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J. Biol. Chem., Vol. 279, Issue 30, 31606-31612, July 23, 2004

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Nuclear Overexpression of NAD(P)H:Quinone Oxidoreductase 1 in Chinese Hamster Ovary Cells Increases the Cytotoxicity of Mitomycin C under Aerobic and Hypoxic Conditions^*

Helen A. Seow, Philip G. Penketh, Michael F. Belcourt, Maria Tomasz¶, Sara Rockwell||, and Alan C. Sartorelli**

From the Departments of Pharmacology and ^||Therapeutic Radiology and the 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

The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr^–) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.

Received for publication, May 3, 2004 , and in revised form, May 17, 2004.

^* This work was supported in part by NCI, National Institutes of Health, United States Public Health Service Grant CA-80845. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Vion Pharmaceuticals, Inc., Four Science Park, New Haven, CT 06511.

^** To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4533; Fax: 203-737-2045; E-mail: alan.sartorelli{at}yale.edu.

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This article has been cited by other articles:

				H. A. Seow, P. G. Penketh, K. Shyam, S. Rockwell, and A. C. Sartorelli PNAS, June 28, 2005; 102(26): 9282 - 9287. [Abstract] [Full Text] [PDF]

				H. A. Seow, M. F. Belcourt, P. G. Penketh, W. F. Hodnick, M. Tomasz, S. Rockwell, and A. C. Sartorelli Nuclear Localization of NADPH:Cytochrome c (P450) Reductase Enhances the Cytotoxicity of Mitomycin C to Chinese Hamster Ovary Cells Mol. Pharmacol., February 1, 2005; 67(2): 417 - 423. [Abstract] [Full Text] [PDF]