ENZYMATIC AND PH MODULATION OF MITOMYCIN C-INDUCED DNA-DAMAGE IN MITOMYCIN C-RESISTANT HCT-116 HUMAN COLON-CANCER CELLS

The effect of pH and oxygen on DNA alkylation by mitomycin C (MMC) was studied with cell fractions and intact cells. The cell lines used wer e the HCT 116 human colon cancer cell line and a MMC-resistant subline (HCT 116-R30A) that has 5% of the quinone reductase activity present in the parent cell line. Microsomal fractions of the two cell lines ca talyzed MMC-DNA adduct formation only under anaerobic conditions with equal efficiency. However, the pH of the reaction controlled the produ ction of four identified and two unidentified adducts. Soluble fractio ns from each cell source catalyzed MMC-DNA adduct formation under aero bic and anaerobic conditions similarly. At higher pH, limited DNA addu cts were produced by MMC activated by soluble fractions from either ce ll source. At lower pH, more DNA adducts were obtained with MMC activa ted by the soluble fraction of HCT 116 cells than with that activated by the soluble fraction of HCT 116-R30A cells. Four of these adducts w ere identified as ,7''-diaminomitosene-1''alpha-yl)-2'-deoxyguanylic a cid, ta,7''-diaminomitosen-1''beta-yl)-2'-deoxyguanylic acid, ',7''-di aminomitosen-1''alpha-yl)-2'-deoxyguanylic acid, and N2-(2''beta,7''-d iamino-1 guanyl-N2-yl-mitosen-1''alpha-yl)-2'-deoxyguanylic acid. Acid ic intracellular pH enhanced the cytotoxicity of MMC for HCT 116 cells , decreasing the IC50 from 0.3 +/- 0.04 mum to 0.1 +/- 0.03 mum, but p H had limited effect on the cytotoxicity of MMC for HCT 116-R30A cells . When intracellular pH was decreased, interstrand DNA cross-linking b y MMC increased to a greater extent in HCT 116 cells than in HCT 116-R 30A cells. Only two DNA adducts, each at low intensity, were detected in HCT 116-R30A cells treated at pH 6.0 and 7.6 and in HCT 116 cells t reated at pH 7.6. However, six radioactive spots were detected in HCT 116 cells treated at pH 6.0. Three of these adducts were identified. T his is the first direct evidence that acidic intracellular pH enhances MMC-DNA adduct formation in tumor cells containing high quinone reduc tase activity. Results from this study further confirm that pH and not enzyme is the determining factor in the distribution of types of MMC- DNA adducts. This study also indicates that low intracellular pH enhan ces the activity of quinone reductase in reducing MMC, which is import ant for aerobic cytotoxicity of MMC against tumor cells with high conc entration of quinone reductase.