BIOCHEMICAL-CHARACTERIZATION OF A MITOMYCIN C-RESISTANT HUMAN BLADDER-CANCER CELL-LINE

This study describes characteristics of a mitomycin C (MMC)resistant h uman bladder cancer cell line, J82/MMC-2, which was established by rep eated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) t o 18 nM MMC. A 9.6-fold higher concentration of MMC was required to ki ll 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT) . NADPH cytochrome P450 reductase and DT-diaphorase activities were si gnificantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impai red drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC- 2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines . Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA po lymerase alpha mRNA expression was comparable in these cell lines, lev els of DNA ligase I mRNA were slightly lower in both MMC-resistant var iants relative to J82/WT. However, the DNA polymerase beta mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J8 2/WT or J82/MMC. Thus, emergence of a higher level of resistance to MM C in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) im paired drug activation through further reduction in DT-diaphorase acti vity and (ii) enhanced DNA repair through over-expression of DNA polym erase beta. (C) 1996 Wiley-Liss, Inc.