild-type BCRP cannot. 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

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

PMID: 20665261 [PubMed] Pozza A et al: "Insect cell versus bacterial overexpressed membrane proteins: an example, the human ABCG2 transporter."

No. Sentence Comment

49 The pcDNA3 plasmid containing R482T-ABCG2 cDNA was kindly provided by Dr. D. Login to comment
52 Site-directed mutagenesis: The R482T-ABCG2 cDNA was mutated to obtain R482-ABCG2 cDNA by site-directed mutagenesis using a "Quick-Change Site-directed mutagenesis" kit (Stratagene, La Jolla, CA). Login to comment
147 Circular dichroism detergent buffer: 10 mM NaPO4 , pH 8, 2% glycerol, 1 mM DTT, 0.05% detergent (either SDS for ABCG2 R482T from bacteria or dodecylmaltoside for the protein from insect cells). Login to comment
151 E. coli strains were transformed with pET21b(+)/ABCG2 R482T using a thermal-shock procedure. Login to comment
193 10. Finally, cured PV6 mutants were transformed with pET21b(+)/ABCG2 R482T, and protein expression and growth kinetics were monitored. Login to comment
211 (■), BL21(DE3)/pET21a(+) ABCG2 R482T; (§), BL21(DE3)/pET21a(+) ABCG2 R482T induced; (▲), PV6/pET21a(+) ABCG2 R482T; ( ), PV6/pET21a(+) ABCG2 R482T induced. Login to comment
374 (b) Effects of substrates and inhibitors on the ATPase activity of ABCG2 within the inverted membrane vesicles from insect cells.TheATPase activity of R482 (black columns) or R482T (gray columns) ABCG2 overexpressed in Sf9 was measured with 2.5 µg of protein, 5 mM ATP, and 20 mM MgCl2 at 37°C for 40 min. Login to comment
395 Inverted membrane vesicles from High-Five cells infected by a baculovirus vector encoding the R482 or R482T transporter were treated with cell solubilization buffer at a final protein concentration of 2 mg/ml, for 30 min with gente shaking at 4°C. Login to comment
424 Lane 1: prestained molecular weight markers; lane 2 : inverted membrane vesicles of High-Five cells infected with the baculovirus vector encoding either R482 or R482T ABCG2; lane 3 : supernatant after solubilization; lane 4: supernatant after centrifugation (15,000 × g, 30 min); lane 5 : detergent-insoluble pellet after centrifugation; lane 6 : supernatant after binding for 2 h; lane 7: Ni-NTA agarose gel after binding for 2 h; lane 8 : washing; lane 9 : Ni-NTA agarose gel after washing; lane 10 : elution; lane 11: Ni-NTA agarose gel after elution; lane 12 : after imidazole removal by gel filtration. Login to comment
443 Measurements were performed under the following conditions: 10 mM NaPi, pH 8, 2% glycerol, 1 mM DTT, 0.05% detergent (SDS for 0.5 µM ABCG2 R482T from bacteria or dodecylmaltoside for 0.24 µM ABCG2 R482T from insect cells), and spectra was scanned from 185-190 to 250 nm with 0.2-nm step and 1-s data acquisition time at 25°C. Login to comment
444 (c) Binding of 6-prenylchrysin on purified ABCG2 R482T from bacteria or insect cells. Login to comment
445 The measurements were performed under the following conditions: 0.5 µM ABCG2 R482T, 0.05% SDS, 0.1 M KPi, 15% glycerol, 0.1 M NaCl, 1 mM DTT at 25°C for ABCG2 from bacteria (■), and 0.6 µM ABCG2 R482T, 50 mM HEPES/NaOH, pH 8, 18 mM CHAPS, 0.5 M NaCl, 20% glycerol ABCG2 for insect cell-expressed transporter (§). Login to comment
449 (d) Inhibition by vanadate of the ATPase activity of purified ABCG2 from insect cells.The inhibition of ATPase activity of R482 (squares) and R482T (triangles) transporter was measured in the presence of 5 mM MgATP, 0.5 µg protein, 50 µg azolectin, and a range of orthovanade concentrations from 0.001 to 10 mM at 37°C for 40 min. Login to comment
451 The ATPase activity of either R482 (squares) or R482T (triangles) purified ABCG2 was measured with 0.5 µg protein in the presence of 50 µg azolectin at 37°C for 40 min. Login to comment
453 Measurements were performed under the following conditions:0.045 µg/µlABCG2 (either R482 or R482T),50 mM HEPES/NaOH,pH 8,18 mM CHAPS,0.5 M NaCl, 20% glycerol. Login to comment
