Pan S S, Yu F, Hipsher C
Division of Developmental Therapeutics, University of Maryland Cancer Center, School of Medicine, Baltimore 21201.
Mol Pharmacol. 1993 Jun;43(6):870-7.
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.
采用细胞组分和完整细胞研究了pH值和氧气对丝裂霉素C（MMC）诱导DNA烷基化的影响。所用细胞系为HCT 116人结肠癌细胞系和一个MMC耐药亚系（HCT 116-R30A），该亚系的醌还原酶活性仅为亲代细胞系的5%。两个细胞系的微粒体组分仅在厌氧条件下以相同效率催化MMC-DNA加合物的形成。然而，反应的pH值控制着四种已鉴定和两种未鉴定加合物的产生。来自每种细胞来源的可溶性组分在需氧和厌氧条件下类似地催化MMC-DNA加合物的形成。在较高pH值下，来自任一细胞来源的可溶性组分激活的MMC产生的DNA加合物有限。在较低pH值下，HCT 116细胞可溶性组分激活的MMC比HCT 116-R30A细胞可溶性组分激活的MMC产生更多的DNA加合物。其中四种加合物被鉴定为N2-（2“β，7”-二氨基丝裂霉素-1“α-基）-2'-脱氧鸟苷酸、N2-（2“β，7”-二氨基丝裂霉素-1“β-基）-2'-脱氧鸟苷酸、N2-（10“-脱氨甲酰基-2”，7”-二氨基丝裂霉素-1“α-基）-2'-脱氧鸟苷酸和N2-（2“β，7”-二氨基-10”-脱氧鸟苷-N2-基-丝裂霉素-1“α-基）-2'-脱氧鸟苷酸。酸性细胞内pH增强了MMC对HCT 116细胞的细胞毒性，使IC50从0.3±0.04微摩尔降至0.1±0.03微摩尔，但pH对MMC对HCT 116-R30A细胞的细胞毒性影响有限。当细胞内pH降低时，MMC诱导的链间DNA交联在HCT 116细胞中比在HCT 116-R30A细胞中增加的程度更大。在pH 6.0和7.6处理的HCT 116-R30A细胞以及pH 7.6处理的HCT 116细胞中仅检测到两种低强度的DNA加合物。然而，在pH 6.0处理的HCT 116细胞中检测到六个放射性斑点。其中三种加合物已被鉴定。这是酸性细胞内pH增强含高醌还原酶活性肿瘤细胞中MMC-DNA加合物形成的首个直接证据。本研究结果进一步证实，pH而非酶是MMC-DNA加合物类型分布的决定因素。本研究还表明，低细胞内pH增强了醌还原酶还原MMC的活性，这对于MMC对高浓度醌还原酶肿瘤细胞的需氧细胞毒性很重要。
