The intracellular location of NADH:cytochrome b5 reductase modulates the cytotoxicity of the mitomycins to Chinese hamster ovary cells

• Published in: The Journal of biological chemistry 1998-04, Vol.273 (15), p.8875
• Main Authors: Belcourt, M F, Hodnick, W F, Rockwell, S, Sartorelli, A C
• Format: Article
• Language: English
• Subjects: Animals; Cell Nucleus - enzymology; Cell Survival - drug effects; Cell Survival - physiology; CHO Cells; Cricetinae; Cytochrome Reductases - biosynthesis; Cytochrome Reductases - metabolism; Cytochrome-B Reductase; Cytoplasm - enzymology; Kinetics; Microsomes - enzymology; Mitochondria - enzymology; Mitomycin - pharmacokinetics; Mitomycin - toxicity; Oxidation-Reduction; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Succinate Dehydrogenase - metabolism; Transfection
• ISSN: 0021-9258
• DOI: 10.1074/jbc.273.15.8875

NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity.