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Mutations in human O6-alkylguanine-DNA alkyltransferase imparting resistance to O6-benzylguanine.

Abstract：

:O6-Benzylguanine is an inactivator of O6-alkylguanine-DNA alkyltransferases (AGT) which is currently entering clinical trials as an agent improving the cancer chemotherapeutic activity of chloroethylnitrosoureas and other alkylating agents. O6-Benzylguanine acts by virtue of its ability to serve as a substrate for the AGT forming S-benzylcysteine at the cysteine acceptor site. The effects of a number of mutations in the human AGT sequence on the reaction with O6-benzylguanine were investigated by two methods: (a) by measuring the loss of the ability of the AGT to repair a methylated DNA substrate after preincubation with O6-benzylguanine; and (b) by measuring the production of guanine from O6-benzylguanine by the AGT proteins. Both assays gave similar results and showed that mutations of the proline residues at positions 138 and 140 and of the glycine residue at position 156 significantly reduced the ability to react with O6-benzylguanine. The combination of these mutations gave even greater resistance. Thus, the 50% effective dose for O6-benzylguanine was increased from 0.25 microM in the control AGT to 29 microM by mutations P138K/P140A, to 60 microM by mutation G156A and to > 300 microM by mutations P140A/G156A. Truncation of the AGT at the carboxyl end, removing either 31 or 23 amino acids did not affect the activity or the ability to react with O6-benzylguanine, but removal of the 36 carboxyl terminal amino acids, which includes a highly conserved glutamic acid residue, led to the loss of all activity. The rate of the reaction between the AGT and O6-benzylguanine was increased when DNA was present. This increase amounted to about 6-fold with the control AGT and the carboxyl-truncated mutants but was reduced to only 2-fold with G156A mutant and increased to 11-18-fold with the mutations of proline residues at 138 and 140. These results indicate that several residues in the AGT sequence affect the access of the active site to O6-benzylguanine and that these residues are located in at least two regions on either side of the active site cysteine, which is located at residue 145. Mutations in these regions may occur during therapy with alkylating agents and O6-benzylguanine. The development of other AGT inactivators which are still able to inactivate the resistant mutants may be necessary to maximize the potential of AGT inhibition for cancer chemotherapy.

journal_name

Cancer Res

journal_title

Cancer research

authors

Crone TM,Goodtzova K,Edara S,Pegg AE

subject

Has Abstract

pub_date

1994-12-01 00:00:00

pages

6221-7

issue

eissn

0008-5472

issn

1538-7445

journal_volume

pub_type

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Mutations in human O6-alkylguanine-DNA alkyltransferase imparting resistance to O6-benzylguanine.