Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine

• Published in: Carcinogenesis (New York) 1991-12, Vol.12 (12), p.2305-2309
• Main Authors: Dolan, M. Eileen, Pegg, Anthony E., Dumenco, Luba L., Moschel, Robert C., Gerson, Stanton L.
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; CHO Cells; Cricetinae; DNA - genetics; Enzyme Activation; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Guanine - analogs & derivatives; Guanine - pharmacology; Humans; Methyltransferases - antagonists & inhibitors; Methyltransferases - genetics; Methyltransferases - metabolism; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; O-Methylguanine-DNA Methyltransferase; Transcription Factors; Transfection
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/12.12.2305

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 μM 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[3H]guanine resulted in a time-dependent production of [3H]guanine. No similar production of [3H]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 μM O6-methylguanine inhibited both alkyltransferases by >85%. In contrast, 2 μM O6-benzylguanine inhibited human alkyltransferase expressed in CHO-MGMT cells by >99% though concentrations as high as 25 μM for 24 h had no inhibitory effects on the bacterial akyltransferase 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.