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Aryl hydrocarbon (benzo[a]pyrene) hydroxylase is inducible by polycyclic hydrocarbons in an epithelial cell strain cloned from Buffalo rat liver. The hydroxylase is NADPH-dependent and carbon monoxide-sensitive. Hydroxylase induction requires both RNA and protein synthesis, and these events can occur independently. The results suggest that the RNA species the synthesis of which is required for induction is not ribosomal RNA and is contained within the “heterogeneous RNA” fraction and that induction requires the synthesis of polyadenylic acid sequences. The hydroxylase is induced by concentrations of polycyclic hydrocarbons as low as 4 x10-10m. Fetal calf serum contains a substance(s) which increases hydroxylase activity. Polycyclic hydrocarbons can also induce the hydroxylase in the absence of serum added to the medium. This cell system may be useful for the study of the molecular biology of hydroxylase induction, for the bioassay of compounds for activity as hydroxylase inducers, and for the study of the role of the hydroxylase in chemical carcinogenesis.

We have analyzed protein-DNA interactionsin vivoat transcriptional control elements for two hypoxia-inducible genes in mouse hepatoma cells. The promoter for the phosphoglycerate kinase 1 (PGK1) gene contains an initiator element, but no TATA sequence, whereas the promoter for the glucose transporter 1 (Glut1) gene contains a TATA element but no initiator sequence. Our findings reveal hypoxia-inducible, Arnt-dependent occupancy of DNA recognition sites for hypoxia-inducible factor 1 (HIF-1) upstream of both target genes. The conserved recognition motif among the five recognition sites is 5′-CGTG-3′. The PGK1 promoter exhibits constitutive occupancy of a binding site for an unknown protein(s); however, we detect no protein-DNA interaction at the initiator element, in either uninduced or induced cells. The Glut1 promoter also exhibits constitutive protein binding; in addition, the TATA element exhibits partial occupancy in uninduced cells and increased occupancy under hypoxic conditions. We find no evidence for hypoxia-induced changes in chromatin structure of either gene. Time-course analyses of the Glut1 gene reveal a temporal relationship between occupancy of HIF-1 sites and TATA element occupancy. Our findings suggest that the promoters for both hypoxia-responsive genes constitutively maintain an accessible chromatin configuration and that HIF-1 facilitates transcription by recruiting and/or stabilizing a transcription factor(s), such as TFIID, at both promoters.

We analyzed mouse hepatoma cells using differential display to discover new genes that respond to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We identified a class I major histocompatibility complex (MHC) gene, which we designated as MHC Q1b, whose expression decreases in the presence of TCDD. TCDD-induced down-regulation of MHCQ1brequires both the aromatic hydrocarbon receptor and the aromatic hydrocarbon receptor nuclear translocator, transcription factors that up-regulate other genes in response to TCDD. Down-regulation of MHC Q1bby TCDD appears to involve both transcriptional and post-transcriptional regulatory events; the post-transcriptional destabilization of MHCQ1bmRNA is probably a secondary response to TCDD. Our findings reveal new mechanistic aspects of gene regulation by TCDD. In addition, our observations suggest a mechanism that might account for some of TCDD's immunotoxic effects.

The microsomal enzyme complex, aryl hydrocarbon (benzo[a]pyrene) hydroxylase, is inducible by benz[a]anthracene in cells cloned from Buffalo rat liver. The enzyme complex can also be induced to high activity, without adding a polycyclic hydrocarbon inducer, by temporarily inhibiting protein synthesis with cycloheximide or puromycin. This increase in enzyme activity is a function of both the degree and the duration of protein synthesis inhibition. It requires RNA synthesis during the period of inhibition of protein synthesis but not subsequent to the removal of the block; and it is prolonged in the presence of actinomycin D, when administered after the release of the block in protein synthesis. Combined exposure to benz[a]anthracene and cycloheximide is followed by an increase in enzyme activity which is greater than the sum of the increases which follow exposure to each compound alone. Qualitatively similar results were observed in four other mammalian cell culture systems. The results suggest two events in the regulation of aryl hydrocarbon hydroxylase induction: the inducer, benz[a]anthracene, acts primarily at the level of transcription, and a labile protein(s) regulates the translation of induction-specific RNA.