ABCMdb

PMID: 20518788

Shigeta J, Katayama K, Mitsuhashi J, Noguchi K, Sugimoto Y
BCRP/ABCG2 confers anticancer drug resistance without covalent dimerization.
Cancer Sci. 2010 Aug;101(8):1813-21. Epub 2010 Apr 28., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser The BCRP-C603S mutant substituting Ser for Cys-603 in the third extracellular domain formed both a 70-75-kDa monomer and 140-150-kDa dimer, suggesting that Cys-603 is an important residue in the covalent bridge. Login to comment 8 ABCG2 p.Cys603Ser Among drug-resistant Cys-mutant BCRP transfectants, PA / C603S, PA / C592SÆC608S, and PA / C592SÆC603SÆC608S were found to be more resistant to the reversal effects of fumitremorgin C than PA / WT, suggesting some alteration in the substrate recognition in Cys-mutant BCRPs. Login to comment 21 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser Among these, BCRP-C603S mutant substituting Ser for Cys-603 was found to migrate both as a 70-75-kDa monomer and as a 140-150-kDa dimer in SDS-PAGE under non-reducing conditions. Login to comment 22 ABCG2 p.Cys603Ser (16) The PA /C603S transfectant showed the drug resistance phenotype. Login to comment 25 ABCG2 p.Cys603Ser However, the existence of a small amount of 150-kDa dimer in the PA/C603S transfectant suggested the involvement of other Cys residues in this process. Login to comment 29 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser In the present study, to elucidate the possible involvement of Cys-592 and Cys-608 in BCRP dimerization and protein activity, we established double (C592SÆC603S, C592SÆC608S, and C603SÆC608S) and triple (C592SÆC603SÆC608S) mutant BCRP transfectants, and compared the expression, dimer formation, and function of these BCRP mutants with wild-type and single (C592S, C603S, and C608S) mutant proteins. Login to comment 33 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser The wild-type and single (C592S, C603S, and C608S) mutant BCRP cDNAs in the same vector were also used as controls. Login to comment 63 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser PA317 cells were transfected with the wild-type, single (C592S, C603S, and C608S), double (C592SÆC603S, C592SÆC608S, and C603SÆC608S), and triple (C592SÆC603SÆC608S) mutant BCRP cDNAs in pHa-IRES-DHFR bicistronic expression vector as described previously. Login to comment 67 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser The resulting mixed populations of the drug-selected cells were designated as PA /WT-mix, PA / C592S-mix, PA /C603S-mix, PA /C608S-mix, PA /C592SÆC603S-mix, PA/ C592SÆC608S-mix, PA /C603SÆC608S-mix, and PA /C592SÆC603SÆC608S-mix. Login to comment 71 ABCG2 p.Cys603Ser Wild-type BCRP and BCRP-C603S both migrated as a 75-kDa molecule. Login to comment 74 ABCG2 p.Cys603Ser ABCG2 p.Cys608Ser A high level of BCRP expression was observed in PA /WT-mix, PA /C603S-mix, and PA /C592SÆ C608S-mix cells (Fig. 2a). Login to comment 76 ABCG2 p.Cys603Ser As reported previously, BCRP-C603S migrated as both a 150-kDa dimer and a 70-75-kDa monomer. Login to comment 77 ABCG2 p.Cys592Ser Breast cancer resistance protein (BCRP) dimer formation was observed in PA /WT-mix, PA /C592S-mix, PA /C592SÆC603S-mix, and