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First published on November 16, 2004; DOI: 10.1124/mol.104.004929

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Received for publication July 13, 2004.
Revised October 22, 2004.
Accepted for publication November 11, 2004.

Nuclear Localization of NADPH:Cytochrome C (P-450) Reductase Enhances the Cytotoxicity of Mitomycin C to Chinese Hamster Ovary Cells

Helen A. Seow 1, Michael F. Belcourt 2, Philip G. Penketh 1, William F. Hodnick 3, Maria Tomasz 4, Sara Rockwell 1, Alan C. Sartorelli 1*

1 Yale University School of Medicine 2 Vion Pharmaceuticals, Inc 3 Athersys, Inc 4 Hunter College- City University of NY

* Address correspondence to: E-mail: alan.sartorelli{at}yale.edu

Abstract

Overexpression of endoplasmic reticulum localized NADPH:cytochrome c (P-450) 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 SV40 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 to 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.


Key words: Cytochrome P450, Enzymology, Mechanisms of cell killing/apoptosis, DNA intercalation


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S.-L. Wang, J.-F. Han, X.-Y. He, X.-R. Wang, and J.-Y. Hong
Genetic Variation of Human Cytochrome P450 Reductase as a Potential Biomarker for Mitomycin C-Induced Cytotoxicity
Drug Metab. Dispos., January 1, 2007; 35(1): 176 - 179.
[Abstract] [Full Text] [PDF]


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