Decreased NADPH:cytochrome P-450 reductase activity and impaired drug activation in a mammalian cell line resistant to mitomycin C under aerobic but not hypoxic conditions.
Hoban PR., Walton MI., Robson CN., Godden J., Stratford IJ., Workman P., Harris AL., Hickson ID.
Mitomycin C (MMC) is regarded as the prototype bioreductive alkylating agent in clinical use. To elucidate the biochemical basis of MMC resistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure to progressively higher concentrations of MMC. CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C. In contrast, CHO-MMC cells showed only a 2-fold level of resistance to BMY 25282, a more easily activated analogue of MMC, and exhibited parental sensitivity to MMC under radiobiologically hypoxic conditions. CHO-MMC cells showed no increased resistance to a range of DNA damaging agents including several other alkylating agents (e.g., melphalan and methyl methanesulfonate). Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vincristine) was present only at very low levels. Using a specific high performance liquid chromatography technique, we examined the rates of reduction of MMC and BMY 25282 in cell extracts from CHO-K1 and CHO-MMC cells under both aerobic (air) and hypoxic (N2) conditions. Reduction rates for both drugs were at least 30-fold faster under nitrogen than in air. Metabolism of MMC was undetectable in air but was readily detectable under nitrogen and was 2- 3-fold slower in CHO-MMC cell extracts than in CHO-K1 cell extracts. Although BMY 25282 was more readily reduced under nitrogen, no difference was detected between extracts from CHO-K1 or CHO-MMC cells in the rate of reduction of BMY 25282 under either air or nitrogen. The activity of NADPH:cytochrome P-450 (cytochrome c) reductase, an enzyme implicated in the bioreductive activation of MMC, was 3-4-fold lower in CHO-MMC cells than in the parental line. These findings suggest that the resistance of CHO-MMC cells to MMC under aerobic conditions may be due to impaired metabolic activation of the drug as a result of a decrease in NADPH:cytochrome P-450 reductase activity. This supports the view that decreased bioreductive enzyme activity may be a significant mechanism for acquired resistance to MMC in tumor cells in vivo and that more readily activated analogues may be potentially useful in overcoming this specific form of resistance.