481 The apparent Kd values were not modified by the R482T point mutation (Fig. 4f), suggesting that the R482T point mutation does not act on binding but on subsequent step to transport. Login to comment
494 The ATPase activity of purified ABCG2 was modified by the R482T point mutation, which both increased Vmax and decreased Km (ATP/Mg2+ ) (Fig. 4e). Login to comment
191 10. Finally, cured PV6 mutants were transformed with pET21b(+)/ABCG2 R482T, and protein expression and growth kinetics were monitored. Login to comment
208 (■), BL21(DE3)/pET21a(+) ABCG2 R482T; (§), BL21(DE3)/pET21a(+) ABCG2 R482T induced; (▲), PV6/pET21a(+) ABCG2 R482T; ( ), PV6/pET21a(+) ABCG2 R482T induced. Login to comment
371 (b) Effects of substrates and inhibitors on the ATPase activity of ABCG2 within the inverted membrane vesicles from insect cells.TheATPase activity of R482 (black columns) or R482T (gray columns) ABCG2 overexpressed in Sf9 was measured with 2.5 µg of protein, 5 mM ATP, and 20 mM MgCl2 at 37°C for 40 min. Login to comment
392 Inverted membrane vesicles from High-Five cells infected by a baculovirus vector encoding the R482 or R482T transporter were treated with cell solubilization buffer at a final protein concentration of 2 mg/ml, for 30 min with gente shaking at 4°C. Login to comment
421 Lane 1: prestained molecular weight markers; lane 2 : inverted membrane vesicles of High-Five cells infected with the baculovirus vector encoding either R482 or R482T ABCG2; lane 3 : supernatant after solubilization; lane 4: supernatant after centrifugation (15,000 × g, 30 min); lane 5 : detergent-insoluble pellet after centrifugation; lane 6 : supernatant after binding for 2 h; lane 7: Ni-NTA agarose gel after binding for 2 h; lane 8 : washing; lane 9 : Ni-NTA agarose gel after washing; lane 10 : elution; lane 11: Ni-NTA agarose gel after elution; lane 12 : after imidazole removal by gel filtration. Login to comment
440 Measurements were performed under the following conditions: 10 mM NaPi, pH 8, 2% glycerol, 1 mM DTT, 0.05% detergent (SDS for 0.5 µM ABCG2 R482T from bacteria or dodecylmaltoside for 0.24 µM ABCG2 R482T from insect cells), and spectra was scanned from 185-190 to 250 nm with 0.2-nm step and 1-s data acquisition time at 25°C. Login to comment
441 (c) Binding of 6-prenylchrysin on purified ABCG2 R482T from bacteria or insect cells. Login to comment
442 The measurements were performed under the following conditions: 0.5 µM ABCG2 R482T, 0.05% SDS, 0.1 M KPi, 15% glycerol, 0.1 M NaCl, 1 mM DTT at 25°C for ABCG2 from bacteria (■), and 0.6 µM ABCG2 R482T, 50 mM HEPES/NaOH, pH 8, 18 mM CHAPS, 0.5 M NaCl, 20% glycerol ABCG2 for insect cell-expressed transporter (§). Login to comment
446 (d) Inhibition by vanadate of the ATPase activity of purified ABCG2 from insect cells.The inhibition of ATPase activity of R482 (squares) and R482T (triangles) transporter was measured in the presence of 5 mM MgATP, 0.5 µg protein, 50 µg azolectin, and a range of orthovanade concentrations from 0.001 to 10 mM at 37°C for 40 min. Login to comment
448 The ATPase activity of either R482 (squares) or R482T (triangles) purified ABCG2 was measured with 0.5 µg protein in the presence of 50 µg azolectin at 37°C for 40 min. Login to comment
450 Measurements were performed under the following conditions:0.045 µg/µlABCG2 (either R482 or R482T),50 mM HEPES/NaOH,pH 8,18 mM CHAPS,0.5 M NaCl, 20% glycerol. Login to comment
478 The apparent Kd values were not modified by the R482T point mutation (Fig. 4f), suggesting that the R482T point mutation does not act on binding but on subsequent step to transport. Login to comment
491 The ATPase activity of purified ABCG2 was modified by the R482T point mutation, which both increased Vmax and decreased Km (ATP/Mg2+ ) (Fig. 4e). Login to comment

PMID: 20705604 [PubMed] Desuzinges-Mandon E et al: "ABCG2 transports and transfers heme to albumin through its large extracellular loop."

