INACTIVATION OF PURIFIED HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE BY ALKYLATING-AGENTS OR ALKYLATED DNA

Abstract

O6-Alkylguanine-DNA alkyltransferase partially purified from cultured human lymphoblasts (CEM-CCRF line) was inactivated with DNA substrates that had been treated separately with five methylating agents or with five chloroethylnitrosoureas. The extent of depletion of the transferase by alkylated DNA was compared with its inactivation by direct reaction with these ten agents. As expected, DNA substrates treated with methylating agents that efficiently produce O6-methylguanine were most effective in depleting the transferase, as was DNA pretreated with 1-(2-chloroethyl)-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and chlorozotocin, agents presumed to form O6-chloroethylguanine as well as O6-hydroxyethylguanine in DNA. Unexpectedly, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-trans-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea were relatively ineffective in producing a DNA substrate that would inactivate the transferase, suggesting that both agents produce low steady-state levels of O6-alkylguanine. All of the agents tested were capable of inactivating the transferase by direct alkylation, although the efficiency of this activity ranges widely. We conclude that simple methylating agents and the cross-linking chloroethylnitrosoureas can inactivate O6-alkylguanine-DNA alkyltransferase both directly and indirectly, affording two mechanisms by which such agents could modulate their own cytotoxicity.