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J. Biol. Chem., Vol. 278, Issue 7, 5029-5034, February 14, 2003

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Nuclear Overexpression of NADH:Cytochrome b₅ Reductase Activity Increases the Cytotoxicity of Mitomycin C (MC) and the Total Number of MC-DNA Adducts in Chinese Hamster Ovary Cells^*

Kathleen M. Holtz, Sara Rockwell^§, Maria Tomasz^¶, and Alan C. Sartorelli

From the Departments of  Pharmacology and ^§ Therapeutic Radiology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520 and the ^¶ Department of Chemistry, Hunter College, City University of New York, New York, New York 10021

NADH:cytochrome b₅ reductase (FpD) is an enzyme capable of converting the prodrug mitomycin C (MC) into a DNA alkylating agent via reduction of its quininone moiety. In this study, Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding rat FpD. Despite the demonstrated ability of this enzyme to reduce MC in vitro, a modest 5-fold level of overexpression of FpD activity in CHO cells did not increase the cytotoxicity of the drug over that seen with the parental cell line under either aerobic or hypoxic conditions. When the enzyme, which is predominantly localized in the mitochondria, was instead directed to the nucleus of cells by the fusion of the SV40 large T antigen nuclear localization signal sequence to the amino terminus of an FpD gene that lacked the membrane anchor domain, drug sensitivity was significantly enhanced at all concentrations of MC examined (2-10 µM) under both aerobic and hypoxic conditions, with greater cell kill occurring under hypoxia. The marked increase in drug sensitivity under hypoxia at 10 µM MC corresponded to a measurable increase in total MC-DNA adducts at the same concentration. The results indicate that the cytotoxicity of MC is modulated by the subcellular location of FpD, with greater cell kill occurring when bioactivation occurs in the proximity of its target, nuclear DNA.

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