Quantitative evaluation of drug resistance profile of cells expressing wild-type or genetic polymorphic variants of the human ABC transporter ABCC4

Abstract

Broad-spectrum resistance in cancer cells is often caused by the overexpression of ABC transporters; which varies across individuals because of genetic single-nucleotide polymorphisms (SNPs). In the present study; we focused on human ABCC4 and established cells expressing the wild-type (WT) or SNP variants of human ABCC4 using the Flp-In™ system (Invitrogen, Life Technologies Corp, Carlsbad, CA, USA) based on Flp recombinase-mediated transfection to quantitatively evaluate the effects of nonsynonymous SNPs on the drug resistance profiles of cells. The mRNA levels of the cells expressing each ABCC4 variant were comparable. 3-(4,5-Dimethyl-2- thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay clearly indicated that the EC50 values of azathioprine against cells expressing ABCC4 (WT) were 1.4–1.7-fold higher than those against cells expressing SNP variants of ABCC4 (M184K; N297S; K304N or E757K). EC50 values of 6- mercaptopurine or 7-Ethyl-10-hydroxy-camptothecin (SN-38) against cells expressing ABCC4 (WT) were also 1.4–2.0- or 1.9-fold higher than those against cells expressing the SNP variants of ABCC4 (K304N or E757K) or (K304N; P403L or E757K); respectively. These results indicate that the effects of nonsynonymous SNPs on the drug resistance profiles of cells expressing ABCC4 can be quantitatively evaluated using the Flp-In™ system.