Disruption of cell cycle kinetics and cyclin-dependent kinase system by ethanol in cultured cerebellar granule progenitors
Disruption of cell cycle kinetics and cyclin-dependent kinase system by ethanol in cultured cerebellar granule progenitors
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DOI：
10.1016/s0165-3806(01)00294-2
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发表时间：
2001-12-14
期刊：
影响因子：
--
通讯作者：
Luo, J
中科院分区：
文献类型：
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作者：
Li, Z;Lin, H;Luo, J
本研究采用小脑颗粒前体细胞（CGPs）原代培养的神经元前体细胞体外模型，探讨乙醇诱导的细胞周期损伤的机制。CGP培养物由3日龄大鼠产生。乙醇显著抑制CGPs的增殖。通过累积5-溴-2 '-脱氧尿苷（BrdU）标记技术进行的细胞周期动力学分析表明，乙醇暴露增加了细胞周期的持续时间，并降低了生长分数（循环群体）。S期和总细胞周期的持续时间显着延长乙醇暴露分别为220%和135%，而生长分数从对照组的44%下降到22%，在乙醇暴露的文化。细胞周期蛋白依赖性激酶2（Cyclin-dependent kinase 2，Cdk 2）是调控细胞从G1期进入S期和S期进程的关键蛋白。体外磷酸化实验和Western blot结果表明，乙醇显著下调Cdk 2的活性和表达。此外，乙醇显著降低Cyclin A和Cyclin D-2的表达。原位TUNEL法和DNA片段化酶联免疫吸附法进一步研究表明，乙醇引起的细胞凋亡是一种延迟性的，即乙醇诱导的细胞凋亡只有在慢性暴露后才明显。另一方面，乙醇不影响坏死指数。总之，乙醇通过诱导细胞周期延迟和促进细胞凋亡来减少CGPs的循环池。乙醇介导的细胞周期蛋白依赖性激酶系统的紊乱可能是神经元前体细胞细胞周期阻滞的一个重要机制。(C)2001爱思唯尔科技有限公司。保留所有权利。
An in vitro model of neuronal precursors, primary culture of cerebellar granule progenitors (CGPs), was used to investigate the mechanisms underlying ethanol-induced cell cycle damage. The CGP cultures were generated from 3-day-old rats. Ethanol significantly inhibited the proliferation of the CGPs in culture. Analysis of cell cycle kinetics by a cumulative 5-bromo-2'-deoxyuridine (BrdU) labeling technique demonstrated that ethanol exposure increased the duration of the cell cycle and decreased the growth fraction (the cycling population). The duration of the S-phase and total cell cycle was significantly prolonged by ethanol exposure by 220% and 135%, respectively, while the growth fraction was decreased from 44% in the control groups to 22% in the ethanol-exposed cultures. Cyclin-dependent kinase 2 (Cdk2) is a key protein that regulates both the passage from G1 into S, and the S phase progression. The results from in vitro phosphorylation assay and Western blot demonstrated that ethanol dramatically down-regulated both the activity and the expression of Cdk2. In addition, ethanol significantly decreased the expression of Cyclin A and Cyclin D-2. Further studies using in situ TUNEL assay and DNA fragmentation ELISA showed that ethanol caused a delayed apoptosis, i.e, the ethanol-induced apoptosis was evident only after chronic exposure. On the other hand, ethanol did not affect the necrotic index. In conclusion, ethanol decreases the cycling pool of CGPs by inducing cell cycle delay and promoting apoptosis. Ethanol-mediated disturbance of the cyclin-dependent kinase system may be an important mechanism to account for cell cycle arrest in neuronal precursor cells. (C) 2001 Elsevier Science B.V. All rights reserved.