ABCMdb

PMID: 15838659

Morisaki K, Robey RW, Ozvegy-Laczka C, Honjo Y, Polgar O, Steadman K, Sarkadi B, Bates SE
Single nucleotide polymorphisms modify the transporter activity of ABCG2.
Cancer Chemother Pharmacol. 2005 Aug;56(2):161-72. Epub 2005 Apr 19., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn ORIGINAL ARTICLE Kuniaki Morisaki Æ Robert W. Robey Csilla O¨ zvegy-Laczka Æ Yasumasa Honjo Orsolya Polgar Æ Kenneth Steadman Bala´ zs Sarkadi Æ Susan E. Bates Single nucleotide polymorphisms modify the transporter activity of ABCG2 Received: 21 July 2004 / Accepted: 1 October 2004 / Published online: 19 April 2005 Ó Springer-Verlag 2005 Abstract Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. Login to comment 2 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. Login to comment 3 ABCG2 p.Gln141Lys FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P=0.0048). Login to comment 4 ABCG2 p.Val12Met ABCG2 p.Asp620Asn Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. Login to comment 6 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Login to comment 7 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Login to comment 8 ABCG2 p.Val12Met ABCG2 p.Val12Met Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. Login to comment 9 ABCG2 p.Gln141Lys These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology. Login to comment 25 ABCG2 p.Arg482Met Wang et al. have also identified an R482M mutation in a doxorubicin-resistant human T-cell line [47]. Login to comment 30 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn We previously sequenced the ABCG2 gene in 90 genomic DNA samples representing a global genetic diversity and identified three nonsynonymous SNPs -34G fi A, substituting a valine for methionine (V12M); 421C fi A, substituting a glutamine for lysine (Q141K); and 1858C fi A, substituting an aspartic acid for asparagine (D620N)-in the coding region of ABCG2 [18]. Login to comment 33 ABCG2 p.Gln141Lys ABCG2 p.Val12Met In a study of SNPs in ABCG2 in the general Japanese population, the V12M and Q141K SNPs were observed at higher allelic frequencies (17.2% and 26.6%, respectively) [19]. Login to comment 34 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Similarly, in other studies, the V12M and Q141K SNPs have also been found to be the most frequent polymorphisms in various ethnic and racial groups including Caucasian, Asian, and Swedish populations [3, 48]. Login to comment 37 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn containing full-length ABCG2 encoding wild-type (R482), mutant (R482T, R482G), or SNP variants (V12M, Q141K, or D620N) of ABCG2. Login to comment 76 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met Cells were also infected with mutant R482G ABCG2 encoding a K86M mutation that has been shown to abolish the function of the resulting protein [34]. Login to comment 95 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly 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 97 ABCG2 p.Val12Met Additionally, we generated an ABCG2 transfectant with the first 11 amino acids deleted and the V12M SNP (1_33del34G fi A, 1_11delV12M) in order to examine whether the substituted methionine at amino acid 12 can serve as the first codon for a functional ABCG2. Login to comment 98 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Val12Met ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn Clones were initially screened using the anti-ABCG2 antibody 5D3 and, from the positive clones obtained, 12 clones transfected with V12M, Q141K, D620N, or 1_11delV12M were selected for further study: V12M-12, -13 and -14; Q141K-5, -8, -13 and -16; D620N-2, -3 and -23; and 1_11delV12M-2 and -8. Login to comment 100 ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn By Northern blot and immunoblot analysis, D620N- 3 and D620N-23 showed relatively low expression levels (Fig. 1a, b). Login to comment 101 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Val12Met Although Northern blot analysis demonstrated higher expression of ABCG2 mRNA in wild-type (482R) ABCG2-transfected clones than in V12M-13 and Q141K-8 clones, immunoblot analysis showed generally comparable (within two- to threefold) ABCG2 protein expression in 482R, V12M-13, and Q141K-8 transfectants. Login to comment 103 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Differential resistance among cells transfected with wild-type, V12M and Q141K ABCG2 To determine whether the SNPs affected the transport activity of ABCG2, 4-day cytotoxicity assays were performed with the ABCG2 substrates mitoxantrone, topotecan, SN-38, and diflomotecan (BN80915) on ABCG2-transfected HEK-293 cells expressing comparable amounts of ABCG2. Login