[CANCER RESEARCH 44, 5638-5643, December 1984] Role of NADPH:Cytochrome c ReducÃ-aseand DT-Diaphorase in the Biotransformation of Mitomycin C1 Susan R. Keyes, Paula M. Fracasso, David C. Heimbrook,2 Sara Rockwell, Stephen G. Silgar,3 and Alan C. Sai-torelli4 Departments of Pharmacology [S. R. K., P. M. F., A. C. S.] and Therapeutic Radiology [S. R.J and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven 06510, and Department of Molecular Biophysics and Biochemistry, [S. G. S., D. C. H.¡Yale University, New Haven 06511, Connecticut ABSTRACT Hypoxie cells of solid tumors are difficult to eradicate by X- irradiation or chemotherapy; as an approach to this problem, our laboratories are investigating the effects of the bioreductive alkylating agent mitomycin C (MC) on hypoxic cells. This anti biotic was preferentially toxic to EMT6 mouse mammary tumor cells and V79 Chinese hamster lung fibroblasts under hypoxic conditions, but it was equitoxic to Chinese hamster ovary cells in the presence and absence of oxygen. All cell lines catalyzed the formation of reactive metabolites under hypoxic conditions and contained NADPH:cytochrome c reducÃ-aseand DT-diaphor- ase, two enzymes which may be responsible for the cellular activation of MC. Although a correlation existed between enzy matic activities and the formation of reactive metabolites from MC, there was no correspondence between these parameters and the degree of cytotoxicity expressed by MC under hypoxic conditions. Purified NADPHrcytochrome c reducÃ-ase reduced MC in the absence of oxygen, with addition of cytochrome P-450 enhanc ing, but noi participating directly in, the reduction reaction. Ad dition of NADP+ lo cell sonicates substantially reduced NADPH:- cylochrome c reducÃ-ase activity, while Ihe formation of reactive metabolites was affected only slightly; converse results were observed using mersalyl. Exposure of cell sonicates lo dicumarol inhibiled DT-diaphorase activity, while the rate of formation of reactive metabolites of MC was enhanced. The findings suggest that NADPH:cytochrome c reducÃ-ase and some as yet to be identified enzyme(s) are important for the reductive activalion of MC. DT-diaphorase and cytochrome P-450 are not directly in volved in the activalion of MC, bul they appear lo modulate the degree of activalion lo reaclive species, which are presumably responsible for Ihe observed cytotoxicity. INTRODUCTION Solid neoplasms contain hypoxic tumor cell populations; since oxygen-deficient cells are relatively refractory lo convenlional radiolherapy and mosl chemolherapy, hypoxic cells have Ihe potential to limit curability (13, 19). As an approach to the circumvention of this problem, we have been studying the natu- 1This work was supported in part by American Cancer Society Grants CH-211 and PDT 145, USPHS Grant CA-02817, NIH Grants GM-31756 and AM-01160, and American Heart Association Fellowship 11-104-812. 2 Present address: Department of Pharmacology, Yate University School of Medicine, New Haven, CT 06510. 3 Present address: Department of Biochemistry, University of Illinois, Urbana, IL 61801. 4 To whom requests for reprints should be addressed. Received May 21, 1984; accepted August 29, 1984. rally occurring antibiolic, MC.5 This drug is aclive against a broad range of transplantable animal (5) and human (2) tumors, is preferentially cytotoxic to some hypoxic tumor cell lines in vitro (11, 23, 27), and is also toxic to hypoxic tumor cells in vivo (25, 26). Early studies have demonslraled lhal enzymalic bioactivalion of MC results in cyloloxicily associated wilh cross-linking of DNA (9). Using liver homogenales, the anaerobic production of reaclive species from MC has been shown lo require NADPH and to be catalyzed primarily by enzymes in the microsomal and nuclear subcellular fractions (12, 28), and some alkylation prod ucts have been identified (31). Limited studies with purified enzymes have indicated that both NADPHxytochrome c reduc Ã-aseand xanthine oxidase actÃ-valeMC under hypoxic conditions, wilh Ihe concomilanl generalion of a reaclive eleclrophile (14, 21 ). Studies in our laboratories have suggested lhal cytochrome P-450 might be involved in the anaerobic metabolism of MC (11, 12), and work by others on DT-diaphorase, an enzyme active in the reduction of quiñones (6, 30), suggests that this enzyme should also be considered as a possible activator of MC. Although the mechanism by which MC is metabolically acti vated has been examined extensively in bacteria and liver, little is known about the bioaclivation of MC by neoplastic cells. Previous investigations in our laboratories have demonslraled that MC is more toxic to EMT6 and Sarcoma 180 tumor cells under hypoxic conditions than in the presence of air, and lhal sonicates of these cells are capable of generating a reactive species from MC (11, 27). However, neilher Ihe enzyme(s) in lumor cells responsible for activating MC nor the exact biochem ical basis of Ihe enhanced cyloloxicily in Ihe absence of oxygen has yet been characterized. The studies described in Ihis paper were designed to address these issues. MATERIALS AND METHODS MC was supplied by the Bristol-Myers Co. (Syracuse, NY). Other materials were obtained from the following sources: NADP*, NADH, NADPH, glucose-6-phosphate dehydrogenase (type XII), glucose 6-phos- phate, dicumarol, mersalyl, 2,6-dichlorophenolindophenol, dilaurylphos- phatidylcholine, DEAE-cellulose, and 2',5'-ADP agarose (Sigma Chemi cal Co., St. Louis, MO); fetal bovine serum and Waymouth's medium (Grand Island Biological Co., Grand Island, NY); DEAE-Trisacryl M and HA-Utrogel (LKB Instruments, Gaithersburg, MD); gases (Presto Welding Service Center, North Haven, CT); and 4-<p-nitrobenzyl)pyridine (Aldrich Chemical Co., Milwaukee, Wl). Chinese hamster V79 cells (subline V79- 8) and CHO cells (subline HA-1) were gifts of Dr. R. Michael Liskay and Dr. Daniel S. Kapp, Department of Therapeutic Radiology, Yale University School of Medicine. 5The abbreviations used are: MC, mitomycin C; CHO, Chinese hamster ovary. 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