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NAD(P)H:Quinone Oxidoreductase Gene Expression in Human Colon Carcinoma Cells: Characterization of a Mutation Which Modulates DT-Diaphorase Activity and Mitomycin Sensitivity¹

University of Southern California, School of Pharmacy [R. D. T., N. W. G.], and Kenneth Norris Jr. Comprehensive Cancer Center [R. D. T., T. H., P. V. D., N. W. G., K. D. D., H. W. S.], Los Angeles, California 90033, and Molecular Toxicology and Environmental Health Sciences Program, University of Colorado, School of Pharmacy, Boulder, Colorado 80309 [D. R.]

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) is an obligate two-electron reductase which may play a role in the bioactivation of antitumor quinones such as mitomycin C (MMC). We studied 10 colon carcinoma cell lines showing different levels of DTD activity (range, 0–3447 nmol/min/mg protein), as measured by the reduction of dichiorophenolindophenol. Expression of the NAD(P)H:quinone reductase gene (NQO1), which codes for the DTD enzyme, as measured by a polymerase chain reaction amplification technique was then correlated with enzymatic activity in all cell lines. HT-29 cells, which have intermediate DTD activity (769 ± 144 nmol/min/mg protein, mean ± SD) and are sensitive to MMC, showed high NQO1 expression relative to ß-actin (taken as 100% here for comparative purposes). BE cells which have no detectable DTD activity and are resistant to MMC showed moderate NQO1 expression (91% of HT-29). RNA single-strand conformational polymorphism analysis and subsequent sequencing of BE complementary DNA revealed a C to T mutation in the NQO1 complementary DNA. This confers a proline to serine substitution in the amino acid sequence of the protein. Additionally, HCT-116 cells showed both moderate DTD activity (390 ± 41 nmol/min/mg protein) and NQO1 expression (41% of HT-29), while resistant subclones of these cells, exposed to MMC during 11 and 44 weeks, showed low gene expression (5 and 9% of HT-29 respectively) and enzymatic activity (11 ± 6 and 36 ± 16 nmol/min/mg protein). These results support the ideas that reductive activation of MMC by DTD may be important in the cytotoxicity of MMC and that polymerase chain reaction may be a useful technique for quantitating the relative expression of genes in human tumors.

¹ This research was supported by USPHS NIH grant CA-51210 and American Cancer Society grant CH-490.

² To whom requests for reprints should be addressed, at 1303 N. Mission Rd., Los Angeles, CA 90033.

Received 7/ 8/91. Accepted 12/ 2/91.

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