ABCMdb

PMID: 17297656

Tamura A, Wakabayashi K, Onishi Y, Takeda M, Ikegami Y, Sawada S, Tsuji M, Matsuda Y, Ishikawa T
Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2.
Cancer Sci. 2007 Feb;98(2):231-9., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Login to comment 6 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Login to comment 7 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. Login to comment 8 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Login to comment 9 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly 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 104 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Characterization of WT ABCG2, V12M and Q141K expressed in Flp-In-293 cells. Login to comment 105 ABCG2 p.Gln141Lys ABCG2 p.Val12Met As shown in Fig. 2A, mRNA levels of WT ABCG2 as well as its major SNP variants (V12M and Q141K) were represented evenly in Flp-In cells. Login to comment 111 ABCG2 p.Gln141Lys (16) As demonstrated in Fig. 2A, the protein expression level of the Q141K variant was approximately half that of the WT level. Login to comment 112 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Figure 2B depicts the immunofluorescence images of Flp-In-293/Mock, Flp-In-293/ABCG2 (WT), Flp-In-293/ABCG2 (V12M) and Flp-In-293/ABCG2 (Q141K) cells. Login to comment 116 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Drug resistance profiles of Flp-In-293 cells expressing WT ABCG2, V12M and Q141K variants toward camptothecin analogs. Login to comment 117 ABCG2 p.Gln141Lys ABCG2 p.Val12Met To examine the drug resistance profiles, we incubated Flp-In-293/ABCG2 (WT), Flp-In-293/ABCG2 (V12M) and Flp-In-293/ABCG2 (Q141K) cells with SN-38 and the new camptothecin analogs at different concentrations for 96 h, after which we observed the cell survival rates. Login to comment 118 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Flp-In-293/ABCG2 (WT), Flp-In-293/ABCG2 (V12M) and Flp-In-293/ABCG2 (Q141K) cells exhibited strong resistance to SN-38, SN-355 and SN-398, as represented by large IC50 values (Fig. 3). Login to comment 119 ABCG2 p.Val12Met Interestingly, the V12M variant provided the cells with higher drug resistance to SN-38, SN-355 and SN-398 than did WT ABCG2 (Fig. 3). Login to comment 120 ABCG2 p.Gln141Lys In contrast, the Q141K variant appeared to render drug resistance at a level only half that of WT. Login to comment 135 ABCG2 p.Gln141Lys ABCG2 p.Val12Met (V12M) or Flp-In-293/ABCG2 (Q141K) cells exhibited resistance toward SN-22, SN-343, SN-348, SN-349, SN-351, SN-352, SN-353, SN-364, SN-397, SN-443 or SN-444. Login to comment 136 ABCG2 p.Gln141Lys ABCG2 p.Val12Met These results suggest that camptothecin analogs lacking the hydroxyl or amino group at position 10 or 11 are not substrates for WT ABCG2 or the V12M and Q141K variants. Login to comment 137 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro Characterization of the F208S, S248P, F431L, S441N and F489L variants expressed in Flp-In-293 cells. Login to comment 138 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro Figure 2C demonstrates the mRNA and protein levels of WT ABCG2 and the F208S, S248P, F431L, S441N and F489L variants expressed in Flp-In-293 cells. Login to comment 139 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn Whereas mRNA levels were almost the same in WT and those variants, the protein levels of the F208S and S441N variants were markedly low. Login to comment 140 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn The immunofluorescence images of Flp-In-293/ABCG2 (F208S) and Flp-In-293/ABCG2 (S441N) cells revealed that those variant proteins were not expressed in the plasma membrane (Fig. 2D). Login to comment 141 ABCG2 p.Ser441Asn The S441N variant appeared to remain in the intracellular space. Login to comment 142 ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro The other variants (S248P, F431L and F489L) were expressed in the plasma membrane, as was WT ABCG2. Login to comment 145 ABCG2 p.Ser441Asn Cells transfected with F 208S or S441N did not exhibit any resistance to SN-38 ormitoxantrone; their