Cellular and in Vitro Transport of Glutathione Conjugates by MRP

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Abstract
MRP is a recently identified ATP-binding cassette transporter. We previously established that MRP confers resistance to a spectrum of natural product cytotoxic drugs [Kruh, G. D., (1994) Cancer Res. 54, 1649−1652], that expression of MRP is associated with enhanced drug efflux [Breuninger, L. M., (1995) Cancer Res. 55, 5342−5347], and that MRP transcript is widely expressed in human tissues and solid tumor cell lines [Kruh, G. D., (1995) J. Natl. Cancer Inst. 87, 1256−1258]. In the present study the relationship between MRP and drug glutathione S-conjugates was examined. We observed that MRP was labeled by azidophenacylglutathione (APA-SG), a photoaffinity analog of glutathione, and that inside-out membrane vesicles prepared from MRP-overexpressing HL60/ADR cells transported this compound. Transport into membrane vesicles was ATP-dependent, sensitive to osmolarity, and saturable with regard to APA-SG and ATP concentrations, with Km values of 15 and 61 μM, respectively. APA-SG transport was competitively inhibited by the natural product cytotoxic drugs daunorubicin, vincristine, and etoposide, with Ki values of 4.8, 3.8, and 5.5 μM, respectively. Oxidized glutathione, the drug−glutathione S-conjugate DNP-SG, the LTD4 antagonist MK571 and arsenate were also competitive inhibitors, with Ki values of 9.0, 23.4, 1.1, and 15.0 μM, respectively. Analysis of the fate of monochlorobimane in MRP transfectants revealed reduced intracellular concentrations of drug-glutathione S-conjugates associated with enhanced efflux and altered intracellular distribution. These results indicate that MRP can transport glutathione conjugates in vitro and in living cells and suggest the possibility that the transporter may represent a link between cellular resistance to some classes of cytotoxic drugs and glutathione-mediated mechanisms of resistance. In addition, the observation that both mildly cationic or neutral natural product cytotoxic drugs and anionic compounds such as DNP-SG, MK571, and arsenate are competitive inhibitors of MRP action suggests that the substrate specificity of the transporter is quite broad.