鼻咽癌细胞中外源性p16表达对CDK4,CyclinD1及pRb的影响

The effects of exogenous p16 expression on CDK4, Cyclin D1 and pRb in nasopharyngeal carcinoma cell lines

目的 :分析外源性p16表达对CDK4,CyclinD1及pRb的影响 ,探讨其抑制细胞生长的机制。方法 :绘制生长曲线 ,比较细胞倍增时间 ,分析HNE1,# 4 - 2及 # 3- 2克隆中细胞增殖状况 ;利用流式细胞仪分析上述细胞中细胞周期分布的改变 ;结合WesternBlot方法 ,分析外源性p16表达对CDK4,CyclinD1及pRb的影响。结果 :生长曲线示 ,HNE 1倍增时间为 2 3 4h ,# 3- 2和 # 4 - 2分别为 2 8 8h和 31 2h。流式细胞仪示 ,HNE1和 # 3- 2细胞周期无明显差别 (P >0 .0 5 ) ,# 4 - 2细胞G0 /G1期细胞数明显增多 ,S期的细胞数显著降低 (P <0 .0 1和P <0 .0 5 )。WesernBlot示外源性p16表达对CDK4表达水平影响并不显著 ,但下调 # 4 - 2中CyclinD1的表达 ,# 3- 2中CyclinD1的表达反而增强 ;各细胞系均可检测到低磷酸化的pRb ,但在 # 4 - 2 ,# 3- 2中表达强于HNE1及Hela细胞 ;HNE1还可检获高磷酸化的pRb。结论 :外源性p16表达使细胞停滞在G1期 ,抑制细胞生长 ,但其主要机制并非改变CDK4表达水平 ;而对CyclinD1的影响 ,随p16表达水平而变动 ,最终表现为抑制pRb磷酸化。

Objective: To investigate the effect of exogenous p16 on Cyclin D1, CDK4 and pRb, and to explore the mechanism of the growth suppression of p16 in nasopharyngeal carcinoma cell lines. Methods: The curve of cell growth rate in three kinds of HNE1, #3-2 and #4-2 cell lines was analyzed and their double time was compared. Then the distribution of the cell cycle was detected by flow cytometry. The expression of p16 and the effect of exogenous p16 expression on CDK4, Cyclin D1 and pRb are studied by means of Western Blot. Results: As compared with HNE1, #3-2 and #4-2 showed a longer double time(23.4?h vs 28.8?h, 31.2?h). #4-2 showed a significant accumulation of cells in G 0/G 1 phase(P<0.01) and decreasing in S phase(P<0.05) while HNE1 and #3-2 had no obvious difference(P>0.05). Cyclin D1 expression was upregulated in #4-2 but downregulated in #3-2 by exogenous expressed p16. No obvious difference on CDK4 expression was found. Hypophosphorylated pRb was detected in three cell lines. The expression was stronger in #4-2, and #3-2 than that in HNE1 and Hela. Hyperphosphorylated pRb was also detected in HNE1. Conclusion: Exogenous p16 expression may arrest cell cycle in G 0/G 1 phase and suppress cell growth. The major mechanism is not to regulate the level of the expression of CDK4. There might be a threshold in p16 regulating Cyclin D1 expression. However, the final result contributes to the inhibition of pRb phosphorylation. [