For understanding of the resistance to topoisomerase! inhibitors, 50 sublines were isolated as single clones from parental glioma cell lines by exposure to VP-16 or m-AMSA. The quantitative aspects of topoisomerase! , multidrug resistant gene (MDR)-1,breastcancer resistance protein (BCRP), and multidrug resistant associated protein (MRP) 1-5 were studied by Northern blotting in 50 resistant cell lines. By understanding the function of MRP2, we picked up three drug resistant sublines (T98G-m1,

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... 8G-m2, and gli36-VP1) that overexpressed MRP2, but did not overexpress MDR-1 or MRP1-5 except 2. Moreover, in the results of northern blot analysis of mRNA for topoisomerase! identical results are observed in parental cell lines and their resistant cell lines, suggesting that alterations in topoisomerase!do not account for the resistance in these cells. To determine whether the cellular sensitivity to anticancer agents was closely associated with the cellular levels of MRP2, we established cell lines with the same levels of MRP2 as their parental cells by introducing the MRP2 antisense expression plasmid into resistant cells. Etoposide (VP-16) accumulation and efflux studies were carried out in the parental cell lines and their drug resistant cell lines. Decreases in the H 3 -VP-16 accumulation and increases in the efflux were observed in these drug resistant cell lines. In the cytotoxicity assay, these drug resistant cell lines were resistant to multiple topoisomerase ! inhibitors with little cross resistance to vincristine, and display efflux of VP-16. We found that the resistant cells transfected with MRP2 antisense cDNA displayed increased cellular levels of VP-16 and enhanced sensitivities to topoisomerase!inhibitors. In this study on the T98G-m1, T98G-m2, and gli36-VP1 cell lines, we showed a high correlation between MRP2 mRNA and VP-16 efflux, suggesting that MRP2 could be a new transporter for topoisomerase !inhibitors.