The Q141K Single-Nucleotide Polymorphism Impacts the Transporter Activity of ABCG2.

2463 The ATP-binding cassette half-transporter ABCG2 has been implicated in oncology as a mediator of drug resistance, in pharmacology as an arbiter of drug absorption, and in normal physiology as a marker of stem cell populations. Three nonsynonymous single nucleotide polymorphisms (SNPs) have been described in the ABCG2 gene, V12M, Q141K and D620N. To determine if the SNPs would have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with ABCG2 coding the SNP mutations V12M, Q141K, or D620N. Northern and immunoblot analysis confirmed expression of ABCG2 mRNA and protein, respectively. Transport and cytotoxicity studies were performed to evaluate the potential impact of the SNPs on protein function. Efflux of both mitoxantrone and Hoechst 33342 inhibitable by fumitremorgin C (FTC) was detected with all three SNP vectors. To detect differences in transport, surface protein expression was quantified using the antibody 5D3. FTC-inhibitable efflux of mitoxantrone was calculated for HEK 293 cells transfected with the SNPs as well as wild-type R482 and the gain-of-function mutants R482G and R482T ABCG2. When FTC-inhibitable mitoxantrone efflux was normalized to cell surface ABCG2 expression, cells transfected with variant Q141K ABCG2 had significantly lower values than wild-type R482 ABCG2 (p=0.0048). Further, in 4-day cytotoxicity assays with mitoxantrone, cells transfected with wild-type R482 ABCG2 had IC50 values 6.0-fold higher than cells expressing comparable levels of Q141K ABCG2, providing support for the observation that the Q141K SNP does impact drug transport. The vanadate-sensitive ATPase activity of ABCG2 in membrane fractions of Sf9 insect cells transfected with recombinant baculoviruses containing wild-type and SNP variants of ABCG2 was subsequently examined, and ATPase activity in the Q141K variant was 1.8-fold lower compared to wild-type ABCG2. The ATPase activities of the V12M and D620N variants were comparable to that of wild-type ABCG2. The results presented suggest that the Q141K SNP affects the transport efficiency of ABCG2 and individuals carrying this ABCG2 variant may be more sensitive to ABCG2 substrate drugs.