Induction of mRNA specific for cytochrome P1-450 in wild type and variant mouse hepatoma cells.

Abstract

We have used a cDNA probe specific for an aromatic hydrocarbon-inducible form of cytochrome P-450 to analyze the accumulation of enzyme-specific mRNA in wild type Hepa 1c1c7 cells and in variant cells defective in aryl hydrocarbon hydroxylase induction. In wild type cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a 25-50-fold increase in the steady state concentration of cytochrome P1-450-specific RNA. This RNA binds to oligo(dT)-cellulose and migrates in agarose gels as a single species of about 2900 nucleotides. Dot hybridization analyses of total RNA indicate that half-maximal induction of P1-450 mRNA occurs at about 10 pM TCDD; maximal induction occurs at about 100 pM TCDD. Parallel increases occur in aryl hydrocarbon hydroxylase activity. RNA induction precedes aryl hydrocarbon hydroxylase induction by 2-4 h. Kinetic analyses of mRNA induction in response to submaximal concentrations of TCDD reveal no change in the degradation of P1-450 mRNA during induction. We detect an increase in P1-450 mRNA 30 min after TCDD administration. Full accumulation of mRNA occurs in cells in which protein synthesis is inhibited by 95-97%. Variant cells with decreased TCDD receptors exhibit decreased basal and inducible levels of P1-450 mRNA. Variant cells with a defect in nuclear localization of the inducer-receptor complex exhibit virtually no basal or inducible levels of P1-450 mRNA. Hybrid cells formed by fusing these different variants contain wild type basal and inducible levels of P1-450 mRNA. We conclude that expression of the cytochrome P1-450 gene is under transcriptional control and requires nuclear localization of the TCDD receptor.