title: Dysfunctional glucocorticoid receptor with a single point mutation ablates the CCAAT/enhancer binding protein-dependent growth suppression response in a steroid-resistant rat hepatoma cell variant.
abstract: We used glucocorticoid-resistant and -sensitive hepatoma cell variants to characterize the mechanism of hepatoma cell resistance to the growth inhibitory effects of glucocorticoids. BDS1 hepatoma cells express transcriptionally active glucocorticoid receptors and undergo a stringent G1 cell cycle arrest in response to glucocorticoids that is dependent on the induced expression of the CCAAT/enhancer binding protein alpha (C/EBPalpha) transcription factor. In contrast, EDR1 hepatoma cells, which express normal levels of glucocorticoid receptors, fail to growth arrest or express C/EBPalpha when treated with glucocorticoids. Ectopic expression of wild-type rat glucocorticoid receptors into EDR1 cells restored the growth suppression response, suggesting a defect in the EDR1 receptor. DNA sequence analysis revealed a single point mutation causing a cysteine-to-tyrosine substitution at amino acid position 457 (C457Y-GR) in the zinc finger region of the glucocorticoid receptor that mediates both receptor-DNA and receptor-protein interactions. Glucocorticoid activation of the alpha1-acid glycoprotein (AGP) promoter, a liver acute-phase response gene, requires receptor-DNA binding as well as an interaction with C/EBPalpha. In contrast to the wild-type glucocorticoid receptor, ectopic expression of C/EBPalpha in EDR1 cells, or coexpression of C/EBPalpha along with the C457Y-GR into receptor-deficient EDR3 cells was required to partially restore glucocorticoid responsiveness of the AGP promoter by the EDR1 glucocorticoid receptor. Constitutive expression of the wild-type glucocorticoid receptor, but not the C457Y-GR mutant, was sufficient to restore the glucocorticoid growth suppression response to receptor-deficient EDR3 cells. Thus, we have identified a glucocorticoid-resistant hepatoma cell variant with a single point mutation in the zinc finger region of the glucocorticoid receptor gene that ablates the glucocorticoid growth suppression response and attenuates transcriptional activation of the AGP promoter.
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Ramos
authors.forename: W J
authors.initials: WJ
authors.lastname: Meilandt
authors.forename: E C
authors.initials: EC
authors.lastname: Wang
authors.forename: G L
authors.initials: GL
authors.lastname: Firestone
mesh_terms: D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D006528:Carcinoma, Hepatocellular; D002455:Cell Division; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D009961:Orosomucoid / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D009687:Nuclear Proteins; D009961:Orosomucoid; D011965:Receptors, Glucocorticoid; D014443:Tyrosine; D003907:Dexamethasone; D004247:DNA; D003545:Cysteine