to comment 105 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met Comparable surface expression of ABCG2 in the 482R-2, Q141K-5, Q141K-8 and V12M-13 clones was confirmed using the 5D3 antibody, as shown in Fig. 2a. Login to comment 106 ABCG2 p.Val12Met This is in contrast to the immunoblot data that showed somewhat higher levels of ABCG2 in the V12M-13 clone. Login to comment 108 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn a Northern blot analysis of ABCG2 expression in representative HEK-293 cells transfected with wild-type, V12M, Q141K, or D620N ABCG2. Login to comment 114 ABCG2 p.Gln141Lys Except for the Q141K-5 clone with diflomotecan, P values for this comparison were <0.05. Login to comment 116 ABCG2 p.Gln141Lys ABCG2 p.Val12Met The V12M-13 and 482R-2 clones were comparably resistant to mitoxantrone, topotecan, SN-38 and diflomotecan, whereas the Q141K-5 and -8 clones had IC50 values that were 3-fold to 5-fold lower for mitoxantrone and topotecan, 2-fold to 3.4-fold lower for SN-38, and 1.2-fold to 2.3-fold lower for diflomotecan. Login to comment 118 ABCG2 p.Gln141Lys These results suggest that the Q141K SNP has functional consequences in the resulting ABCG2 protein. Login to comment 119 ABCG2 p.Gln141Lys Correlation between FTC-inhibitable mitoxantrone efflux and surface expression in ABCG2 transfectants To confirm the impaired transport of mitoxantrone in cells expressing Q141K ABCG2, we selected two to six Fig. 2 Impact of SNPs on ABCG2-mediated resistance. Login to comment 121 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met b Cytotoxicity assays were performed with mitoxantrone, topotecan, SN-38, or diflomotecan on HEK-293 cells transfected with empty vector (filled circles), or the 482R-2 (open circles), V12M-13 (filled triangles), Q141K-5 (open squares), and Q141K-8 (hatched squares) clones from a. Login to comment 122 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn Representative results are shown clones each of ABCG2-transfected cells expressing R482, R482T, R482G, V12M, Q141K or D620N ABCG2. Login to comment 132 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly 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 133 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn Efflux and expression values for cells transfected with V12M and D620N ABCG2 fell close to the line, while values for cells transfected with Q141K ABCG2 fell predominantly below the line. Login to comment 135 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn Among the transfectants, Q141K variants showed significantly lower values compared to the transfectants with wild-type ABCG2 and the other SNP variants, V12M and D620N (P=0.0048, 0.0005, and 0.0126, respectively), suggesting that Q141K ABCG2 transports mitoxantrone less efficiently than wild-type ABCG2. Login to comment 136 ABCG2 p.Val12Met ABCG2 p.Asp620Asn Although V12M and D620N variants showed somewhat higher efficiency of mitoxantrone transport than 482R, no statistically significant difference was found. Login to comment 138 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly 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 140 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn We next examined the ATPase activity of V12M, Q141K, and D620N variants using this system. Login to comment 143 ABCG2 p.Gln141Lys Despite similar levels of ABCG2 expression, the basal ATPase activity was significantly lower in the case of the Q141K variant than in wild-type ABCG2, whereas in the other SNP variants, the ATPase activity was similar to that observed in wild-type ABCG2 (Fig. 5b). Login to comment 146 ABCG2 p.Gln141Lys The lower basal ATPase activity in the Q141K variant persisted in the presence of mitoxantrone (Fig. 5b). Login to comment 148 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met At 1 lM Clone Mitoxantrone Topotecan SN-38 Diflomotecan IC50 RR IC50 RR IC50 RR IC50 RR pcDNA3-10 0.6±0.3 - 8.7±1.2 - 1.2±0.7 - 0.3±0.2 - 482R-2 34±5.4 57 275±150 32 123±68 103 1.4±0.6 5 V12M-13 44±15 73 250±71 29 100±1 83 1±0.1 3 Q141K-5 7.4±1.8 12 55±7 6 36±11 30 0.6±0.07 2 Q141K-8 10.8±5.3 18 90±14 10 66±11 55 0.9±0.1 3 Table 1 Relative resistance (RR) of ABCG2-transfected cells to ABCG2 substrates. Login to comment 151 ABCG2 p.Gln141Lys In all cases P<0.05 except for the Q141K-5 clone with diflomotecan where P=0.09 (IC50 values are in nanomoles) Ko143, ATPase activity in membrane protein from cells expressing any of the ABCG2 proteins was almost completely abrogated. Login to comment 157 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Effect of SNPs on cellular localization of ABCG2 To determine if the SNPs affected membrane localization