MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines

Multidrug resistant prostate cancer cell lines DU 0.03 and PC 0.03 were est ablished from the parental prostate cancer cell lines DU145 and PC-3 respec tively by stepwise selection in doxorubicin (DOX) from 0.001 to 0.03 mu g/m l. As cells adapted to each concentration of DOX. the drug concentration wa s increased by 0.001 mu g/ml. The chemosensitivity of each line was determi ned by growth inhibition assay. The DU 0.03 and PC 0.03 lines exhibit a 5-1 0-fold and 1.3-2.8-fold increase in resistance to anthracyclines, vinblasti ne (VLB) and mitozantrone (Mito), respectively. Verapamil (5 mu M) partiall y reversed the resistance to the anthracycline and completely reversed the resistance to VLB and Mito. Drug kinetic studies measured by intracellular accumulation of H-3-daunorubicin demonstrated a 3 fold decrease in the leve l of intracellular H-3-daunorubicin in the PC 0.03 and DU 0.03 resistant li nes compared with their respective parental line. This effect was partially reversed by 5 mu M verapamil. The expression of MDR1 and MRP genes was ana lysed by Northern blotting and RT-PCR. P-glycoprotein (Pgp) and MRP protein were tested by immunocytochemistry staining using the monoclonal antibodie s J-SB1. C219 and MRK16 (Pgp) and MRPm6 and MRPr1 (MRP). Neither Northern b lot analysis nor the more sensitive RT-PCR demonstrated detectable MDR1 tra nscripts in any of the prostate cancer cell lines and the three Pgp monoclo nal antibodies failed to reveal expression of Pgp. A 2-4-fold increase in MRP1 mRNA levels in the drug resistant DU 0.03 and P C 0.03 lines were demonstrated by both Northern blotting and RT-PCR consist ent with the findings observed after staining by the true specific monoclon al antibodies, MRPm6 and MRPr1. Southern blot analysis demonstrated a 2-fol d increase in the MRP1 gene copy number in the PC 0.03 line but not in the DU 0.03 line, suggesting that the overexpression of the MRP gene was regula ted at the level of transcription in the latter line. We conclude that MRP1 not MDR1 overexpression. contributes to acquired drug resistance in these two prostate cancer cell lines.