We have isolated cisplatin-resistant human liver carcinoma (7404-CP20) cells with reduced accumulation of cisplatin and other drugs (methotrexate, arsenate, and arsenite) to which these cells are cross-resistant. To determine whether the reduction of drug accumulation in cisplatin-resistant cells results from impaired uptake or from active efflux, [(14)C]carboplatin was used for kinetic analysis of drug uptake and efflux. We demonstrate here that the uptake of [(14)C]carboplatin in 7404 parental cells is time, temperature, and energy dependent, and that the rate of uptake is reduced in 7404-CP20 cells. Efflux of [(14)C]carboplatin in cisplatin-resistant cells was comparable to efflux in the parental cisplatin-sensitive cells. There was little effect of temperature (between 37 degrees C and 4 degrees C) on efflux in cisplatin-resistant cells. Immunoblotting with specific antibodies directed to MRP1 and MRP2 (cMOAT) also showed that expression of these two ABC transporter genes was considerably reduced in 7404-CP20 cells and another cisplatin-resistant cell line KB-CP20, in contradistinction to previous studies suggesting that MRP might be responsible for cisplatin efflux. To rule out a generalized defect in uptake of small molecules, fluorescence-activated cell sorter (FACS) analysis of rhodamine 123 uptake showed that there was no difference between cisplatin-sensitive and -resistant cells. The presence of a pleiotropic defect in uptake of [(14)C]carboplatin, [(3)H]methotrexate, [(73)As]arsenate, and [(73)As]arsenite in cisplatin-resistant cells, in association with reduced expression of related cell surface proteins as demonstrated in our previous work, suggests a novel mechanism for acquisition of resistance to cisplatin associated with reduced activity of many different specific uptake systems.
Copyright 2000 Wiley-Liss, Inc.