EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN IN NIH 3T3 CELLS CONFERS MULTIDRUG-RESISTANCE ASSOCIATED WITH INCREASED DRUG EFFLUX AND ALTERED INTRACELLULAR DRUG DISTRIBUTION/

Multidrug resistance is a major obstacle to cancer treatment. Using an expression cDNA library transfer approach to elucidating the molecula r basis of non-P-glycoprotein-mediated multidrug resistance, we previo usly established that expression of multidrug resistance protein (MRP) , an ATP-binding cassette superfamily transporter, confers multidrug r esistance (G. D. Kruh et al., Cancer Res., 54: 1649-1652, 1994). In th e present study, we generated NIH/3T3 MRP transfectants without using chemotherapeutic drugs to facilitate the pharmacological analysis of t he MRP phenotype. MRP transfectants displayed increased resistance to several lipophilic drugs, including doxorubicin, daunorubicin, etoposi de, actinomycin D, vincristine, and vinblastine. However, increased re sistance was not observed for Taxol, a drug for which transfection of MDR1 confers high levels of resistance. Verapamil increased the sensit ivity of MRP transfectants relative to control transfectants, but reve rsal was incomplete for doxorubicin and etoposide, the drugs for which MRP conferred the highest resistance levels. For the latter two drugs , MRP transfectants, which were similar to 8- and similar to 10-fold m ore sensitive than control cells in the absence of verapamil, exhibite d 3.8- and 3.3-fold relative sensitization with 10 mu M verapamil, res pectively, but remained similar to 2 and similar to 3-fold more resist ant than control cells. Analysis of drug kinetics using radiolabeled d aunorubicin revealed decreased accumulation and increased efflux in MR P transfectants. Confocal microscopic analysis of intracellular daunor ubicin in MRP transfectants was consistent with reduced intracellular drug concentrations, and also revealed an altered pattern of intracell ular drug distribution characterized by the initial accumulation of dr ug in a perinuclear location, followed by the development of a punctat e pattern of drug scattered throughout the cytoplasm. This pattern was suggestive of a process of drug sequestration, possibly followed by v esicle transport. Both increased drug efflux and perinuclear drug accu mulation are consistent with the reported localization of MRP in plasm a and cytosolic membranes (N. Krishnamachary and M. S. Center, Cancer Res., 53: 3658-3663, 1993; hi. J. Flens et at, Cancer Res., 54: 4557-4 563, 1994). These results thus indicate that the drug specificity of M RP is quite similar to that of,MDR1, but also suggest potential differ ences in Taxol specificity and the level of verapamil sensitivity. In addition, these results indicate that MRP functions to extrude drug fr om the cell, but additionally suggest the intriguing possibility that drug sequestration contributes to drug resistance by protecting cellul ar targets and/or contributing to drug efflux.