PA/C592SÆC608S-mix cells. Login to comment 78 ABCG2 p.Cys608Ser The BCRP expression levels in PA /C608S-mix, PA /C603SÆC608S-mix, and PA /C592SÆC603SÆC608S-mix cells were too low to evaluate dimer /monomer formation (Fig. 2b). Login to comment 82 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser The BCRP expression levels and monomer /dimer formation in PA/ WT and single mutant BCRP transfectants (PA /C592S, PA/C603S, and PA/C608S) were essentially the same as those in our previous report (Fig. 3a-d). Login to comment 94 ABCG2 p.Cys603Ser PA /WT cells expressed 75-kDa BCRP only, and PA /C603S cells mostly expressed the 75-kDa molecule. Login to comment 95 ABCG2 p.Cys592Ser The ratio of 75-kDa BCRP in PA/C592S, PA /C592SÆC608S, and PA/C592SÆC603SÆC608S cells was two-third to half of the total BCRP. Login to comment 97 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser ABCG2 p.Cys608Ser +SH -SH PA/WT-mix PA/C592S-mix PA/C603S-mix PA/C608S-mix PA/C592S·C603S-mix PA/C592S·C608S-mix PA/C603S·C608S-mix PA/C592S·C603S·C608S-mix PA317 PA/WT-mix PA/WT-mix(0.5) PA/C592S-mix PA/C603S-mix PA/C608S-mix PA/C592S·C603S-mix PA/C592S·C608S-mix PA/C603S·C608S-mix PA/C592S·C603S·C608S-mix PA317 150 75 kDa 150 75 kDa GAPDH (b)(a) Fig. 2. Login to comment 102 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser -mix -cl.1 -cl.8 PA317 PA/WT PA/C592S PA/C603S PA/WT-mix -mix -cl.3 -cl.7 PA317 PA/WT-mix -mix -cl.5 -cl.7 PA317 PA/C608S PA/WT-mix -mix -cl.6 -cl.7 PA317 BCRP (-SH) 75 150 kDa GAPDH (+SH) BCRP (+SH) PA/WT-mix -mix -cl.1 -cl.4 PA317 PA/WT-mix -mix -cl.6 -cl.7 PA317 PA/WT-mix -mix -cl.6 -cl.7 PA317 PA/C592S ·C603S PA/C592S ·C608S PA/C603S ·C608S PA/C592S ·C603S·C608S PA/WT-mix -mix -cl.2 -cl.7 PA317 BCRP (-SH) 75 150 kDa GAPDH (+SH) BCRP (+SH) (a) (b) (c) (d) (e) (g)(f) (h) Fig. 3. Login to comment 105 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser ABCG2 p.Cys608Ser (a) PA / WT cells; (b) PA / C592S cells; (c) PA / C603S cells; (d) PA / C608S cells; (e) PA / C592SÆC603S cells; (f) PA / C592SÆC608S cells; (g) PA / C603SÆC608S cells; (h) PA / C592SÆC603SÆ C608S cells. Login to comment 109 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser PA /WT, PA/C603S-cl.3, and PA /C603S-cl.7 transfectants showed high levels of resistance to SN-38. Login to comment 110 ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser PA /C592S transfectants exhibited intermediate levels of resistance to SN-38 whereas PA /C608S transfectants showed only marginal levels of resistance to this drug. Login to comment 120 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser Based on the results of drug sensitivity assay, we selected PA /WT-mix, PA/C592S-cl.6, PA /C603S-cl.3, PA /C592SÆC608S-cl.7, and PA/C592SÆC603SÆC608S-cl.7 cells as representative drug-resistant transfectants for further study. Login to comment 122 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser As shown in Figure 7(a), intracellular mitoxantrone was at low levels in PA /WT-mix and PA /C603S-cl.3 cells and at intermediate levels in PA /C592S-cl.6, PA /C592SÆC608S-cl.7, and PA /C592SÆC603SÆC608S-cl.7 cells. Login to comment 134 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser Immunofluorescent microscopy analysis