Multidrug Resistance Associated with Increased Drug Efflux and Altered

Multidrug resistance is a major obstacle to cancer treatment. Using an expression cDNA library transfer approach to elucidating the molecular basis of non-P-glycoprotein-mediated multidrug resistance, we previously established that expression of multidnag resistance protein (MRP), an ATP-binding cassette superfamily transporter, confers multidrug resist ance (G. D. Kruh et aL, Cancer Res., 54: 1649—1652,1994). In the present study, we generated NIHI3T3 MRP transfectants without using chemo therapeutic drugs to facilitate the pharmacological analysis of the MRP phenotype. MRP transfectantS displayed increased resistance to several lipophilic drugs, induding doxorubicin, daunorubicin, etoposide, actino mycin D, vincristine, and vinblastine. However, increased resistance was not observed for Taxol, a drug for which transfectionof MDR1 confers high levels of resistance. Verapamil increased the sensitivity of MRP transfectants relative to control transfectants, but reversal was incomplete for doxorubicinand etoposide, the drugs for which MRP conferred the highest resistance levels. For the latter two drugs, MRP transfectants, which were —S. and @“1O-fold more sensitive than control cells in the absence ofverapamil, exhibited 3.8- and 3.3-fold relative sensitization with 10JIM verapamil, respectively, butremained“‘2 and @3-fold moreresist ant than control cells. Analysisof drug kineticsusing radiolabeleddauno rubicin revealed decreased accumulation and increased effiux in MRP transfectants. Confocal microscopic analysis ofmtracellular daunorubicin in MRP transfectants was consistent with reduced intracellular drug concentrations, and also revealed an altered pattern of intracellular drug distribution characterized by the initial accumulation of drug in a perinu clear location,followedby the developmentof a punctatepatternof drug scattered throughout the cytoplasm. This pattern was suggestive of a process ofdrug sequestration, possibly followed by vesicle transport. Both increased drug emux and perinuclear drug accumulation are consistent with the reported localization of MRP in plasma and cytosolic membranes (N. Krishnamachary and M. S. Center, Cancer Res., 53: 3658—3663, 1993; M. J. Fleas et aL, Cancer Res., 54: 4557-4563, 1994). These results thus indicate that the drug specificity of MRP is quite similar to that of MDR1, butalsosuggestpotentialdifferences in Taxolspecificity andthelevelof verapamil sensitivity. In addition, these results indicate that MRP func dons to extrude drug from the cell, but additionally suggest the intriguing possibility that drug sequestration contributes to drug resistance by pro tecting cellular targets and/or contributing to ding efflux.