REGULATION OF EXPRESSION OF THE DNA-REPAIR GENE O-6-METHYLGUANINE-DNAMETHYLTRANSFERASE VIA PROTEIN-KINASE C-MEDIATED SIGNALING

O-6-Alkylguanine is the major mutagenic and cytotoxic DNA lesion induc ed by alkylating agents, including 2-chloroethyl-N-nitrosourea-based a ntitumor drugs. This lesion is repaired by O-6-methylguanine-DNA methy ltransferase (MGMT), the expression of which is highly variable in bot h normal tissues and in tumor cells. The promoter of the human MGMT ge ne was found to contain two putative activator protein (AP)-1 sites. H ere, we show that the level of MGMT mRNA in HeLa S3 cells was increase d 3-5-fold by phorbol-12-myristate-13-acetate (TPA) and 1,2-diacyl-sn- glycerol (DAG), which are activators of protein kinase C (PKC), as wel l as by okadaic acid, an inhibitor of protein phosphatases. The PKC in hibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine-HCl eliminated MGMT activation by TPA and DAG but not by OA, Prior down-regulation of PKC abolished subsequent effects of TPA or DAG, The results indicate AP-1 to be involved in regulation of MGMT expression. This hypothesis was supported by showing AP-1 binding to two target sequences of the M GMT promoter and transactivation of the MGMT promoter upon cotransfect ion with c-fos and c-jun in Fg cells. That TPA-mediated induction of M GMT caused increased cellular resistance to 2-chloroethyl-N-nitrosoure a suggests a therapeutic significance for PKC-mediated MGMT modulation .