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 microM 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 microM O6-methylguanine inhibited both alkyltransferases by greater than 85%. In contrast, 2 microM O6-benzylguanine inhibited human alkyltransferase expressed in CHO-MGMT cells by greater than 99% though concentrations as high as 25 microM 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.
Dolan
M E
ME
Division of Hematology-Oncology, University of Chicago Medical Center, IL 60637.
Pegg
A E
AE
Dumenco
L L
LL
Moschel
R C
RC
Gerson
S L
SL
D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014157:Transcription Factors; D014162:Transfection
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C064976:O(6)-benzylguanine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase