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

FASEB J. 1999 Jan;13(1):169-80. doi: 10.1096/fasebj.13.1.169.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • CCAAT-Enhancer-Binding Proteins
  • Carcinoma, Hepatocellular
  • Cell Division
  • Cysteine / genetics
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • Dexamethasone / metabolism
  • Dexamethasone / pharmacology
  • Gene Expression Regulation
  • Glucocorticoids / metabolism
  • Glucocorticoids / pharmacology
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / metabolism*
  • Orosomucoid / genetics
  • Point Mutation*
  • Promoter Regions, Genetic
  • Rats
  • Receptors, Glucocorticoid / genetics*
  • Receptors, Glucocorticoid / metabolism*
  • Transcriptional Activation
  • Tumor Cells, Cultured
  • Tyrosine / genetics
  • Zinc Fingers

Substances

  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins
  • Glucocorticoids
  • Nuclear Proteins
  • Orosomucoid
  • Receptors, Glucocorticoid
  • Tyrosine
  • Dexamethasone
  • DNA
  • Cysteine