In vitro inactivation of human O6-alkylguanine DNA alkyltransferase by antitumor triazene compounds.
The cytotoxic and mutagenic properties of antitumor triazene compounds (TZC) have been mainly attributed to their ability to form DNA adducts at the O6 position of guanine. Repair of these lesions is mediated by O6-alkylguanine DNA alkyltransferase (OGAT) in an autoinactivating reaction. Therefore when lesion repair has occurred, cells are depleted of OGAT until synthesis of new enzyme molecules takes place. In this study, we have evaluated the ability of DNA alkylated by different TZC to deplete OGAT activity. Moreover, we have also investigated whether these compounds might inactivate the OGAT enzyme by a direct reaction with the protein. Human OGAT protein was partially purified from insect cells infected with a recombinant baculovirus containing the human OGAT coding sequences. Thereafter human OGAT protein was exposed directly to TZC or to TZC-alkylated DNA. Among the TZC tested, p-(3-methyl-1-triazeno)benzoic acid was the most effective OGAT inactivator by direct interaction with the protein. Moreover DNA substrates treated with methylating TZC, such as temozolomide or p-(3-methyl-1-triazeno)benzoic acid, were more effective in depleting the repair enzyme, compared to DNA pretreated with the chloroethylating TZC mitozolomide. In conclusion, our results show that TZC inactivate in vitro OGAT activity by either direct or indirect mechanisms. Therefore TZC are good candidates for 1) increasing their own cytotoxicity, if used according to appropriate dose and treatment schedules and 2) reversing tumor cell resistance to O6-guanine alkylating agents.
Lacal
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PM
Istituto Dermopatico dell'Immacolata (IDI, IRCCS, Rome, Italy.
D'Atri
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S
Orlando
L
L
Bonmassar
E
E
Graziani
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G
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D004351:Drug Resistance; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D018411:Spodoptera; D000077204:Temozolomide; D014226:Triazenes / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018906:Antineoplastic Agents, Alkylating; D011994:Recombinant Proteins; D014226:Triazenes; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide