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ORIGINAL ARTICLE

Nuclear Localization of NADPH:Cytochrome c (P450) Reductase Enhances the Cytotoxicity of Mitomycin C to Chinese Hamster Ovary Cells

Departments of Pharmacology (H.A.S., M.F.B., P.G.P., W.F.H., S.R., A.C.S.) and Therapeutic Radiology (S.R.) and the Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut; and Department of Chemistry (M.T.), Hunter College, City University of New York, New York, New York

Abstract

Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence. Immunofluorescent imaging of transfected cell lines expressing the fusion protein confirmed the nuclear location of the enzyme. Regardless of the oxygenation state of the cell, mitomycin C (MC) cytotoxicity was enhanced in cells with overexpressed NPR localized to the nuclear compartment compared with cells overexpressing an endoplasmic reticulum localized enzyme. Enhanced cytotoxicity in cells treated under hypoxic conditions correlated with increases in genomic DNA alkylations, with more MC-DNA adducts being formed when the enzyme was expressed closer to its DNA target. No change was observed in the hypoxic/aerobic differential toxicity as a function of enzyme localization. These findings indicate that drug efficacy is increased when the subcellular site of drug activation corresponds to its site of action.

Address correspondence to: Dr. Alan C. Sartorelli, Department of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. E-mail: alan.sartorelli{at}yale.edu

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