[CANCER RESEARCH 50, 7789-7792, December 15, 1990] Activation of Mitomycin C by NADPH:Cytochrome P-450 ReducÃ-ase1 H. Frances J. Bligh, Agnieszka Bartoszek,2 Craig N. Robson, Ian D. Hickson, Charles B. Kasper, Jean D. Beggs, and C. Roland Wolf3 Department of Molecular Biology, Kings Buildings, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom [H. F. J. B., J. D. B.J; Imperial Cancer Research Fund, Molecular Pharmacology Group, Department of Biochemistry, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom [A. B., C. R. W.j; Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom [C. N. R., I. D. H.J; and McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 [C. B. K.J ABSTRACT Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reducÃ-ase.In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reduc- tase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reducÃ-aseactivity is a factor in the resistance mechanism, we incorporated NADPH:cytochronte P-450 reducÃ-aseinto cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reducÃ-asecDNA in a Salmonella ty- phimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reducÃ-aseshowed increased sensitivily to mitomycin C when incubated with Ihis compound under aerobic condilions. However, under hypoxic condilions increased sensitivity was not observed. This parallels Ihe previous finding wilh milomycin ( -resistant Chinese ham ster ovary cells. These dala provide direct evidence for Ihe role of NADPH:cylochrome P-450 reducÃ-asein Ihe cyloloxic aclion of Ihis milomycin C under aerobic bui noi hypoxic condilions and suggesl thai reduced levels of Ihis enzyme can lead lo drug resislance. P-450 reducÃ-ase expressed in S. typhimurium may provide a valuable tool for evaluating the role of Ihis enzyme in Ihe loxicily of drugs activaled Ihrough a one electron reduction palhway. INTRODUCTION In order to exert its cytotoxic effects, the anticancer antibiotic MMC4 appears to require reductive activation to products that either bind covalently to DNA or induce the formation of DNA cross-links (1-3). Several enzymes have been implicated in this activation pathway including xanthine oxidase, DT diaphorase, NADPHxytochrome P-450 reducÃ-ase, and mitochondrial NADPH reducÃ-ase (4-6). The cytotoxic effect of MMC has been shown to be often greater in cells grown under hypoxic conditions (7, 8). Hypoxia is considered to be an important factor in the treatment of solid tumors with ionizing radiation (9). Enhanced toxicity in the absence of oxygen has been attributed to the preferential activation to reactive metabolites under these conditions (10). Under aerobic conditions the semi- Received6/11/90;accepted9/10/90. 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. 1This work was supported by funds from the Science and Engineering Research Council and the Imperial Cancer Research Fund. J. D. B. holds a Royal Society E.P.A. Cephalosporin Fund Senior Research Fellowship. 1On leave from the Department of Pharmaceutical Technology and Biochem istry, The Technical University of Gdansk, Poland. 3To whom requests for reprints should be addressed, at Imperial Cancer Research Fund, Hugh Robson Building, George Square, Edinburgh EH8 9XD, United Kingdom. 4 The abbreviations used are: MMC, mitomycin C; P-450 reducÃ-ase, NADPH:cytochrome P-450 reducÃ-ase;CHO, Chinese hamster ovary; cDNA, complementary DNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide. quinone free radical generated by one electron reduction reacts with molecular oxygen to generate Superoxide, and as a conse quence the MMC then reverts to the parent molecule. The recovery of unmetabolized drug and the proteclive effect of oxygen radical scavengers against MMC cytotoxicity under aerobic conditions support this reaction pathway (2, 11). These data provide circumstantial evidence that the products gener ated, e.g., by P-450 reductase-mediated one electron reduction, are cytotoxic. However, this has not been formally shown to be the case. Recently a CHO cell line (CHO-MMC') was established, which exhibited a 17-fold resistance to MMC under aerobic conditions but had the same sensitivity as the parental cell line in the absence of oxygen (8). The reduced level of MMC- induced cytotoxicity in CHO-MMC' appeared to be due to a reduced rate of metabolic activation. This possibility was sub stantiated by the finding that the drug-resistant cell line con tained lower cytochrome P-450 reducÃ-aseactivity. In order to show unequivocally that P-450 reducÃ-ase activates MMC to cytotoxic products and to determine whether the change in P- 450 reducÃ-asemay be directly involved in the resistance mech anism, we have added P-450 reducÃ-aseexogenously in cytotox icity assays. In addition, we have expressed rat liver P-450 reducÃ-ase in Salmonella typhimurium and have shown that these cells become significantly more sensitive to MMC-in- duced toxicity. This effect is only observed under aerobic con ditions, which parallels the findings obtained using mammalian cells. MATERIALS AND METHODS Escherìchiacoli strain MM294 (end A, thi A, hsd R) was used to propagate recombinant plasmid DNA. 5. typhimurium strain LR5000 [met A, met B, Irp B, leu, val (unstable), sir (rps L) hsd LT, hsd SA, hsd SB] was used to express P-450 reductase cDNA for cytoloxicity lesls. The mammalian cell lines used were Ihe wild lype, Chinese hamster ovary cell line (CHO-K1), the mitomycin C-resistanl variant (CHO- MMC1) (5), and Ihe human mammary carcinoma cell line, MCF-7. CHO-K1 and CHO-MMC' cells were cultured as described previously (8). MCF-7 cells were cultured in RPMI 1640 (Gibco) supplemenled wilh 10% felal calf serum, 100 units ml"1 penicillin, and 100 fig/ml slreplomycin. All chemicals were obtained from Sigma Chemical Com pany. Reslriction enzymes and T4 DNA ligase were obtained from Boehringer-Mannheim Corporation. Cytoloxicily Assays. MCF-7 cells (7-9 x 103/well) were plaled out in 180 fil of medium in 96-well microtiler plates and left al 37'C for 16h to adhere. Cells were ihen treated for 3 h at 37°Cwith appropriate concentrations of MMC plus NADPH plus P-450 reductase all added in a volume of 20 n\. The total volume of culture medium was 200 n\. Final concentrations were NADPH, 1 HIM,and P-450 reductase, 250 units/ml. In control experimenls, either NADPH, P-450 reductase, or MMC was omitted. After 3 h, the medium was replaced and the cells allowed to grow for 72 h. Cell survival was then assessed using the MTT assay as described previously (12). In experiments using CHO cells, the cells were plated out in 30-mm l'etri dishes at a density of 250-1000 cells/plate and left for 4 h at 37'C to adhere. 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