Abstract
Mitomycin C (MMC) is a prototype bioreductive drug employed to treat a variety of cancers including head and neck cancer. Among the various enzymes, dicoumarol inhibitable cytosolic NAD(P)H:quinone oxidoreductase1 (NQO1) was shown to catalyse bioreductive activation of MMC leading to cross-linking of the DNA and cytotoxicity. However, the role of NQO1 in metabolic activation of MMC has been disputed. In this report, we present cellular and animal models to demonstrate that NQO1 may play only a minor role in metabolic activation of MMC. We further demonstrate that bioreductive activation of MMC is catalysed by a unique cytosolic activity which is related but distinct from NQO1. Chinese hamster ovary (CHO) cells were developed that permanently express higher levels of cDNA-derived NQO1. These cells showed significantly increased protection against menadione toxicity. However, they failed to demonstrate higher cytotoxicity due to exposure to MMC under oxygen (normal air) or hypoxia, as compared to the wild-type control CHO cells. Disruption of the NQO1 gene by homologous recombination generated NQO1–/– mice that do not express the NQO1 gene resulting in the loss of NQO1 protein and activity. The cytosolic fractions from liver and colon tissues of NQO1–/– mice showed similar amounts of DNA cross-linking upon exposure to MMC, as observed in NQO1+/+ mice. The unique cytosolic activity that activated MMC in cytosolic fractions of liver and colon tissues of NQO1–/– mice was designated as cytosolic MMC reductase. This activity, like NQO1, was inhibited by dicoumarol and immunologically related to NQO1. © 2000 Cancer Research Campaign
Similar content being viewed by others
Article PDF
Change history
16 November 2011
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Dulhanty AM and Whitmore GF (1991) Chinese hamster ovary cells resistant to mitomycin C under aerobic but not hypoxic conditions are deficient in DT-diaphorase. Cancer Res 51: 1860–1865
Fitzsimmons SA, Workman P, Grever M, Paull K, Camalier R and Lewis AD (1996) Reductase enzyme expression across the National Cancer Institute Tumor Cell Line panel: correlation with sensitivity to mitomycin C and EO9. J Natl Cancer Inst 21: 1161–1163
Gustafson DL, Beall HD, Bolton EM, Ross D and Waldren CA . Expression of human NAD(P)H:quinone oxidoreductase (DT-diaphorase) in Chinse hamster ovary cells: effect on the toxicity of antitumor quinones. Mol Pharmacol 50: 728–735
Iyer VN and Szybalski W (1964) Mitomycins and porfiromycin: chemical mechanism of activation and cross-linking of DNA. Science 145: 55–58
Jaiswal AK, McBride OW, Adensik M and Nebert DW (1988) Human dioxin-inducible cytosolic NAD(P)H:quinone oxidoreductase. J Biol Chem 263: 13572–13578
Jaiswal AK, Burnett P, Adesnik M and McBride OW (1990) Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6. Biochemistry 29: 1899–1906
Joseph P, Xu Y and Jaiswal AK (1996) Non-enzymatic and enzymatic activation of mitomycin C. Int J Cancer 65: 263–271
Kaufman RJ, Davies MV, Wasley LC and Michnick D (1991) Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res 19: 4485–4490
Kennedy KA, Teicher BA, Rockwell S and Sartorelli AC (1980) The hypoxic tumor cell: a target for selective cancer chemotherapy. Biochem Pharmacol 29: 1–8
Knox RJ, Boland MP, Friedlos F, Coles B, Southan C and Roberts JJ (1988) The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxyl-amino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2). Biochem Pharmacol 37: 4671–4677
Marshall RS, Paterson MC and Rauth AM (1991) DT-diaphorase and mitomycin C sensitivity in nontransformed cell strains derived from members of a cancer prone family. Carcinogenesis 12: 1175–1180
Plumb JA and Workman P (1994) Unusually marked hypoxic sensitization to indoloquinone EO9 and mitomycin C in a human colon-tumour cell line that lacks DT diaphorase activity. Int J Cancer 56: 134–139
Powis G, Gasdaska PY, Gallegos A, Sherrill K and Goodman D (1995) Over-expression of DT-diaphorase in transfected NIH 3T3 cells does not lead to increased anticancer quinone drug sensitivity: a questionable role for the enzyme as a target for bioreductively activated anticancer drugs. Anticancer Res 15: 1141–1146
Radjendirane V, Joseph P, Lee H, Kimura S, Klein-Szanto AJP, Gonzalez FJ and Jaiswal AK (1998) Disruption of the DT diaphorase (NQO1) gene in mice leads to increased menadione toxicity. J Biol Chem 273: 7382–7389
Rauth AM, Goldberg Z and Misra V (1997) DT-diaphorase: possible roles in cancer chemotherapy and carcinogenesis. Oncol Res 9: 339–349
Riley RJ and Workman P (1992) DT-diaphorase and cancer chemotherapy. Biochem Pharm 43: 1657–1669
Robertson N, Stratford IJ, Houlbrook S, Carmichael J and Adams GE (1992) The sensitivity of human tumor cells to quinone bioreductive drugs: what role for DT-diaphorase? Biochem Pharmacol 44: 409–412
Ross D, Beall H, Traver RD, Siegel D, Phillips RM and Gibson NW (1994) Bioactivation of quinones by DT-diaphorase, molecular, biochemical, and chemical studies. Oncol Res 6: 493–500
Sakai A, Miyata N and Takahashi A (1995) Initiating activity of quinones in the two-stage transformation of Balb/3T3 cells. Carcinogenesis 16: 477–481
Sartorelli AC (1988) Therapeutic attack of solid tumors. Cancer Res 48: 775–778
Shaw PM, Reiss A, Adesnik M, Nebert DW, Schembri J and Jaiswal AK (1991) The human dioxin-inducible NAD(P)H:quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells in identical to diaphorase 4. Eur J Biochem 195: 171–176
Siegel D, Gibson NW, Perusch PC and Ross D (1990) Metabolism of diaziquone by NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase): role in diaziquone-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res 50: 7293–7300
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S and Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107–1118
Stratford IJ and Stephens MA (1989) The differential hypoxic cytotoxicity of bioreductive agents determined in vitro by the MTT assay. Int J Radiation Oncol Biol Phys 16: 973–976
Talalay P, Fahey JW, Holtzclaw WD, Prestera T and Zhang Y (1995) Chemoprotection against cancer by phase 2 enzyme induction. Toxicol Lett 82–83: 173–179
Verweij J and Pinedo HH (1990) Cancer Chemotherapy and Biological Modifyers, Annual 11, Pinedo HM, Cabner BA, Longo DL (eds), pp. 63–73. Elsevier Science: Amsterdam
Workman P (1994) Enzyme-directed bioreductive drug development revisited: a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase. Oncol Res 6: 461–475
Wu K, Knox R, Sun XZ, Joseph P, Jaiswal AK, Zhang D, Deng PSK and Chen S (1997) Catalytic properties of NAD(P)H:quinone oxidoreductase2 (NQO2), a dihydronicotinamide riboside dependent oxidoreductase. Arch Biochem Biophys 345: 221–228
Zilfou JT and Smith CD (1995) Differential interactions of cytochalasins with P-glycoprotein. Oncol Res 7: 435–443
Author information
Authors and Affiliations
Rights and permissions
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
About this article
Cite this article
Joseph, P., Jaiswal, A. A unique cytosolic activity related but distinct from NQO1 catalyses metabolic activation of mitomycin C. Br J Cancer 82, 1305–1311 (2000). https://doi.org/10.1054/bjoc.1999.1096
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1054/bjoc.1999.1096
