Cytotoxic effects associated with targeting the subcellular distribution of the bioreductive enzyme, DT -diaphorase, and mitomycin C activation

The effects of subcellular distribution of the bioreductive enzyme, DT-diaphorase (DTD) on mitomycin C (MC) activation were examined. Chinese hamster ovary (CHO K1 dhfr-) cells were transfected to produce cells that highly overexpressed DTD in the nucleus or cytoplasm. Proper subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence techniques. We observed that nuclear localization of DTD sensitized cells to the cytotoxic effects of MC. We found that sensitization was not due to elevations in endogenous enzyme expression associated with hypoxic treatment, high cell density, or plateau phase cell growth. Additionally, sensitization to MC was not attributable to increases in MC metabolism between the cytosolic overexpressing and nuclear overexpressing cells, but accounted for the sensitization observed between both overexpressing cells compared the parental cells. Furthermore, CHO K1 dfhr- transfectant cells that overexpress nuclear localizing DTD showed a positive correlation between MC-associated cytotoxicity and increases in numbers of MC-DNA adducts in excess of both cytosolic overexpressing transfectant and parental CHO K1 dfhr- cells under both aerobic and hypoxic conditions. These findings, taken together, indicate that activation of MC proximal to nuclear DNA by nuclear localizing DTD sensitizes cells to the cytotoxic effects of MC irrespective of oxygenation and suggest that interactions between MC reactive intermediates and DNA are diffusion limited. Gene therapy approaches that permit the delivery of prodrug activating enzymes to tumor cells may permit therapeutic gains in MC therapy. Selection of the best possible bioreductive enzyme and prodrug pair is important for therapeutic success. Therefore, in addition to overexpression, nuclear localization of DTD should be considered in gene therapy approaches to sensitize tumor cells to MC.