Differential sensitivities of the human ATP-binding cassette transporters ABCG2 and P-glycoprotein to cyclosporin A

Several ATP-binding cassette ( ABC) transporters can confer multidrug resistance to cancer cells by functioning as energy-dependent efflux pumps. The half-transporter ABCG2 and the widely studied P-glycoprotein (P-gp) are two ABC transporters that, when overexpressed, are capable of extruding a variety of structurally unrelated chemotherapy agents from cells. In this study, we demonstrate that human ABCG2 and P-glycoprotein, despite overlapping substrate specificities, differ in sensitivity to the immunomodulator cyclosporin A. In this study, we used human ABCG2 and human P-gp, each expressed separately in drug-selected MCF-7 sublines and transiently transfected HeLa cells. By flow cytometric analysis using the fluorescent substrates rhodamine 123 and mitoxantrone, we showed that cyclosporin A inhibits P-gp function at low micromolar concentrations, whereas ABCG2 function was unaffected. Furthermore, P-gp, but not ABCG2, was able to transport [H-3] cyclosporin A directly in intact cells. We also demonstrated, for the first time, that [I-125] iodoarylazidoprazosin, a photoaffinity analog of the substrate prazosin, labels multiple variants of ABCG2 specifically and that this labeling, although competed by some ABCG2 substrates, is unaffected by cyclosporin A. These labeling data also suggest the presence of multiple drug binding sites in ABCG2. In addition, cyclosporin A had no effect on the basal or prazosin-stimulated ATPase activity of ABCG2, whereas both the basal and verapamil-stimulated ATPase activities of P-gp were inhibited markedly. Together, our results suggest that cyclosporin A is neither a substrate nor an inhibitor of the human ABCG2 transporter, under the conditions and concentrations examined.