Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2.
Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. 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. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. 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. 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. 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. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.
Tamura
Ai
A
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan
Wakabayashi
Kanako
K
Onishi
Yuko
Y
Takeda
Misako
M
Ikegami
Yoji
Y
Sawada
Seigo
S
Tsuji
Masahisa
M
Matsuda
Yoichi
Y
Ishikawa
Toshihisa
T
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002166:Camptothecin / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance; D015870:Gene Expression / Q000187:drug effects; D015894:Genome, Human / Q000235:genetics; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D014633:Valine / Q000235:genetics / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D009363:Neoplasm Proteins; D005973:Glutamine; D000251:Adenosine Triphosphatases; D014633:Valine; D002166:Camptothecin