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 r eductase (P450R), In cancer cell lines, MMC cytotoxicity is correlated with DTD and P450R expression levels, The present study investigated the relati onship between enzyme expression/activity and MMC cytotoxicity in patient b ladder 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 p- actin levels, varied from 0 to 90% and 0 to 29%, respectively. The DTD expr ession 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 evaluat e tumor sensitivity to MMC, The concentration of MMC required to produce 50 % inhibition (IC50) of DNA precursor incorporation for a 2-h treatment rang ed from 0.17 to 18.1 <mu>g/ml, indicating a 110-fold intertumor variation, with the high-grade and more invasive tumors being less chemosensitive comp ared with the low-grade and less invasive tumors, Tumor sensitivity to MMC, as indicated by the inverse of IC,, 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 fa ctors (e,g,, tumor grade, stage, and labeling index) that have been shown t o significantly correlate with MMC activity, Eleven tumors were studied for the relation- ship between gene expression level and enzyme activity of DT D and P450R, The DTD activity was significantly correlated with the gene ex pression level (r(2), 0.84; P < 0.001). For P450R, there is a trend of a co rrelation between enzyme activity and its mRNA level, but the correlation w as not statistically significant (r(2), 0.28; P = 0,09), These data indicat e 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 expres sion of these enzymes in the high-grade and more invasive tumors is a cause of the lower efficacy of intravesical MMC in these tumors.