CELLULAR AND IN-VITRO TRANSPORT OF GLUTATHIONE CONJUGATES BY MRP

MRP is a recently identified ATP-binding cassette transporter. We prev iously established that MRP confers resistance to a spectrum of natura l product cytotoxic drugs [Kruh, G. D., (1994) Cancer Res. 54, 1649-16 52], that expression of MRP is associated with enhanced drug efflux [B reuninger, L. M., (1995) Cancer Res. 55, 5342-5347], and that MRP tran script is widely expressed in human tissues and solid tumor cell lines [Kruh, G. D., (1995) J. Natl. Cancer Inst. 87, 1256-1258]. In the pre sent study the relationship between MRP and drug glutathione S-conjuga tes was examined. We observed that MRP was labeled by azidophenacylglu tathione (APA-SG), a photoaffinity analog of glutathione, and that ins ide-out membrane vesicles prepared from MRP-overexpressing HL60/ADR ce lls transported this compound. Transport into membrane vesicles was AT P-dependent, sensitive to osmolarity, and saturable with regard to APA -SG and ATP concentrations, with K-m values of 15 and 61 mu M, respect ively. APA-SG transport was competitively inhibited by the natural pro duct cytotoxic drugs daunorubicin, vincristine, and etoposide, with K- i values of 4.8, 3.8, and 5.5 mu M, respectively. Oxidized glutathione , the drug-glutathione S-conjugate DNP-SG, the LTD(4) antagonist MK571 and arsenate were also competitive inhibitors, with K-i values of 9.0 , 23.4, 1.1, and 15.0 mu M, respectively. Analysis of the fate of mono chlorobimane in MRP transfectants revealed reduced intracellular conce ntrations of drug-glutathione S-conjugates associated with enhanced ef flux and altered intracellular distribution. These results indicate th at MRP can transport glutathione conjugates in vitro and in living cel ls and suggest the possibility that the transporter may represent a li nk between cellular resistance to some classes of cytotoxic drugs and glutathione-mediated mechanisms of resistance. In addition, the observ ation that both mildly cationic or neutral natural product cytotoxic d rugs and anionic compounds such as DNP-SG, MK571, and arsenate are com petitive inhibitors of MRP action suggests that the substrate specific ity of the transporter is quite broad.