cell survival curves were very similar to that of Flp-In-293/Mock cells. Login to comment 146 ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro On the contrary, the S248P, F431L and F489L variants contributed drug resistance; however, their contribution was not so large as that of WT. Login to comment 150 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys It is important to note that the IC50 values of Q141K-expressing cells toward doxorubicin and daunorubicin were greater than that of WT-expressing cells, whereas Q141K-expressing cells were more sensitive to SN-38 and mitoxantron compared with WT-expressing cells. Login to comment 151 ABCG2 p.Gln141Lys These results suggest that the substrate specificity of the Q141K variant differs delicately from that of WT ABCG2. Login to comment 152 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Considered overall, the minor SNP variants were less effective in drug resistance than the V12M and Q141K variants. Login to comment 154 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly 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 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro 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 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Continued markedly stimulated with prazosin in those acquired mutants (R482G and R482T). Login to comment 159 ABCG2 p.Gln141Lys ABCG2 p.Val12Met Honjo et al. first identified non-synonymous SNP of V12M and Q141K. Login to comment 160 ABCG2 p.Val12Met (23) The V12M polymorphism in exon 2 (34G >A) affects the N-terminal intracellular region of the protein. Login to comment 162 ABCG2 p.Val12Met The V12M polymorphism was found in all ethnic groups tested, with the highest allele frequency in Mexican-Indians (90% of only five individuals tested), but only 1.7% in a Swedish population. Login to comment 164 ABCG2 p.Val12Met Thus, there is a large difference in the allele frequency of the V12M polymorphism among different ethnic groups. Login to comment 166 ABCG2 p.Gln141Lys The Q141K polymorphism located in exon 5 (421C > A) leads to the replacement of the negatively charged glutamic acid residue with a positively charged lysine residue. Login to comment 168 ABCG2 p.Gln141Lys The Q141K variant was also detected in all ethnic groups tested, with the allele frequency ranging from 0 to 35% (Africans North Fig. 3. Login to comment 169 ABCG2 p.Gln141Lys ABCG2 p.Val12Met The effect of camptothecin analogs on the growth of Flp-In-293/ABCG2 (wild type), Flp-In-293/ABCG2 (V12M) or Flp-In-293/ABCG2 (Q141K) cells. Login to comment 176 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro Resistance profile (IC50) of ABCG2 Compound IC50 (nM) Mock Wild type V12M Q141K F208S S248P F431L S441N F489L SN-38 0.9 40.0 (44.4) 40.0 (44.4) 17.0 (18.9) 0.6 (0.7) 3.0 (3.3) 10.0 (11.1) 0.7 (0.8) 3.1 (3.4) Mitoxantorone 5.2 >100 (>19) 92.0 (17.7) 45.0 (8.7) 4.5 (0.9) 11.0 (2.1) 21.0 (4.0) 4.6 (0.9) 11.0 (2.1) Doxorubicin 32.0 78.0 (2.4) 100.0 (3.1) 110.0 (3.4) 20.0 (0.6) 20.0 (0.6) 40.0 (1.3) 21.0 (0.7) 45.0 (1.4) Daunorubicin 12.0 30.0 (2.5) 50.0 (4.2) 50.0 (4.2) 12.0 (1.0) 21.0 (1.8) 14.0 (1.2) 12.0 (1.0) 19.0 (1.6) Etoposide 110.0 200.0 (1.8) 220.0 (2.0) 200.0 (1.8) 110.0 (1.0) 120.0 (1.1) 120.0 (1.1) 130.0 (1.2) 170.0 (1.5) Vincristine 1.4 4.0 (2.9) 5.0 (3.6) 4.5 (3.2) 0.6 (0.4) 4.0 (2.9) 1.4 (1.0) 0.8 (0.6) 2.8 (2.0) Relative resistances to mock cells are described in parentheses. Login to comment 179 ABCG2 p.Gln141Lys (22,25,27,29) An investigation of the expression level of ABCG2 in 99 Japanese placenta samples revealed that individuals homozygous for the Q141K variant showed significantly lower expression levels of this transporter protein, whereas the heterozygous samples displayed an intermediate expression level. Login to comment 180 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys (27) It has been reported that the SNP Q141K may cause increased plasma concentrations of orally administered diflomotecan in cancer patients who express low levels of ABCG2 (Q141K) protein in the small intestine. Login to comment 182 ABCG2 p.Gln141Lys By using an in-vitro system, Imai et al. showed that ABCG2 Q141K variant-transfected PA317 cells had a low-level of drug resistance associated with decreased protein expression. Login to comment 183 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys (25) This finding is apparently in accordance with a report by Kondo et al.