No. Sentence Comment

266 Bound compounds are restricted to porphyrins because nonporphyrin molecules, such as mitoxantrone, riboflavin, or doxorubicin (the latter binds to ABCG2 (12) but is only transported by the mutated R482T/G trans- FIGURE 8. Login to comment

PMID: 20739628 [PubMed] Ni Z et al: "Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport."

No. Sentence Comment

338 Likewise, R482T with a similar "gain-of-function" also showed decreased Km and increased Vmax/Km values for ATP hydrolysis (21). 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

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
264 Alqawi et al. [123] showed that wild-type BCRP was more intensely photo-labeled with a photo-active analog of rhodamine 123 than R482T even though wild-type BCRP does not transport rhodamine 123. 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

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: 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

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

PMID: 21480930 [PubMed] Takeuchi Y et al: "Biological effect of tyrosine kinase inhibitors on three canine mast cell tumor cell lines with various KIT statuses."

No. Sentence Comment

163 Rh123 is extruded by another ABC transporter, such as Arg482 Thr mutant ABCG2 (Honjo et al., 2001). 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: 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: 22778153 [PubMed] Sen R et al: "The novel BCR-ABL and FLT3 inhibitor ponatinib is a potent inhibitor of the MDR-associated ATP-binding cassette transporter ABCG2."

No. Sentence Comment

27 Doxorubicin- and verapamil-selected MCF7/AdrVp breast carcinoma cells, overexpressing ABCG2 with the R482T mutation (25), were obtained from Dr. Douglas Ross, University of Maryland Greenebaum Cancer Center, Baltimore, MD, and flavopiridol-selected MCF-7/Flv1000 cells (26), overexpressing wild-type ABCG2, from Dr. Susan Bates, National Cancer Institute. Login to comment
69 The effect in MCF7/AdrVp was less than in 8226/MR20 and K562/ABCG2, likely because of a greater degree of resistance in the solid tumor in relation to hematopoietic cell lines, rather than to the presence of the R482T mutation in MCF7/AdrVp, though the latter is also possible. Login to comment
70 Because the R482T ABCG2 mutation is not clinically relevant, we did not pursue this distinction. Login to comment
25 Doxorubicin- and verapamil-selected MCF7/AdrVp breast carcinoma cells, overexpressing ABCG2 with the R482T mutation (25), were obtained from Dr. Douglas Ross, University of Maryland Greenebaum Cancer Center, Baltimore, MD, and flavopiridol-selected MCF-7/Flv1000 cells (26), overexpressing wild-type ABCG2, from Dr. Susan Bates, National Cancer Institute. Login to comment
67 The effect in MCF7/AdrVp was less than in 8226/MR20 and K562/ABCG2, likely because of a greater degree of resistance in the solid tumor in relation to hematopoietic cell lines, rather than to the presence of the R482T mutation in MCF7/AdrVp, though the latter is also possible. Login to comment
68 Because the R482T ABCG2 mutation is not clinically relevant, we did not pursue this distinction. 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: 22426010 [PubMed] Heffeter P et al: "Impact of terminal dimethylation on the resistance profile of alpha-N-heterocyclic thiosemicarbazones."

No. Sentence Comment

47 Center, Kansas State University, USA), the small cell lung carcinoma cell line GLC-4 and its ABCC1- and LRP-overexpressing subline GLC-4/adr (from E.G. deVries, Groningen, The Netherlands); the breast adenocarcinoma cell line MDA-MB-231 with its respective ABCG2(R482T)-transfected subclone MDA-MB-231/bcrp (from D. Ross, University of Maryland, Greenbaum Cancer Centre, USA), the non-small cell lung carcinoma cell line SW1573 with its ABCC1- and LRP-overexpressing subline 2R120 as well as its ABCB1- and ABCC1-overexpressing subline 2R160 (H. Broxterman, Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands). Login to comment

PMID: 22414725 [PubMed] Sodani K et al: "GW583340 and GW2974, human EGFR and HER-2 inhibitors, reverse ABCG2- and ABCB1-mediated drug resistance."

