GLYCINE N-METHYLTRANSFERASE IS A MEDIATOR OF CYTOCHROME P4501A1 GENE-EXPRESSION

Cytochrome P4501A1, the isozyme most closely approximating aryl hydroc arbon hydroxylase activity under conditions of induction, is thought t o be regulated by several trans-acting factors, including the 4S polyc yclic aromatic hydrocarbon-binding protein; this protein has recently been identified as glycine N-methyltransferase (Raha et al, (1994) J. Biol. Chem, 269, 5750-5756), Previous studies had shown that partially purified liver preparations containing the 4S binding protein interac ted with 5'-flanking regions of the cytochrome P4501A1 gene, Consequen tly, the ability of the 4S binding protein to serve as a mediator in t he regulation of the cytochrome P4501A1 gene was investigated further, Introduction of an antisense 24-mer oligonucleotide to glycine N-meth yltransferase cDNA into rat hepatoma H4IIE cells by lipofectin resulte d in a 60% reduction in the benzo(a)pyrene-mediated induction of ethox yresorufin-O-deethylase activity and protein over the sense and scramb led antisense oligonucleotide controls. In addition, the antisense oli gonucleotide caused a marked reduction in the steady-state level of cy tochrome P4501A1 mRNA; no such effect was observed with the sense olig onucleotide. Introduction of GNMT polyclonal antibodies into H4IIE cel ls by a streptolysin-O permeabilization technique markedly reduced bot h benzo(a)pyrene-binding and benzo(a)pyrene-induced ethoxyresorufin-O- deethylase activities, but had no effect on 2,3,7,8-tetrachlorodibenzo -p-dioxin induction, Collectively, these findings suggest that, in add ition to the Ah (dioxin) receptor, glycine N-methyltransferase appears to be both a polycyclic aromatic hydrocarbon-binding protein and a me diator of the induction of the cytochrome P4501A1 gene by polycyclic h ydrocarbons such as benzo(a)pyrene. (C) 1995 Academic Press, Inc.