Gao Ai, Liu Bing-ci, Shen Fu-hai, Du Hong-ju, Huang Chuan-shu, Jia Xiao-wei, You Bao-rong, Ye Meng
National Institute of Occupation Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2006 Mar;40(2):79-83.
To study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4.
The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle.
B (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P < 0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P < 0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P < 0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone.
Vitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.
研究E2F1/4信号通路在维生素C逆转苯并(a)芘[B(a)P]诱导的人胚肺成纤维细胞(HELF)细胞周期变化中的作用以及E2F1与细胞周期蛋白D1/细胞周期蛋白依赖性激酶4(CDK4)之间的关系。
构建稳定转染反义细胞周期蛋白D1和反义CDK4的HELF细胞株，以检测信号通路间的关系。细胞培养后，在B(a)P刺激前用维生素C预处理24小时。采用蛋白质免疫印迹法检测细胞周期蛋白D1、CDK4、E2F1和E2F4的表达水平，并用Gel-Pro 3.0软件分析条带强度，以相对于对照的相对值表示。采用流式细胞术分析检测细胞周期分布。
B(a)P显著升高HELF细胞中细胞周期蛋白D1、CDK4、E2F1和E2F4的表达水平。维生素C降低了B(a)P刺激的HELF细胞中上述蛋白的表达水平。B(a)P处理上述转染细胞后，这些蛋白的表达水平低于B(a)P处理的HELF细胞。与单独使用反义细胞周期蛋白D1相比，维生素C联合反义细胞周期蛋白D1处理后上述蛋白的表达水平降低。与对照组相比，B(a)P增加了S期细胞百分比[(41.1±0.2)% vs (33.5±3.2)%, P<0.05]。维生素C[(33.2±0.6)% vs (41.1±0.2)%, P<0.05]和反义细胞周期蛋白D1[(31.2±1.3)% vs (41.1±0.2)%, P<0.05]均抑制了B(a)P诱导的细胞周期变化。与单独使用反义CDK4相比，维生素C联合反义CDK4显著抑制了B(a)P诱导的细胞周期变化。
维生素C可能通过细胞周期蛋白D1-CDK4/E2F-1/4细胞内信号通路逆转B(a)P诱导的细胞周期变化。