No. Sentence Comment

135 Therefore, we used both wild-type (R482) and mutant (R482T) forms of ABCG2 in the present study. Login to comment
264 Our results showed that GW583340 and GW2974 can inhibit the activity of both wild-type (R482) and mutant variant (R482T) of ABCG2. Login to comment
388 [38] Alqawi O, Bates S, Georges E. Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. Login to comment
136 Therefore, we used both wild-type (R482) and mutant (R482T) forms of ABCG2 in the present study. Login to comment
268 Our results showed that GW583340 and GW2974 can inhibit the activity of both wild-type (R482) and mutant variant (R482T) of ABCG2. Login to comment

PMID: 22039929 [PubMed] Valdameri G et al: "Methoxy stilbenes as potent, specific, untransported, and noncytotoxic inhibitors of breast cancer resistance protein."

No. Sentence Comment

34 (25% and 10%, respectively), in agreement with other data published for compound 9.31 Rhodamine 123 efflux could also be studied using the R482T ABCG2 mutant: a maximum of 75% inhibition was then obtained at 10-20 μM, with an IC50 value of 0.45 μM (Figure 1B). Login to comment
46 Inhibition of rhodamine 123 efflux in HEK293 cells transfected by either wild-type ABCB1 or R482T ABCG2 constructs. Login to comment
50 (B) Effects of compound 9 (○) and GF120918 (■) on R482T-ABCG2-mediated efflux. Login to comment
71 The interaction at this transport site was more sensitive to the R482T mutation than the interaction at the ABCG2-specific site, as monitored here by the relative loss in efficiency against rhodamine 123 and mitoxantrone efflux.26 Methoxy stilbenes probably bind to the same site as 6-prenylchrysin, since both types of ABCG2-specific inhibitors display the same partial inhibition (around 75%), possibly due to their rather small size. Login to comment
128 All cells were maintained in Dulbecco`s modified Eagle`s medium (DMEM high glucose), supplemented with 10% fetal bovine serum (FBS), 1% penicilin/streptomycin, and drug supplemented in some cases with either 0.75 mg mL-1 G418 (HEK293-pcDNA3.1, HEK293-R482 or R482T), 2 mg mL-1 G418 (HEK293-MDR1), or 5 μM etoposide (HEK293-MRP1). Login to comment
129 ABCG2- and ABCB1-Mediated Drug Transport. HEK293 cells were seeded at a density of 1 × 105 cells/well into 24-well culture plates and, after 48 h, exposed to 10 μM mitoxantrone (HEK293-R482 and HEK293-MDR1 cells) or 10 μM rhodamine 123 (HEK293-R482T and HEK293-MDR1 cells) for 30 min at 37 °C, in the absence or presence of compounds at various concentrations. Login to comment
132 The Hill number of mitoxantrone efflux in HEK293 cells transfected by either wild-type ABCG2 or R482T ABCG2 contructs was determined from the curves (sigmoidal -3 parameters) fitted in the SigmaPlot 11.0 software and calculated by the equation f = axb /(cb + xb ), where b is the Hill number. 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

57 Cytotoxicity assays Cytotoxicity assays were performed using PLB cells expressing the wild-type or the mutant (R482G, R482T) ABCG2 proteins. Login to comment
133 In cytotoxicity assays VX710 (biricodar) showed higher reversing effect in cell lines expressing the wild type than the R482T variant of ABCG2 (Minderman et al., 2004). Login to comment
134 GF120918 (elacridar), strongly inhibited the ATPase activity of the wild type ABCG2 in Sf9 membranes, whereas the same compound rather stimulated this activity in the case of the R482T mutant (Ahmed-Belkacem et al., 2005). 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
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

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
247 Some HIV protease inhibitors including ritonavir, saquinavir, nelfinavir, and iopinavir could effectively inhibit the transport activity of wild type ABCG2 but with less effect on R482T/G mutants [151, 152]. Login to comment
570 [116] Alqawi O, Bates S and Georges E. Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. Login to comment
569 [116] Alqawi O, Bates S and Georges E. Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. 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: 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
603 Interestingly, the gene was found to be amplified 10 to 12-fold in the MCF-7 AdVp3000 cells, but TABLE 5 A partial list of substrates transported by human BCRPa Anticancer drugs Anthracycline antibiotics (daunorubicin, doxorubicin, epirubucin; transported by R482T mutant form of BCRP) Anthracenes (mitoxantrone, bisantrene) Camptothecin derivatives (topotecan, irinotecan, SN-38, methotrexate) Epipodophyllotoxin derivatives (plant alkaloid) (etoposide, teniposide) Tyrosine kinase inhibitor (imatinib) Nucleoside drugs AZT Lamivudine Fluoroquinolone Antibioticsb Ciprofloxacin Ofloxacin Norfloxacin Conjugates Estrone-3-sulfate Taurocholate 3-sulfate (TLC-S) Estradiol-17β-D-Glucuronide (E217βG) Dinitrophenyl-S-glutathione (DNP-SG) Natural compounds Flavonoids Porphyrins Flurophores Rhodamine 123 (transported by R482T mutant form of BCRP) Hoechst 33342 a Source: Krishnamurthy and Schuetz (2005) and Mao and Unadkat (2005). 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
617 Wild-type BCRP does not transport anthracyclines but the R482T mutant BCRP confers higher anthracycline resistance. Login to comment

