ABCMdb

PMID: 14566825

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

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Asn557Asp Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Login to comment 6 ABCG2 p.Asn557Glu ABCG2 p.His630Leu ABCG2 p.His630Glu Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Login to comment 8 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3 H]mitoxantrone than the wild-type BCRP-transfected cells. Login to comment 13 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Met ABCG2 p.Arg482Met 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 42 ABCG2 p.Arg482Ala ABCG2 p.Arg482Lys ABCG2 p.Arg482Trp Because FACS analysis showed that certain populations of the methotrexate-resistant PA/R482A and PA/R482W cells did not express BCRP (data not shown), all the R482-mutant BCRP transfectants (except R482K-BCRP transfectant) were further selected with 1 ng/ml of mitoxantrone for 5 days to eliminate untransfected cells. Login to comment 43 ABCG2 p.Arg482Lys These mitoxantrone-selected cells (or R482K-BCRP transfectant) were termed PA/R482X2 (X2 ϭ Y, N, C, M, S, T, V, A, G, E, W, D, Q, H, or K). Login to comment 48 ABCG2 p.Arg482Lys ABCG2 p.Arg482Asp ABCG2 p.Glu446Asp Among PA/E446X1 transfectants, PA/E446D expressed a small amount of BCRP. Among PA/R482X2, PA/R482D and PA/R482K expressed lesser amounts of BCRP than the other transfectants. Login to comment 49 ABCG2 p.His630Glu ABCG2 p.Asn557Arg Among PA/ N557X3, PA/N557R expressed little BCRP. Among PA/H630X4 transfectants, moderate BCRP expression was observed in PA/ H630E. Login to comment 50 ABCG2 p.His630Leu ABCG2 p.His630Asp ABCG2 p.His630Ser PA/H630L, PA/H630D and PA/H630S expressed low amounts of BCRP. Login to comment 51 ABCG2 p.His630Arg ABCG2 p.His630Lys BCRP expression was not detected in PA/ H630K and PA/H630R. Login to comment 56 ABCG2 p.Asn557Asp PA/N557D showed a similar resistance to mitoxantrone but 5-fold lower resistance to SN-38 than PA/WT1 (Fig. 2c). Login to comment 57 ABCG2 p.Asn557Glu PA/ N557E showed similar degrees of resistance to mitoxantrone and SN-38 (Fig. 2c). Login to comment 58 ABCG2 p.His630Leu ABCG2 p.His630Glu PA/H630E and PA/H630L also showed similar degrees of resistance to mitoxantrone and SN-38 (Fig. Login to comment 61 ABCG2 p.Arg482Lys ABCG2 p.Arg482Asp Most of the PA/ R482X2 cells (except for PA/R482D and PA/R482K) showed similar or somewhat lower levels of SN-38 resistance as compared to PA/WT2. Login to comment 63 ABCG2 p.Arg482Tyr PA/WT2 (R482) and PA/R482Y (Group 1) showed higher degrees of resistance to SN-38 than to mitoxantrone. Login to comment 64 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Met ABCG2 p.Arg482Ala ABCG2 p.Arg482Trp ABCG2 p.Arg482Asp ABCG2 p.Arg482Val ABCG2 p.Arg482Cys ABCG2 p.Arg482Glu ABCG2 p.Arg482Asn 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 65 ABCG2 p.Arg482Asp The drug resistance of PA/R482D was weak because of the low expression level of BCRP protein. Login to comment 66 ABCG2 p.Arg482Gln ABCG2 p.Arg482His PA/R482Q and PA/R482H (Group 3) showed similar degrees of resistance to mitoxantrone and SN-38. Login to comment 67 ABCG2 p.Arg482Lys PA/R482K (Group 4) did not show drug resistance. Login to comment 76 ABCG2 p.Arg482Gly ABCG2 p.Asn557Asp ABCG2 p.His630Leu ABCG2 p.Glu446Gly As shown in Figure 3, E446G-, R482G-, N557D- and H630L-BCRP were expressed on the