of ABCG2, we performed immunofluorescence studies on HEK-293 cells stably transfected with wild-type, V12M or Q141K ABCG2. Login to comment 159 ABCG2 p.Val12Met Similarly, cells expressing V12M ABCG2 also had ABCG2 localized primarily to the cell surface (Fig. 6b). Login to comment 160 ABCG2 p.Gln141Lys In contrast, HEK-293 cells expressing Q141K ABCG2 demonstrated high intracellular staining with the BXP-21 antibody, as well as cell surface staining (Fig. 6c). Login to comment 161 ABCG2 p.Gln141Lys These results, despite the selection of clones expressing comparable levels of cell surface ABCG2, suggest impaired membrane trafficking or incorrect membrane insertion of Q141K ABCG2. Login to comment 163 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn To examine whether the nonsynonymous SNPs in ABCG2 affect the transport of this compound, Hoechst 33342 dye transport was measured in intact Sf9 cells expressing wild-type, V12M, Q141K, or D620N ABCG2, as well as the nonfunctional mutant, R482G/K86M. Login to comment 164 ABCG2 p.Gln141Lys ABCG2 p.Asp620Asn Immunoblot analysis of protein obtained from the infected cells is shown in Fig. 7a. Hoechst 33342 transport was comparable in cells expressing wild-type, Q141K or D620N ABCG2. Login to comment 165 ABCG2 p.Val12Met ABCG2 p.Val12Met In contrast, Hoechst 33342 transport was significantly lower in cells expressing V12M ABCG2 following normalization to the higher ABCG2 expression in these cells, suggesting less efficient Hoechst 33342 transport by V12M ABCG2 (Fig. 7b). Login to comment 167 ABCG2 p.Arg482Gly ABCG2 p.Lys86Met In Sf9 cells expressing the R482G/K86M ATP binding-site mutant, Hoechst 33342 uptake was comparable to the level of Hoechst 33342 accumulation found in the Ko143-inhibited cells. Login to comment 168 ABCG2 p.Val12Met To evaluate the possibility that Hoechst 33342 transport was impaired in human cells expressing V12M ABCG2, we performed Hoechst accumulation studies with HEK-293 cells transfected with the wild-type or SNP variants of ABCG2. Login to comment 172 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn Representative histograms for 482R-9, 482G-1, V12M-13, D620N-2, Q141K-5, and 1_11delV12M-8 are shown substrate-free medium for 60 min continuing with or without FTC. Login to comment 174 ABCG2 p.Gln141Lys ABCG2 p.Val12Met No significant difference in FTC-inhibitable Hoechst efflux was observed between cells expressing wild-type (R482), V12M or Q141K ABCG2 (Fig. 7c, right column). Login to comment 175 ABCG2 p.Val12Met Cytotoxicity assays with Hoechst 33342 confirmed marked resistance conferred by ABCG2, with an IC50 for the 482R-2 clone of 16.3±15.8 lM and for the V12M-13 clone of 56.7±5.8 lM. Login to comment 176 ABCG2 p.Val12Met Since the IC50 for empty vector-transfected cells was 26.3±15.8 n M, this translates to a relative resistance of 621 for 482R-2 and 2155 for V12M-13. Login to comment 177 ABCG2 p.Val12Met These data are not consistent with impaired transport of Hoechst 33342 by V12M ABCG2 and are in contrast to the results obtained with intact Sf9 insect cells. Login to comment 178 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn Discussion We and others have recently identified several polymorphisms in ABCG2, including three nonsynonymous SNPs resulting in amino acid substitution in the coding region of ABCG2: V12M, Q141K, D620N [4, 19, 20, 50]. Login to comment 180 ABCG2 p.Gln141Lys Our results suggest that the Q141K SNP affects the transport efficiency of ABCG2. Login to comment 189 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn 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 199 ABCG2 p.Gln141Lys Imai et al. have previously reported that the Q141K variant is associated with decreased protein expression in transfected cells and therefore results in increased sensitivity to chemotherapeutic agents [20]. Login to comment 200 ABCG2 p.Gln141Lys In contrast, we did not observe decreased expression of ABCG2 in HEK-293 cells transfected with Q141K ABCG2. Login to comment 201 ABCG2 p.Gln141Lys Supporting our findings, Zamber et al. have found no correlation between the Q141K SNP and level of expression of ABCG2 protein [50]. Login to comment 202 ABCG2 p.Gln141Lys The Q141K SNP does not appear to prevent expression of the protein on the cell surface. Login to comment 204 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met Four-day cytotoxicity assays demonstrated that, among HEK-293 cells transfected with wild-type, V12M, or Q141K ABCG2, those expressing Q141K ABCG2 had IC50 values for mitoxantrone, topotecan, SN-38 and diflomotecan that were as much as fivefold lower than those for cells expressing comparable levels Fig. 5 