(31) However, Morisaki et al.(30) reported that the protein expression level of ABCG2 Q141K varied among clones of HEK293 cells where ABCG2 Q141K cDNA was expressed stably with the conventional pcDNA3.1(+) vector. Login to comment 184 ABCG2 p.Gln141Lys In contrast, Mizuarai et al.(28) described that approximately equal levels of ABCG2 were expressed in Q141K clones compared to the expression in WT, although they did not present any data in this respect. Login to comment 191 ABCG2 p.Gln141Lys Based on the new method, our present study (Fig. 2A) supports the original report of Imai et al.,(25) suggesting that the protein stability of the Q141K variant is reduced without any detectable changes in its mRNA levels. Login to comment 192 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro As clearly demonstrated in this study, the F208S, S248P, F431L, S441N and F489L variants exhibited greatly altered protein expression levels (Fig. 2C) or drug resistance profiles (Fig. 4 and Table 1). Login to comment 193 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn In particular, expression levels of the F208S and S441N variants were markedly low (Fig. 2C). Login to comment 195 ABCG2 p.Phe208Ser ABCG2 p.Phe208Ser In fact, when Flp-In-293/ ABCG2 (F208S) cells were treated with MG132, a proteasome inhibitor, the protein level recovered up to approximately 50% of the WT level, suggesting the involvement of proteasomes in degradation of the F208S variant (data not shown). Login to comment 199 ABCG2 p.Phe208Ser ABCG2 p.Ser248Pro The most recent version of NCBI dbSNP does not appear to contain validation for F208S and S248P as bona fide SNP. Login to comment 202 ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro As one of the specific aims of the present study, we functionally classified the non-synonymous polymorphisms (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) in terms of their protein expression level, drug resistance profile and prazosin-stimulated ATPase activity. Login to comment 203 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly As summarized in Fig. 5A, the functional properties of these variants were compared with acquired mutations of R482G and R482T. Login to comment 204 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The drug resistance profiles and prazosin-stimulated ATPase activity of R482G and R482T were reported previously. Login to comment 206 ABCG2 p.Gln141Lys ABCG2 p.Val12Met As shown in Fig. 5B, it is obvious that WT, V12M and Q141K form one group where protein expression, methotrexate and porphyrin transport, and resistance to SN-38 and mitoxantrone are positive, but contribution to doxorubicin- and daurorubicin- Fig. 4. Login to comment 207 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro Drug resistance profiles of Flp-In-293 cells expressing the wild-type (WT) BCRP/MXR1/ABCP (ABCG2), F208S, S248P, F431L, S441N or F489L variants toward (A) SN-38 and (B) mitoxantrone. Login to comment 212 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly 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 214 ABCG2 p.Phe208Ser ABCG2 p.Ser441Asn (16) Both F208S and S441N belong to the third group where protein expression levels were extremely low (Fig. 2C). Login to comment 216 ABCG2 p.Phe489Leu ABCG2 p.Phe431Leu ABCG2 p.Ser248Pro Finally, S248P, F431L and F489L appear to form the fourth heterogeneous group, where those variant proteins are expressed at normal levels but exhibit different profiles of drug resistance and transport activity. Login to comment 221 ABCG2 p.Gln141Lys ABCG2 p.Val12Met (32,33) As demonstrated in Fig. 3, the new camptothecin analogs that were non-substrates for ABCG2 circumvented ABCG2-mediated drug resistance without any influence from major SNP (i.e. V12M and Q141K). Login to comment