using an anti-BCRP antibody BXP-21 was performed to examine whether 250 200 70-kDa BCRP 75-kDa BCRP 100 150 0 50 NormalizedBCRPexpression(%ofPA/WT) PA317 -mix -cl.1 -cl.8 -mix -cl.6 -cl.7 -mix -cl.3 -cl.7 -mix -cl.5 -cl.7 -mix -cl.1 -cl.4 -mix -cl.2 -cl.7 -mix -cl.6 -cl.7 -mix -cl.6 -cl.7 PAC592S ·C603S PA/C592S ·C608S PA/C603S ·C608S PA/C592S ·C603S·C608S PA/WT PA/C592S PA/C603S PA/C608S Fig. 4. Login to comment 138 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser ABCG2 p.Cys608Ser ABCG2 p.Cys608Ser SN-38 resistance of mutant BCRP transfectants Cell line IC50 to SN-38† (nM) Degree of resistance‡ (x-fold) Relative expression level Total BCRP§ 75-kDa BCRP- PA317 3.7 ± 0.2 1 - - PA / WT-mix 90 ± 4.4 24 100 100 PA / WT-cl.1 172 ± 6.1 47 207 206 PA / WT-cl.8 77 ± 10.3 21 56 56 PA / C592S-mix 12.4 ± 0.8 3.4 34 24 PA / C592S-cl.6 28.5 ± 1.4 7.7 52 37 PA / C592S-cl.7 13.7 ± 0.5 3.7 16 11 PA / C603S-mix 9.7 ± 0.5 2.6 54 51 PA / C603S-cl.3 409 ± 15.9 110 146 132 PA / C603S-cl.7 158 ± 17.7 43 135 122 PA / C608S-mix 4.8 ± 0.3 1.3 27 3 PA / C608S-cl.5 5.6 ± 0.2 1.5 26 7 PA / C608S-cl.7 6.3 ± 0.6 1.7 43 12 PA / C592SÆC603S-mix 3.6 ± 0.2 1.0 27 10 PA / C592SÆC603S-cl.1 5.1 ± 1.1 1.4 27 10 PA / C592SÆC603S-cl.4 6.2 ± 0.8 1.7 43 26 PA / C592SÆC608S-mix 16.7 ± 1.8 4.6 92 67 PA / C592SÆC608S-cl.2 22.8 ± 2.0 6.2 163 111 PA / C592SÆC608S-cl.7 34.5 ± 4.9 9.4 151 106 PA / C603SÆC608S-mix 2.9 ± 0.2 0.8 21 3 PA / C603SÆC608S-cl.6 4.4 ± 0.4 1.2 57 3 PA / C603SÆC608S-cl.7 3.4 ± 0.1 0.9 51 4 PA / C592SÆC603SÆC608S-mix 4.1 ± 0.2 1.1 13 7 PA / C592SÆC603SÆC608S-cl.6 4.0 ± 0.4 1.1 25 13 PA / C592SÆC603SÆC608S-cl.7 11.1 ± 1.5 3.0 145 102 †The cells were incubated for 4 days at 37°C in the presence or absence of various concentrations of 7-ethyl-10-hydroxycamptothecin (SN-38). Login to comment 149 ABCG2 p.Cys603Ser To examine the sensitivity of mutant BCRPs to a BCRP inhibitor, namely FTC, the reversal effect of FTC on the mutant BCRP transfectants was examined. PA /C603S-cl.7 was added to the panel of drug-resistant transfectants in this experiment. Login to comment 150 ABCG2 p.Cys592Ser As shown in Table 2, FTC (1 lM) completely reversed the resistance of PA /WT-mix and PA /C592S-cl.6 cells to SN-38 and mitoxantrone. Login to comment 151 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser Fumitremorgin C (FTC) reversed the drug resistance of PA /C603S-cl.3 and PA /C603S-cl.7 cells, although these clones still showed 4-to 15-fold higher levels of resistance to SN-38 and mitoxantrone in the presence of FTC. Login to comment 162 ABCG2 p.Cys603Ser Consistent with our previous study, PA/C603S cells expressed both monomeric and dimeric BCRP. Login to comment 163 ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser (16) PA /C608S cells also expressed a BCRP monomer whereas PA/ C592S cells expressed a BCRP dimer. Login to comment 170 ABCG2 p.Cys603Ser Single mutation on Cys-603 loses this homo-dimerization site, and it can therefore be concluded that BCRP-C603S protein mainly existed as a monomer. Login to comment 171 ABCG2 p.Cys603Ser The intramolecular disulfide bridge between Cys-592 and Cys-608 is partially disrupted for some reason, and small amount of the BCRP dimer could be detected in PA /C603S. Login to comment 172 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser Introduction of the C592S mutation into BCRP-C603S extinguishes the counter partner against Cys-608 in the intramolecular disulfide bridge. Login to comment 175 ABCG2 p.Cys608Ser In PA /C608S cells, Cys-603 can also construct an intramolecular disulfide bridge with Cys-592, and therefore a single mutation on Cys-608 may weaken dimerization of BCRP. Login to comment 195 ABCG2 p.Cys592Ser PA/C592S-cl.6 4. Login to comment 196 ABCG2 p.Cys603Ser PA/C603S-cl.3 5. Login to comment 208 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser (1) PA317 cells; (2) PA / WT-mix cells; (3) PA / C592S-cl.6 cells; (4) PA / C603S-cl.3 cells; (5) PA / C592SÆC608S-cl.7 cells; (6) PA / C592SÆC603SÆC608S-cl.7 cells. Login to comment 226 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser PA/WT, PA /C592S, PA/C603S, PA /C592SÆC608S, and PA /C592SÆC603SÆC608S-cl.7 cells exhibited drug resistance and low accumulation of mitoxantrone. Login to comment 227 ABCG2 p.Cys603Ser PA/WT and PA /C603S cells expressed a 75-kDa BCRP product (Fig. 2). Login to comment 228 ABCG2 p.Cys592Ser ABCG2 p.Cys608Ser Significant expression of a 75-kDa BCRP was also found in PA /C592S, PA /C592SÆC608S, and PA/ C592SÆC603SÆC608S-cl.7 cells, whereas the PA/C608S cells mainly expressed a 70-kDa BCRP PA317 PA/WT-mix PA/C592S·C603S ·C608S-cl.7 BCRP Nuclei Merge 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm 20 µm Fig. 8. Login to comment 234 ABCG2 p.Cys603Ser ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser Effect of FTC on the SN-38 and mitoxantrone resistance of mutant BCRP transfectants Cell line SN-38 Mitoxantrone Relative expression level of total BCRP§ Degree of resistance† RI‡ Degree of resistance† RI‡ )FTC +FTC )FTC + FTC PA317 1.0 ± 0.1 0.9 ± 0.1 - 1.0 ± 0.1 0.9 ± 0.1 - - PA / WT-mix 19.3 ± 1.7 1.4 ± 0.1 13.8 9.0 ± 0.3 1.2 ± 0.1 7.5 100 PA / C592S-cl.6 4.2 ± 0.3 1.2 ± 0.1 3.5 3.7 ± 0.1 1.3 ± 0.1 2.8 52 PA / C603S-cl.3 106 ± 7.3 7.9 ± 0.3 13.4 57 ± 5.9 15 ± 0.7 3.8 146 PA / C603S-cl.7 18 ± 2.0 4.1 ± 0.2 4.4 28 ± 3.9 12 ± 1.0 2.3 135 PA / C592SÆC608S-cl.7 8.4 ± 0.5 4.0 ± 0.2 2.1 3.3 ± 0.2 3.3 ± 0.1 1.0 151 PA / C592SÆC603SÆC608S-cl.7 4.1 ± 0.3 4.6 ± 0.3 0.9 7.5 ± 0.2 6.0 ± 0.8 1.3 145 †The cells were incubated for 4 days at 37°C in the presence or absence of various concentrations of anticancer agents in the presence or absence of 1 lM fumitremorgin C (FTC). Login to comment 245 ABCG2 p.Cys603Ser ABCG2 p.Cys592Ser Fumitremorgin C (FTC) strongly reversed the resistance of PA /WT and PA /C592S cells to SN-38 and mitoxantrone, and partially reversed the drug resistance of PA /C603S cells. Login to comment 260 ABCG2 p.Phe208Ser ABCG2 p.Phe431Leu We have also reported that two germ line mutations of BCRP in our laboratory, namely 623T>C (F208S) and 1291T>C (F431L), also resulted in the loss of function or severely reduced the function of BCRP. Login to comment 261 ABCG2 p.Phe431Leu (44) Sunitinib clearly reversed the SN-38 resistance of PA /WT cells but not of BCRP-F431L transfectants. Login to comment 262 ABCG2 p.Phe431Leu In addition, sunitinib did not inhibit [125 I]iodoarylazidoprazosin-binding to BCRP-F431L. Login to comment