ATPase activity in Sf9 insect cells infected with ABCG2-bearing baculovirus. Login to comment 205 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn a Immunoblot detection of human wild-type, D620N, Q141K and V12M ABCG2 expressed in Sf9 insect cells. Login to comment 206 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Val12Met ABCG2 p.Asp620Asn ABCG2 p.Asp620Asn Membranes of Sf9 cells (1.5 lg total protein from V12M/Sf9, Q141K/Sf9 and D620N/Sf9, 1.0 lg from wild-type ABCG2/Sf9, and 1.2 lg from b-galactosidase/Sf9) dissolved in disaggregation buffer were subjected to electrophoresis on 7.5% Laemmli-type gels and blotted onto PVDF membranes, followed by immunodetection with the BXP-21 antibody. b ATPase activity measured in membranes of Sf9 cells expressing the wild-type, V12M, Q141K, and D620N variants of human ABCG2. Login to comment 210 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Val12Met HEK-293 cells transfected with wild-type (482R-2) (a), V12M (V12M-13) (b), or Q141K (Q141K-8) (c) ABCG2 were fixed, permeabilized, and then incubated with the mouse monoclonal anti-ABCG2 antibody, BXP-21. Login to comment 211 ABCG2 p.Val12Met After washing, cells were incubated with Alexa Fluor 488-labeled secondary antibody and subjected to confocal laser scanning microscopy of wild-type or V12M ABCG2. Login to comment 212 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn When surface ABCG2 expression was used to normalize the FTC-inhibitable mitoxantrone efflux, we found that HEK-293 cells expressing Q141K ABCG2 transported mitoxantrone less efficiently than cells expressing wild-type, V12M or D620N ABCG2. Login to comment 213 ABCG2 p.Gln141Lys These findings are in agreement with those of Mizuarai et al., who found that polarized LLC/ PK1 cells transfected with Q141K ABCG2 are more sensitive to mitoxantrone, topotecan and an indolocarbazole topoisomerase I inhibitor, all of which are known ABCG2 substrates [30]. Login to comment 214 ABCG2 p.Val12Met However, in contrast to the results of Mizuarai et al., we did not observe enhanced sensitivity to ABCG2 substrate drugs in cells transfected with V12M ABCG2. Login to comment 217 ABCG2 p.Val12Met Indeed, we noted impaired Hoechst 33342 transport in Sf9 cells expressing V12M ABCG2. Login to comment 218 ABCG2 p.Val12Met This was in contrast to cytotoxicity assays with transfected human cells that displayed comparable resistance to Hoechst 33342 in cells expressing wild-type or V12M ABCG2. Login to comment 219 ABCG2 p.Val12Met It is of note that Imai et al., using a human expression system, did not find increased sensitivity to mitoxantrone in PC3 cells transfected with V12M ABCG2 compared to cells transfected with wild-type [20]. Login to comment 220 ABCG2 p.Val12Met These contrasting results suggest that the glycosylation status of the protein may be significant for the V12M SNP. Login to comment 221 ABCG2 p.Gln141Lys The Q141K SNP has also recently been shown to significantly affect the pharmacokinetics of diflomotecan. Login to comment 223 ABCG2 p.Gln141Lys Despite the relatively poor transport of diflomotecan by ABCG2, Sparreboom et al. have reported that, in patients heterozygous for the Q141K SNP, plasma diflomotecan levels are approximately threefold higher than in patients expressing the wild-type allele [44]. Login to comment 225 ABCG2 p.Gln141Lys These results suggest that the Q141K SNP may alter the pharmacokinetic profile of other ABCG2 substrate drugs. Login to comment 226 ABCG2 p.Gln141Lys Basal ATPase activity was determined to be 1.8-fold lower in membrane protein isolated from Sf9 insect cells infected with recombinant baculoviruses containing full-length Q141K ABCG2 compared to protein from cells expressing wild-type or the other SNP variant ABCG2 forms. Login to comment 227 ABCG2 p.Gln141Lys Our results are in agreement with those of Mizuarai et al. who reported that the ATPase activity of Q141K ABCG2 is 1.3-fold lower than wild-type in the Sf9 system [30]. Login to comment 241 ABCG2 p.Gln141Lys Consistent with other reports, we found that the Q141K polymorphism impaired the activity of the ABCG2 protein. Login to comment 243 ABCG2 p.Val12Met In contrast to results in non-mammalian systems, no impairment of the V12M variant was observed in the HEK-293 transfectants. Login to comment 244 ABCG2 p.Gln141Lys Taken together, the results presented here support the hypothesis that the Q141K variant has the potential to alter the pharmacokinetics of ABCG2 substrates. Login to comment 245 ABCG2 p.Gln141Lys Whether the presence of the Q141K polymorphism in clinical tumors could promote chemosensitivity is unknown, but remains a question for further study. Login to comment