A simplified assay for O6-methylguanine-DNA methyltransferase activity and its application to human neoplastic and non-neoplastic tissues

Abstract

A rapid assay of O ⁶ -MeG-DNA methyltransferase activity is described. Following incubation of cell extracts with O ⁶ -[ ³ H]MeG-containing DNA, remaining radioactive DNA was hydrolyzed in trichloroacetic acid and separated from methylated radioactive protein by filtration or centrifugation. Transfer of radioactive methyl from DNA to protein was proportional to the amount of protein added, and was not linear with time. More than 90% of the radioactivity precipitated after acid hydrolyses was in S-methyl cysteine residues. The method was used to measure O ⁶ -MeG-DNA methyltransferase activity in extracts of 24 neoplastic tissues from human organs. Although five tumor tissues had 28–84% lower activity of O ⁶ -MeG-DNA methyltransferase than the corresponding normal tissue from the same patient, higher or similar levels of activity were found more frequently. Thus, a lack of O ⁶ -MeG-DNA methyltransferase activity in human tumors appears not to be a frequent event. The DNA repair enzyme uracil-DNA glycosylase was also measured in the same extracts. Most frequently the level of uracil-DNA glycosylase activity was essentially similar in tumors and normal tissues but significantly higher or lower levels were also observed.