Two polymorphic variants of ABCC1 selectively alter drug resistance and inhibitor sensitivity of the multidrug and organic anion transporter multidrug resistance protein 1.
In this study we compared the in silico predictions of the effect of ABCC1 nonsynonymous single nucleotide polymorphisms (nsSNPs) with experimental data on MRP1 transport function and response to chemotherapeutics and multidrug resistance protein 1 (MRP1) inhibitors. Vectors encoding seven ABCC1 nsSNPs were stably expressed in human embryonic kidney (HEK) cells, and levels and localization of the mutant MRP1 proteins were determined by confocal microscopy and immunoblotting. The function of five of the mutant proteins was determined using cell-based drug and inhibitor sensitivity and efflux assays, and membrane-based organic anion transport assays. Predicted consequences of the mutations were determined by multiple bioinformatic methods. Mutants C43S and S92F were correctly routed to the HEK cell plasma membrane, but the levels were too low to permit functional characterization. In contrast, levels and membrane trafficking of R633Q, G671V, R723Q, A989T, and C1047S were similar to wild-type MRP1. In cell-based assays, all five mutants were equally effective at effluxing calcein, but only two exhibited reduced resistance to etoposide (C1047S) and vincristine (A989T; C1047S). The GSH-dependent inhibitor LY465803 (LY465803 [N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide)] was less effective at blocking calcein efflux by A989T, but in a membrane-based assay, organic anion transport by A989T and C1047S was inhibited by MRP1 modulators as well as wild-type MRP1. GSH accumulation assays suggest cellular GSH efflux by A989T and C1047S may be impaired. In conclusion, although six in silico analyses consistently predict deleterious consequences of ABCC1 nsSNPs G671V, changes in drug resistance and inhibitor sensitivity were only observed for A989T and C1047S, which may relate to GSH transport differences.
Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario, Canada.
Gwenaëlle
G
Conseil
Susan P C
SP
Cole
D001692:Biological Transport / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005978:Glutathione / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D021381:Protein Transport / Q000235:genetics
D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010447:Peptide Hydrolases; C421109:multidrug resistance-associated protein 1