Expression of DT-diaphorase and cytochrome P450 reductase correlates with mitomycin C activity in human bladder tumors.

Mitomycin C (MMC) is activated by DT-diaphorase (DTD) and cytochrome P450 reductase (P450R). In cancer cell lines, MMC cytotoxicity is correlated with DTD and P450R expression levels. The present study investigated the relationship between enzyme expression/activity and MMC cytotoxicity in patient bladder tumors. DTD and P450R expression was detected by competitive reverse transcription-PCR and their activity was measured by bioreductive assays. The expression of DTD and P450R in patient tumors (n = 29), as ratios to beta-actin levels, varied from 0 to 90% and 0 to 29%, respectively. The DTD expression was significantly correlated with P450R expression (r(2), 0.32; P < 0.01), whereas the average DTD level was 2-fold higher than that of P450R (P < 0.01). Among the 29 tumors, 21 provided sufficient materials to evaluate tumor sensitivity to MMC. The concentration of MMC required to produce 50% inhibition (IC(50)) of DNA precursor incorporation for a 2-h treatment ranged from 0.17 to 18.1 microg/ml, indicating a 110-fold intertumor variation, with the high-grade and more invasive tumors being less chemosensitive compared with the low-grade and less invasive tumors. Tumor sensitivity to MMC, as indicated by the inverse of IC(50) values, was positively correlated with the expression of DTD (r(2), 0.28; P < 0.05) and P450R (r(2), 0.26; P < 0.05). Multivariate analysis indicates DTD expression and P450R expression as better determinants of MMC activity compared with other pathobiological factors (e.g., tumor grade, stage, and labeling index) that have been shown to significantly correlate with MMC activity. Eleven tumors were studied for the relationship between gene expression level and enzyme activity of DTD and P450R. The DTD activity was significantly correlated with the gene expression level (r(2), 0.84; P < 0.001). For P450R, there is a trend of a correlation between enzyme activity and its mRNA level, but the correlation was not statistically significant (r(2), 0.28; P = 0.09). These data indicate that the sensitivity of patient bladder tumors to MMC is correlated with the expression of DTD and P450R in tumors and suggest that the lower expression of these enzymes in the high-grade and more invasive tumors is a cause of the lower efficacy of intravesical MMC in these tumors.