MUTATIONS IN HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE IMPARTING RESISTANCE TO O-6-BENZYLGUANINE

O-6-Benzylguanine is an inactivator of O-6-alkylguanine-DNA alkyltrans ferases (AGT) which is currently entering clinical trials as an agent improving the cancer chemotherapeutic activity of chloroethylnitrosour eas and other alkylating agents. O-6-Benzylguanine acts by virtue of i ts ability to serve as a substrate for the AGT forming S-benzylcystein e at the cysteine acceptor site. The effects of a number of mutations in the human AGT sequence on the reaction with O-6-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 wi th O-6-benzylguanine; and (b) by measuring the production of guanine f rom O-6-benzylguanine by the AGT proteins. Both assays gave similar re sults and showed that mutations of the proline residues at positions 1 38 and 140 and of the glycine residue at position 156 significantly re duced the ability to react with O-6-benzylguanine. The combination of these mutations gave even greater resistance. Thus, the 50% effective dose for O-6-benzylguanine was increased from 0.25 mu M in the control AGT to 29 mu M by mutations P138K/P140A, to 60 mu M by mutation G156A and to >300 pill by mutations P140A/G156A. Truncation of the AGT at t he carboxyl end, removing either 31 or 23 amino acids did not affect t he activity or the ability to react with O-6-benzylguanine, but remova l of the 36 carboxyl terminal amino acids, which includes a highly con served glutamic acid residue, led to the loss of all activity. The rat e of the reaction between the AGT and O-6-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 mutatio ns of proline residues at 138 and 140. These results indicate that sev eral residues in the AGT sequence affect the access of the active site to O-6-benzylguanine and that these residues are located in at least two regions on either side of the active site cysteine, which is locat ed at residue 145. Mutations in these regions may occur during therapy with alkylating agents and O-6-benzylguanine. The development of othe r AGT inactivators which are still able to inactivate the resistant mu tants may be necessary to maximize the potential of AGT inhibition for cancer chemotherapy.