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 were 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 catalyzed MMC-
DNA adduct formation only under anaerobic conditions with equal efficiency. However, the pH of the reaction controlled the production of four identified and two unidentified adducts. Soluble fractions from each cell source catalyzed MMC-
DNA adduct formation under aerobic and anaerobic conditions similarly. At higher pH, limited
DNA adducts were produced by MMC activated by soluble fractions from either cell source. At lower pH, more
DNA adducts were obtained with MMC activated 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 were identified as N2-(2" beta,7"-diaminomitosene-1" alpha-yl)-2'-deoxyguanylic
acid, N2-(2" beta,7"-diaminomitosen-1" beta-yl)-2'-deoxyguanylic
acid, N2-(10"-decarbamoyl-2",7"-diaminomitosen-1" alpha-yl)-2'-deoxyguanylic
acid, and N2-(2" beta,7"-diamino-10"-deoxyguanyl-N2-yl-mitosen-1" alpha-yl)-2'- deoxyguanylic
acid. Acidic intracellular pH enhanced the cytotoxicity of MMC for HCT 116 cells, decreasing the IC50 from 0.3 +/- 0.04 microM to 0.1 +/- 0.03 microM, but pH had limited effect on the cytotoxicity of MMC for HCT 116-R30A cells. When intracellular pH was decreased, interstrand
DNA cross-linking by MMC increased to a greater extent in HCT 116 cells than in HCT 116-R30A 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 treated 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. This is the first direct evidence that acidic intracellular pH enhances MMC-
DNA adduct formation in
tumor cells containing high
quinone reductase 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 enhances the activity of
quinone reductase in reducing MMC, which is important for aerobic cytotoxicity of MMC against
tumor cells with high concentration of
quinone reductase.