Ramos R A, Nishio Y, Maiyar A C, Simon K E, Ridder C C, Ge Y, Firestone G L
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.
Mol Cell Biol. 1996 Oct;16(10):5288-301. doi: 10.1128/MCB.16.10.5288.
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 alpha (C/EBP alpha) transcription factor is required for the steroid-mediated G1 cell cycle arrest of minimal-deviation rat hepatoma cells. Comparison of C/EBP alpha transcript and active protein levels induced by the synthetic glucocorticoid dexamethasone in glucocorticoid growth-suppressible (BDS1), nonsuppressible receptor-positive (EDR1) and nonsuppressible receptor-deficient (EDR3) hepatoma cell proliferative variants revealed that the stimulation of C/EBP alpha expression is a rapid, glucocorticoid receptor-mediated response associated with the G1 cell cycle arrest. Consistent with the role of C/EBP alpha 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/EBP alpha protein expression and DNA-binding activity by transfection of an antisense C/EBP alpha expression vector blocked the dexamethasone-induced G1 cell cycle arrest of hepatoma cells but did not alter general glucocorticoid responsiveness. Transforming growth factor beta induced a G1 cell cycle arrest in C/EBP alpha antisense transfected cells, demonstrating the specific involvement of C/EBP alpha in the glucocorticoid growth suppression response. Constitutive expression of a conditionally activated form of C/EBP alpha caused a G1 cell cycle arrest of BDS1 hepatoma cells in the absence of glucocorticoids. In contrast, overexpression of C/EBP beta or C/EBP delta had no effect on hepatoma cell growth. Taken together, these results demonstrate that the steroid-induced expression of C/EBP alpha 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.
通过与生长抑制表型的遗传相关性及直接功能测试，我们证明，CCAAT/增强子结合蛋白α（C/EBPα）转录因子的糖皮质激素刺激表达是类固醇介导的最小偏差大鼠肝癌细胞G1期细胞周期阻滞所必需的。比较合成糖皮质激素地塞米松在糖皮质激素生长抑制型（BDS1）、非抑制型受体阳性（EDR1）和非抑制型受体缺陷（EDR3）肝癌细胞增殖变体中诱导的C/EBPα转录本和活性蛋白水平，发现C/EBPα表达的刺激是一种与G1期细胞周期阻滞相关的快速、糖皮质激素受体介导的反应。与C/EBPα作为生长抑制反应关键中间物的作用一致，地塞米松处理2小时内转录因子mRNA达到最大诱导，而类固醇处理24小时后观察到[3H]胸苷掺入的最大抑制。作为一种直接功能方法，通过转染反义C/EBPα表达载体消除C/EBPα蛋白表达和DNA结合活性，可阻断地塞米松诱导的肝癌细胞G1期细胞周期阻滞，但不改变一般糖皮质激素反应性。转化生长因子β在反义C/EBPα转染细胞中诱导G1期细胞周期阻滞，证明C/EBPα特异性参与糖皮质激素生长抑制反应。条件性激活形式的C/EBPα的组成型表达在无糖皮质激素的情况下导致BDS1肝癌细胞G1期细胞周期阻滞。相反，C/EBPβ或C/EBPδ的过表达对肝癌细胞生长无影响。综上所述，这些结果表明，类固醇诱导的C/EBPα表达对于介导大鼠肝癌细胞的糖皮质激素G1期细胞周期阻滞是必需的，并暗示该转录因子在肝源性上皮肿瘤细胞的生长控制中起作用。