cell surface. Login to comment 77 ABCG2 p.Arg482Gly ABCG2 p.Glu446Gly Expression levels of BCRP in PA/ WT2, PA/E446G and PA/R482G were similar. This suggests that trafficking problem is not a main reason for the loss of BCRP function in E446-mutants. Login to comment 78 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser Anthracycline resistance of R482-mutant BCRP transfectants Resistance to anthracycline antibiotics of PA/R482G and PA/ R482S, typical members of Group 2 transfectants, was examined (Fig. 4). Login to comment 80 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser PA/R482G and PA/R482S showed resistance to doxorubicin, epirubicin and daunorubicin, but not to aclarubicin (Fig. 4). Login to comment 84 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Asn557Asp ABCG2 p.His630Leu ABCG2 p.His630Glu ABCG2 p.Asn557His Estrone is the first physiological substrate that was shown to circumvents BCRP-mediated drug resistance.17 As shown in Table I, estrone effectively reversed mitoxantrone resistance and SN-38 resistance of PA/R482G, PA/R482S, PA/N557H, PA/N557D, PA/H630E and PA/H630L. Login to comment 85 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser Estrone also reversed doxorubicin resistance of PA/ R482G and PA/R482S (Table II). Login to comment 86 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Asn557Asp ABCG2 p.His630Leu ABCG2 p.His630Glu ABCG2 p.Asn557His Similarly, fumitremorgin C, a well-known BCRP inhibitor, strongly reversed the mitoxantrone resistance and SN-38 resistance of PA/R482G, PA/R482S, PA/ N557H, PA/N557D, PA/H630E and PA/H630L (Table III). Login to comment 88 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Ser ABCG2 p.Arg482Ser Uptake of [3 H]mitoxantrone To confirm that the high mitoxantrone resistance of PA/R482G and PA/R482S is attributable to decreased drug accumulation, the uptake of [3 H]mitoxantrone in PA317, PA/WT2, PA/R482G and PA/R482S cells was examined. As shown in Figure 6, intracellular accumulation of [3 H]mitoxantrone was significantly lower in PA/ R482G and PA/R482S than in PA/WT2. Login to comment 89 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser The [3 H]mitoxantrone uptake reached plateau levels in 4 hr. The results clearly showed that steady-state levels of mitoxantrone accumulation in PA/ R482S and R482G were lower than that in PA/WT2 (Fig. 6). Login to comment 104 ABCG2 p.Arg482Tyr These 13 PA/R482X2 and PA/R482Y cells exhibited significantly greater resistance to doxorubicin than PA/WT2. Login to comment 105 ABCG2 p.Asn557Asp PA/N557D cells exhibited similar resistance to mitoxantrone but less resistance to SN-38 than PA/WT1. Login to comment 106 ABCG2 p.Asn557Glu ABCG2 p.His630Leu ABCG2 p.His630Glu PA/N557E, PA/H630E and PA/H630L showed similar degrees of resistance to mitoxantrone and SN-38. Login to comment 110 ABCG2 p.Asn557Asp In addition, we constructed N557-mutants because N557D-BCRP isolated by PCR-based mutagenesis was found to have altered cross resistance patterns (unpublished result of the authors). Login to comment 112 ABCG2 p.Glu446Asp ABCG2 p.Asn557Arg ABCG2 p.His630Arg ABCG2 p.His630Lys Such transfectants include PA/E446D, PA/R486D, PA/R486K, PA/N557R, H630K and H630R. Login to comment 115 ABCG2 p.His630Leu In contrast, most BCRP mutants except H630L were stable when charged amino acids were converted to hydrophobic amino acids. Login