COMPARISON OF THE INACTIVATION OF MAMMALIAN AND BACTERIAL O6-ALKYLGUANINE-DNA ALKYLTRANSFERASES BY O6-BENZYLGUANINE AND O6-METHYLGUANINE

The inactivation of human and Escherichia coli O6-alkylguanine-DNA alkyltransferase by O6-methylguanine and O6-benzylguanine was compared. When HT29 cell extracts or E.coli Ada protein were incubated in the presence of 200-mu-M O6-methylguanine for 1 h, alkyltransferase activity was reduced to 44 and 39% of control levels respectively. However, under the same conditions O6-benzylguanine completely depleted alkyltransferase activity in the extract from human cells but had virtually no effect on the Ada protein. Incubation of the HT29 cell alkyltransferase with O6-benzyl[H-3]guanine resulted in a time-dependent production of [H-3]guanine. No similar production of [H-3]guanine was observed in the presence of the Ada protein. In CHO cells transfected with the bacterial ada gene (CHO-ada) or the human alkyltransferase cDNA (CHO-MGMT), treatment with 500-mu-M O6-methylguanine inhibited both alkyltransferases by > 85%. In contrast, 2-mu-M O6-benzylguanine inhibited human alkyltransferase expressed in CHO-MGMT cells by > 99% though concentrations as high as 25-mu-M for 24 h had no inhibitory effects on the bacterial alkyltransferase expressed in CHO-ada cells. This selective inhibition was also observed in vivo in transgenic mice expressing ada in the liver where O6-benzylguanine caused a decrease of only 40% in total hepatic alkyltransferase activity compared to 95% in non-transgenic mice, consistent with inhibition of only the mammalian alkyltransferase and maintenance of bacterial alkyltransferase activity in these animals. Thus, while O6-methylguanine at high concentrations inactivates both bacterial and mammalian alkyltransferases, O6-benzylguanine is a substrate only for the mammalian protein and is unable, perhaps due to steric hindrance, to inhibit the Ada protein.