Glucocorticoid-Stimulated CCAAT/Enhancer-Binding Protein α Expression Is Required for Steroid-Induced G₁ Cell Cycle Arrest of Minimal-Deviation Rat Hepatoma Cells

Abstract

By genetic correlation with the growth-suppressible phenotype and direct functional tests, we demonstrate that the glucocorticoid-stimulated expression of the CCAAT/enhancer-binding protein α (C/EBPα) transcription factor is required for the steroid-mediated G₁ cell cycle arrest of minimal-deviation rat hepatoma cells. Comparison of C/EBPα transcript and active protein levels induced by the synthetic glucocorticoid dexameth-asone in glucocorticoid growth-suppressible (BDS1), nonsuppressible receptor-positive (EDR1) and nonsup-pressible receptor-deficient (EDR3) hepatoma cell proliferative variants revealed that the stimulation of C/EBPα expression is a rapid, glucocorticoid receptor-mediated response associated with the G₁ cell cycle arrest. Consistent with the role of C/EBPα as a critical intermediate in the growth suppression response, maximal induction of transcription factor mRNA occurred within 2 h of dexamethasone treatment whereas maximal inhibition of [³H]thymidine incorporation was observed 24 h after steroid treatment. As a direct functional approach, ablation of C/EBPα protein expression and DNA-binding activity by transfection of an antisense C/EBPα expression vector blocked the dexamethasone-induced G₁ cell cycle arrest of hepatoma cells but did not alter general glucocorticoid responsiveness. Transforming growth factor β induced a G₁ cell cycle arrest in C/EBPα antisense transfected cells, demonstrating the specific involvement of C/EBPα in the glucocorticoid growth suppression response. Constitutive expression of a conditionally activated form of C/EBPα caused a G₁ cell cycle arrest of BDS1 hepatoma cells in the absence of glucocorticoids. In contrast, overexpression of C/EBPβ or C/EBPδ had no effect on hepatoma cell growth. Taken together, these results demonstrate that the steroid-induced expression of C/EBPα is necessary to mediate the glucocorticoid G₁ cell cycle arrest of rat hepatoma cells and implicates a role for this transcription factor in the growth control of liver-derived epithelial tumor cells.