to comment 117 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser Mitoxantrone uptake in PA317 cells was 10-fold higher than that in PA/R482S or PA/R482G cells. Login to comment 119 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser PA/R482S and PA/R482G cells show approximately 100-200-fold more resistance to mitoxantrone than the parental cells. Login to comment 122 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser This concentration was much higher than the IC50 values of FIGURE 4 - Resistance to mitoxantrone, SN-38 and anthracyclines of R482-mutant BCRP transfectants. PA/WT2, PA/R482G and PA/ R482S were mixed populations of the transfected cells established after the 2-step selection with 120 ng/ml methotrexate and 1 ng/ml mitoxantrone. Login to comment 123 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser PA317 (closed circle), PA/WT2 (open triangle), PA/ R482G (open square) or PA/R482S (open lozenge) cells were cultured for 5 days with increasing concentrations of anthracyclines. Login to comment 131 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser PA/R482S or PA/R482G and transporter function might not be so efficient in the presence of such a high concentration of mitoxantrone. Login to comment 135 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Ser ABCG2 p.Arg482Met ABCG2 p.Arg482Met ABCG2 p.Arg482Met MCF-7 AdVp3000 established by treating MCF-7 cells with 3 ␮g/ml of doxorubicin and 5 ␮g/ml of verapamil overexpressed R482T-BCRP.5,13 S1-M1-80 established by treating human colon carcinoma S1 cells with 80 ␮M mitoxantrone overexpressed R482G-BCRP.6,13 These resistant cells exhibited high resistance to doxorubicin and mitoxantrone.5,6,13 We found recently that MT-4/DOX500 cells established by treating human T cell MT-4 cells with 500 ng/ml of doxorubicin overexpressed R482M-BCRP.23 Two doxorubicin-resistant murine fibroblast lines 88.6/D800-A and 88.6/D800-B overexpressed R482M-BCRP and R482S-BCRP, respectively.14 Another doxorubicin-resistant cell line KOT52/D800 from mouse fibroblasts co-expressed wild-type BCRP and R482M-BCRP.14 In addition to anthracyclines and mitoxantrone, cells transfected with R482-mutant BCRP cDNAs also showed high resistance to methotrexate.28 Theoretically, 9 FIGURE 6 - Accumulation of [3 H]mitoxantrone in R482-mutant BCRP transfectants. PA/WT2, PA/R482G and PA/R482S were mixed populations of the transfected cells established after the 2-step selection with 120 ng/ml methotrexate and 1 ng/ml mitoxantrone. Login to comment 136 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser PA317 (closed circle), PA/WT2 (open triangle), PA/R482S (open lozenge) or PA/R482G (open square) cells were incubated at 37°C with 50 nM [3 H]mitoxantrone. Login to comment 140 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Asn557Asp ABCG2 p.His630Leu ABCG2 p.His630Glu ABCG2 p.Asn557His TABLE I - REVERSAL OF MITOXANTRONE RESISTANCE AND SN-38 RESISTANCE BY ESTRONE1 Cell line Degree of Mitoxantrone resistance Reversal index Degree of SN-38 resistance Reversal index Control 10 ␮M estrone Control 10 ␮M estrone PA/WT1 7.1 Ϯ 0.6 2.2 Ϯ 0.4 3.2 28 Ϯ 1 4.7 Ϯ 0.3 6.0 PA/R482S 120 Ϯ 20 6.8 Ϯ 0.4 18 37 Ϯ 2 4.4 Ϯ 1.1 8.4 PA/R482G 84 Ϯ 17 3.7 Ϯ 0.3 23 22 Ϯ 1 3.3 Ϯ 0.1 6.7 PA/N557H 3.3 Ϯ 0.1 1.0 Ϯ 0.1 3.3 4.8 Ϯ 0.5 3.4 Ϯ 0.1 1.4 PA/N557D 7.4 Ϯ 0.2 1.4 Ϯ 0.1 5.3 3.8 Ϯ 0.6 2.9 Ϯ 0.2 1.3 PA/H630E 5.8 