PMID: 19631619 [PubMed] Tai LM et al: "Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123."

No. Sentence Comment

245 Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding. 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

21 In contrast, zosuquidar, a highly specific P-gp inhibitor, which does not interact with other transporters including MRP1, MRP2 and mutant BCRP (R482T) [12], has been developed in an attempt to avoid significant pharmacokinetic interactions and therefore allow co-administration of standard dosing of cytotoxic chemotherapy. Login to 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
89 It has been shown that zosuquidar did not affect on the mutant BCRP (R482T) mediated drug transport [21]. Login to comment
120 It has been shown that zosuquidar did not affect on the mutant BCRP (R482T) mediated drug transport [21]. Login to comment

PMID: 16568474 [PubMed] Chaoui D et al: "JC-1, a sensitive probe for a simultaneous detection of P-glycoprotein activity and apoptosis in leukemic cells."

No. Sentence Comment

255 Alqawi O, Bates S, Georges E. Arginine 482 to threonine 482 mutation in breast cancer resistance protein (ABCG2) inhibits rhodamine 123 transport while increasing binding. Login to comment

PMID: 15868961 [PubMed] Krasznai ZT et al: "Quantitative and functional assay of MDR1/P170-mediated MDR in ascites cells of patients with ovarian cancer."

No. Sentence Comment

155 It is also supported by the facts that R-123 can be effluxed only by cells expressing mutant BCRP with glycine or threonine instead of arginine at position 482 (20) and R-123 is transported approx. Login to comment

PMID: 12535578 [PubMed] Dantzig AH et al: "Considerations in the design and development of transport inhibitors as adjuncts to drug therapy."

No. Sentence Comment

46 acid reside 482 from arginine to threonine or glycine, It also cotransports glutathione with certain un- confers resistance to the anthracyclines [28]. Login to comment

PMID: 22695718 [PubMed] Stolarczyk EI et al: "Casein kinase 2alpha regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249."

No. Sentence Comment

257 We considered the expression of other ABC proteins that transport doxorubicin, [e.g., ABCB1 and a mutant form of ABCG2 (R482T/G)]. 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
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
19 In these cells the BCRP gene has been shown to be mutated in the exon 5 leading to the substitution of the arginine 482 for a threonine (R482T). 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
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
91 In contrast, resistance to etoposide in HEK/ R482T cell line remains in the same range of HEK/R482 cell line. 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
110 Novobiocin was not able to modulate resistance to etoposide in HEK/R482T cell line. 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
117 The [3 H]-etoposide uptake is also reduced in HEK/R482T cells as compared to HEK/R482 cells, although Table III. 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
148 However, this does not reach stastical significance when HEK/R482T cells were compared to HEK/R482 cells. 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
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
163 Nevertheless, HEK/R482 and HEK/R482T cells were only 5and 8-fold resistant, respectively, to etoposide (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

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

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: 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: 23742976 [PubMed] Tian Y et al: "The interaction between human breast cancer resistance protein (BCRP) and five bisbenzylisoquinoline alkaloids."

No. Sentence Comment

225 Alqawi et al. reported that wild-type BCRP was more intensely photolabelled by iodoaryl-azido-rhodamine 123 (IAARh123, a photoreactive drug analog of rhodamine 123) than mutant BCRP in which the Arg 482 was mutated to Thr, but could not transport it (Alqawi et al., 2004). Login to comment

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

No. Sentence 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

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

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
110 These results suggest that masitinib enhances the sensitivity of ABCG2 substrates in both wild-type and R482T/G mutant ABCG2 overexpressing cells, i.e. it selectively reverses MDR. 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

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

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
156 While taxanes act only against WT BCRP, no effect was found on the Arg482 Thr mutant. 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: 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: 26515463 [PubMed] Anreddy N et al: "A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo."

No. Sentence Comment

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