Abstract
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.
Original language | English (US) |
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Pages (from-to) | 2305-2309 |
Number of pages | 5 |
Journal | Carcinogenesis |
Volume | 12 |
Issue number | 12 |
DOIs | |
State | Published - Dec 1991 |
All Science Journal Classification (ASJC) codes
- Cancer Research