Ϯ 0.2 1.1 Ϯ 0.1 5.3 20 Ϯ 2.8 6.0 Ϯ 0.1 3.3 PA/H630L 3.3 Ϯ 0.1 0.9 Ϯ 0.1 3.7 8.6 Ϯ 0.4 2.7 Ϯ 0.1 3.2 1 Cells were cultured in the absence or presence of 10 ␮M estrone with increasing concentrations of mitoxantrone or SN-38. Degree of resistance is the ratio of the IC50 value for BCRP-expressing cells divided by that for parental PA317. Login to comment 141 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser The reversal index is calculated by dividing degree of resistance without estrone by that with estrone. TABLE II - REVERSAL OF DOXORUBICIN RESISTANCE BY ESTRONE Cell line Degree of Doxorubicin resistance Reversal index Control 10 ␮M Estrone PA/WT1 1.2 Ϯ 0.1 1.0 Ϯ 0.0 1.2 PA/R482S 10 Ϯ 1 1.3 Ϯ 0.1 7.7 PA/R482G 8.0 Ϯ 0.6 1.2 Ϯ 0.1 6.7 These cells were cultured in the absence or presence of 10 ␮M estrone with increasing concentrations of doxorubicin. Login to comment 143 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Asn557Asp ABCG2 p.His630Leu ABCG2 p.His630Glu ABCG2 p.Asn557His The reversal index is calculated by dividing degree of resistance without estrone by that with estrone. TABLE III - REVERSAL OF MITOXANTRONE RESISTANCE AND SN-38 RESISTANCE BY FUMITREMORGIN C1 Cell line Degree of Mitoxantrone resistance Reversal index Degree of SN-38 resistance Reversal index Control 3 ␮M Fumitremorgin C Control 3 ␮M Fumitremorgin C PA/WT1 11 Ϯ 1 1.0 Ϯ 0.1 11 23 Ϯ 1 1.1 Ϯ 0.1 21 PA/R482S 140 Ϯ 10 1.2 Ϯ 0.1 120 41 Ϯ 1 0.9 Ϯ 0.1 46 PA/R482G 89 Ϯ 21 0.9 Ϯ 0.1 99 17 Ϯ 2 0.9 Ϯ 0.1 19 PA/N557H 3.3 Ϯ 0.1 1.0 Ϯ 0.1 3.3 4.8 Ϯ 0.5 1.1 Ϯ 0.1 4.4 PA/N557D 7.4 Ϯ 0.2 0.8 Ϯ 0.0 9.3 3.8 Ϯ 0.6 1.2 Ϯ 0.1 3.2 PA/H630E 5.8 Ϯ 0.2 1.1 Ϯ 0.1 5.3 20 Ϯ 2.8 1.4 Ϯ 0.1 14 PA/H630L 3.3 Ϯ 0.1 0.9 Ϯ 0.1 3.7 8.6 Ϯ 0.4 1.4 Ϯ 0.1 6.1 1 These cells were cultured in the absence or presence of 3 ␮M fumitremorgin C with increasing concentrations of mitoxantrone or SN-38. Degree of resistance is the ratio of IC50 value for BCRP-expressing cells divided by that for parental PA317. Login to comment 147 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Met 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 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Ser ABCG2 p.Arg482Met ABCG2 p.Arg482Met ABCG2 p.Arg482Lys ABCG2 p.Arg482Lys ABCG2 p.Arg482Trp ABCG2 p.Arg482Trp 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 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Met ABCG2 p.Arg482Trp ABCG2 p.Arg482Trp 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 150 ABCG2 p.Arg482Lys PA/R482K did not show drug resistance. Login to comment 151 ABCG2 p.Arg482Lys ABCG2 p.Arg482Trp The low-level drug resistance of the transfectants would explain why R482W- and R482K-BCRP were not expressed in drug-selected cell lines. Login to comment 160 ABCG2 p.Arg482Tyr Group 1 member transfectants (PA/WT and PA/R482Y) showed higher degrees of resistance to SN-38 than to mitoxantrone. Login to comment 163 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Arg482Met ABCG2 p.Arg482Ala ABCG2 p.Arg482Trp ABCG2 p.Arg482Asp ABCG2 p.Arg482Val ABCG2 p.Arg482Cys ABCG2 p.Arg482Glu ABCG2 p.Arg482Asn 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 165 ABCG2 p.Arg482Gln ABCG2 p.Arg482His Group 3 members (PA/R482Q and PA/R482H) showed similar degrees of resistance to mitoxantrone and SN-38. Login to comment 167 ABCG2 p.Arg482Lys PA/R482K (Group 4) did not show drug resistance. Login to comment