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目的:探讨Fas介导的细胞周期特异性细胞凋亡发生过程中,细胞周期蛋白(cyclins)和细胞周期依赖性蛋白激酶(cyclin-dependent kinases,CDKs)的变化规律,及其可能的调节机制。方法:以急性淋巴细胞白血病Molt-4细胞株为靶细胞,建立Fas介导的细胞周期特异性凋亡模型;采用cyclin/DNA双参数流式细胞术和Western印迹法检测cyclins的表达规律;应用Western印迹法检测CDK1的Thr-161、Tyr-15位点和CDK2的Thr-160位点磷酸化水平的变化。结果:重组人Fas配体(re-combinant human Fas ligand,rhFasL)诱导Molt-4细胞的凋亡定位在G1期。在发生细胞凋亡效应前,cyclin D3水平明显升高,而cyclin E水平和CDK2的Thr-160位点磷酸化水平均明显下降;发生凋亡效应后,cyclin D3水平明显下降,而cyclin E水平升高,CDK2的Thr-160位点磷酸化水平则明显下降,CDK1的Thr-161、Tyr-15位点磷酸化水平稍有下降。Cyclin A和Cyclin B1水平在诱导细胞凋亡过程中无明显变化。Roscovitine在特定浓度(5μmol/L)下可下调CDK2 Thr-160位点和CDK1 Thr-161位点的磷酸化水平,与rhFasL共同作用后细胞凋亡的发生率明显升高。结论:Fas介导的细胞凋亡具有细胞周期特异性,并始动于晚G1期,是G1期cyclin E/CDK2活性下降以及晚G1期检测点监督的结果;cyclin D3/CDK复合体可能是决定细胞凋亡能否发生的关键。
OBJECTIVE: To investigate the changes of cyclins and cyclin-dependent kinases (CDKs) induced by Fas in cell cycle-specific apoptosis and its possible regulatory mechanism. Methods: Fas-mediated cell cycle-specific apoptotic model was established by Molt-4 cell line of acute lymphoblastic leukemia; cyclin / DNA double-flow cytometry and Western blotting were used to detect the expression of cyclins; Western blotting was used to detect the changes of Thr-161, Tyr-15 and CDK2 Thr-160 phosphorylation of CDK1. Results: The apoptosis of Molt-4 cells induced by recombinant human Fas ligand (rhFasL) was localized in G1 phase. Cyclin D3 levels were significantly increased before cyclin E level and phosphorylation level of Thr-160 phosphorylation of CDK2 significantly decreased after apoptosis. Cyclin D3 levels decreased significantly after apoptosis, but cyclin E level Phosphorylation of Thr-160 in CDK2 was significantly decreased. The phosphorylation levels of Thr-161 and Tyr-15 in CDK1 decreased slightly. Cyclin A and Cyclin B1 levels did not change significantly during apoptosis. Roscovitine down-regulated the phosphorylation level of CDK2 Thr-160 and CDK1 Thr-161 at specific concentration (5μmol / L). The apoptosis rate of Roscovitine significantly increased after treated with rhFasL. CONCLUSIONS: Fas-mediated apoptosis is cell-cycle-specific and begins in late G1 phase, which is the result of the decrease of cyclin E / CDK2 activity in G1 phase and the surveillance of late G1 phase. The cyclin D3 / CDK complex may be The key to determine whether apoptosis occurs.