title: Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the Ah receptor.
abstract: The Ah receptor (AhR) is a ligand-dependent transcription factor subunit that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD activates target genes in xenobiotica metabolism in many cell lines and, more specifically, delays G1-S progression of 5L hepatoma cells. Here we describe transient and stable AhR-expression analysis in AhR-deficient subclones of the TCDD-sensitive 5L cells. We tested the integrity of the AhR-signaling system beyond the lack of the receptor in the variant subclone and analyzed the role of AhR in cell cycle regulation. Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so-called XREs, when transfected into receptor-deficient variant cells compared to wild-type cells. Single- and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to an XRE. Hybrid receptors also show the high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR-activating signal acts directly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor. Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target-gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid. Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity. In vitro ligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR-binding endogenous ligand. In conclusion, AhR is causally responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity.
authors.affiliation: Research Center Karlsruhe, Institute of Genetics, Karlsruhe, D-76021, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Weiss
authors.forename: S K
authors.initials: SK
authors.lastname: Kolluri
authors.forename: F
authors.initials: F
authors.lastname: Kiefer
authors.forename: M
authors.initials: M
authors.lastname: Göttlicher
mesh_terms: D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive / Q000502:physiology; D006528:Carcinoma, Hepatocellular; D002453:Cell Cycle / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D017930:Genes, Reporter / Q000502:physiology; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014157:Transcription Factors; D014316:Tritium / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000187:drug effects
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014316:Tritium