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

M. Eileen Dolan, Anthony Pegg, Luba L. Dumenco, Robert C. Moschel, Stanton L. Gerson

Research output: Contribution to journalArticle

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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 languageEnglish (US)
Pages (from-to)2305-2309
Number of pages5
JournalCarcinogenesis
Volume12
Issue number12
DOIs
StatePublished - Dec 1 1991

Fingerprint

Alkyl and Aryl Transferases
DNA
Proteins
Protein
Cell
Escherichia coli
CHO Cells
Guanine
Escherichia Coli
HT29 Cells
Transgenic Mice
Level control
Cell Extracts
Liver
CDNA
Animals
Genes
Cells
O-(6)-methylguanine
O(6)-benzylguanine

All Science Journal Classification (ASJC) codes

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Physiology
  • Behavioral Neuroscience
  • Cancer Research

Cite this

Dolan, M. Eileen ; Pegg, Anthony ; Dumenco, Luba L. ; Moschel, Robert C. ; Gerson, Stanton L. / Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine. In: Carcinogenesis. 1991 ; Vol. 12, No. 12. pp. 2305-2309.
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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.",
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Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine. / Dolan, M. Eileen; Pegg, Anthony; Dumenco, Luba L.; Moschel, Robert C.; Gerson, Stanton L.

In: Carcinogenesis, Vol. 12, No. 12, 01.12.1991, p. 2305-2309.

Research output: Contribution to journalArticle

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T1 - Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine

AU - Dolan, M. Eileen

AU - Pegg, Anthony

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N2 - 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.

AB - 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.

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