Characterisation of the DNA repair enzyme for O(6)-methylguanine in cirrhosis

The reason(s) why cirrhosis is a major risk factor for the development of hepatocellular carcinoma worldwide are largely unknown. In a previous preliminary study, we reported deficiency of the repair enzyme for the highly promutagenic and potentially carcinogenic DNA base lesion, O(6)-methylguanine, in cirrhotic human liver. The aims of the present study were: (i) to confirm this observation in an extended series of cirrhosis patients, using a new DNA repair enzyme assay approach, (ii) to characterise the enzyme, in particular to seek physicochemical differences between control and cirrhotic liver that might account for the enzyme defect in cirrhosis, and (iii) to examine the relationship between magnitude of DNA repair deficiency in cirrhotic liver and aetiology of cirrhosis or male sex, both of which are independent risk factors of hepatocellular carcinoma. Tissue extracts containing DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase, were prepared from liver biopsy samples from 41 patients with cirrhosis (10 viral (HBV and HCV), 17 alcohol and 14 autoimmune), 17 patients with non-cirrhotic diseased liver and 4 patients with histologically normal liver. Repair enzyme levels and electrophoretic profiles were both obtained using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in conjunction with fluorography and densitometry. We have demonstrated the feasibility of both analysing and reproducibly measuring DNA repair enzyme in small liver biopsy samples using the gel electrophoresis/densitometry approach for enzyme assay. Using the new densitometric assay approach, levels of O(6)-methylguanine-DNA methyltransferase were found to be significantly lower in cirrhotic compared to non-cirrhotic liver, with very low values obtained for two individuals who were incidentally found to have small hepatocellular carcinomas at the time of liver transplantation. There were no significant differences in enzyme levels between patients with cirrhosis of different aetiology, and between male and female patients. Methyltransferase was found to be present in all liver extracts as a major 23.1 kDA protein, along with other less abundant enzyme forms of both slightly higher and lower molecular weight; there were no obvious differences in size and relative abundance of these enzyme forms between liver samples from any of the patient and control groups. Our findings of enzyme instability and multiple forms led us to examine methyltransferase's amino acid sequ