Bioreductive chemotherapy is based on reductive activation of drugs by enzymes such as quinone oxidoreductase (NQO1) and P450 reductase, identification of tumors rich in those enzymes and difference in oxygen and pH between normal and tumor tissues. Mitomycin C (MMC) is a prototype bioreductive drug employed to treat head and neck cancer. Recently, we have identified a unique but NQO1 related cytosolic activity in wild type (NQO1+/+) and knockout (NQO1-/-) mouse tissues (liver and colon) and several mammalian cells (human colon carcinoma HT29, human hepatoblastoma Hep-G2 and rat hepatoma H4II that catalyzes metabolic activation of MMC leading to inter strand DNA cross-linking, a major determinant of cell death. This activity was absent in mouse kidney and monkey kidney COS1 cells. The goals of this proposal are to purify and clone the unique but NQO1 related cytosolic activity, investigate its role in activation of MMC, MMC analogue BMY 25067 and indoloquinone EO9, and determine its level of expression in various normal and tumor tissues. To this effect, we plan to purify the unique cytosolic activity to homogeneity from NQO1-/- mice liver by dicoumarol affinity column chromatography and other biochemical techniques. The antibodies raised against the purified activity and/or the mixed oligonucleotides designed from peptide sequences will be used as probes to clone the cDNA encoding unique cytosolic mitomycin C reducing activity. The cDNA will be sequenced and subcloned in eukaryotic expression vector pED4 to develop chinese hamster ovary (CHO) cells permanently expressing higher levels of unique cytosolic activity. These cells will be used to analyze the role of unique cytosolic activity in activation of quinone bioreductive drugs by cytotoxicity assays using colony formation assays. The untreated and drugs treated cells will also be analyzed for drug induced DNA cross-linking and membrane and membrane damage as targets of drug action leading to cytotoxicity and cell death. The results obtained with over-expressing CHO cells will be confirmed by inhibiting the expression of unique cytosolic activity by specific antisense oligonucleotide in HT29 and Hep-G2 cells. Norther, Western and immunohistochemistry assays will be performed to measure the levels of the unique activity in normal and tumor tissues. These experiments may lead to the identification of tumors highly rich in unique not NQO1 related cytosolic activity which may be benefitted most from bioreductive therapy based on activation of drugs by unique cytosolic activity.
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