Non-enzymatic and enzymatic activation of mitomycin C: identification of a unique cytosolic activity.
Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductaseI (NQOI or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQOI because of a lack of correlation between NQOI (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH-cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).
Joseph
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Department of Pharmacology, Fox Chase Center, Philadelphia, PA 19111, USA.
Xu
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Jaiswal
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D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D016685:Mitomycin / Q000378:metabolism*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D018076:DNA, Complementary; D016685:Mitomycin; D004247:DNA; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D016660:NAD(P)H Dehydrogenase (Quinone)