INDUCTION OF THE HUMAN CYP1A2 ENHANCER BY PHORBOL ESTER

Induction of cytochrome (CYP) P4501A2 by such polycyclic aromatic hydr ocarbons as 3-methylcholanthrene (3MC) can lead to the bioactivation o f carcinogenic aromatic amines and heterocyclic amines, A 3MC response element was recently identified approximately 2.2 kb upstream of the transcription start site of the human CYP1A2 gene, Sequence analysis o f this enhancer identified, in addition to a binding site for the aryl hydrocarbon receptor, two other sequences, referred to as 5'AP1 and 3 'AP1, each with complete homology to the phorbol 12-O-tetradecanoate 1 3-acetate (TPA) response element consensus sequence. Nuclear extracts from TPA-treated HepG2 cells protected both the 5'AP1 and 3'AP1 sequen ces against digestion with DNase I, Gel mobility shift and supershift assays revealed that TPA treatment of HepG2 results in increased bindi ng activity of the AP-1 proteins, c-Jun, JunD, and c-Fos, to both site s, We transiently expressed, in HepG2, either a fragment containing bo th the 5'AP1 and 3'AP1 sites (-2.3pT81Luc) or only the 3'AP1 site (-2. 2pT81Luc) cloned into a plasmid containing the luciferase gene under t ranscriptional control of the thymidine kinase promoter, TPA treatment of cells transfected with -2.3pT81Luc resulted in an approximately th reefold induction of luciferase activity over untreated control cells, while the -2.2pT81Luc construction containing only the 3'AP1 site dis played an approximately sixfold induction, These studies suggest that the human CYP1A2 gene may be regulated by tumor promoters in addition to polycyclic aromatic hydrocarbons. (C) 1998 Academic Press.