钙调素拮抗剂对HeLa细胞S期进程的影响

THE EFFECTS OF ANTAGONIST OF CaM ON THE PROGRESSION OF S PHASE IN HeLa CELLS

目的 探讨钙调素拮抗剂三氟拉嗪（ＴＦＰ）对ＨｅＬａ细胞Ｓ期进程的影响及其分子机理。 方法 通过ＴｄＲ双阻断法获得同步化的Ｓ期ＨｅＬａ细胞，以３Ｈ－ＴｄＲ掺入法和Ｗｅｓｔｅｒｎ 技术分别观察了ＴＦＰ对ＨｅＬａ 细胞Ｓ期进程和胸苷激酶（ｔｈｙｍ ｉｄｉｎｅ ｋｉｎａｓｅ，ＴＫ）活性及细胞周期引擎分子ＣｙｃｌｉｎＡ、Ｃｄｋ２ 和细胞周期调节蛋白ｐ８０ｃｄｃ２５Ｂ、ＰＣＮＡ、ｐ２１ 蛋白表达水平的影响。 结果 ＴＦＰ（２０μｍ ｏｌ／Ｌ）能使３ Ｈ－ＴｄＲ的掺入峰值后移，并抑制了Ｃｙ－ｃｌｉｎＡ、ＰＣＮＡ、ｐ８０ｃｄｃ２５Ｂ的表达和提高了ｐ２１ 蛋白的水平，但对Ｃｄｋ２的表达几乎无影响。 结论 ＣａＭ 除了能通过影响ＰＣＮＡ的水平直接作用于ＤＮＡ 合成，同时又可通过作用于ＣｙｃｌｉｎＡ、ｐ８０ｃｄｃ２５Ｂ、ｐ２１ 等周期引擎分子和调控因子水平正调ＨｅＬａ 细胞Ｓ期进程。

Objective\ To study the effects of antagonist of CaM(TFP) on the progression of S phase and the molecular mechanism in HeLa cells. Methods The synchronized S phase cells were obtained by the method of thymidine(TdR)double block.The progression of S phase and the activity of thymidine kinase were determined through 3H\|TdR incorporation assay.Western blotting was used to analyze the levels of CyclinA,Cdk2,PCNA,p80 cdc25B and p21 proteins. Results The TFP(20μmol/L)put off the appearing of the peak of 3H\|TdR incorporation and inhibited S phase progression.The activity of thymidine kinase decreased after treatment with TFP.The TFP(20μmol/L)decreased the level of CyclinA,PCNA,p80 cdc25B increased the expression of p21 protein,but not affected on the expression of Cdk2. Conclusion CaM could positively regulate the S phase progression in HeLa cells not only by affecting on the synthesis of DNA through PCNA,but also by influencing the expression of cell cycle engine molecules and related regulators including CyclinA,p80 cdc25B ,and p21 proteins.This may be one of the molecular mechanisms which is involved in the positive regulation of S phase progression in HeLa cells by CaM.