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The Subcellular Localization of cyclin A Regulates Apoptosis in Cardiomyocytes

细胞周期蛋白 A 的亚细胞定位调节心肌细胞凋亡

基本信息

批准号：
15590727
负责人：
ADACHI Susumu
金额：
$ 1.79万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590727/
关键词：
cardiomyocytes apoptosis nitricoxide cyclin caspase S-nitrosylation 酸化窒素サイクリンA

项目摘要

Doxorubicin (Dox) is an anticancer agent which has the side effect of cardiac toxicity. However, the precise molecular mechanism of doxorubicin-induced myocardial apoptosis is still unknown. We have previously reported that cyclin A/cdk2 kinase activity, which is one of the cell cycle regulators, is mediated hypoxia-induced apoptosis in cardiomyocytes. Because apoptosis typically begins in the cytoplasm in the proliferating cells, we tested the hypothesis that the subcellular localization of cyclin A determines the apoptotic fate. Primary rat cardiomyocytes were exposed to doxorubicin and increased apoptosis dose-dependently (10-5 to 10-7M) evaluated by TUNEL method and the number of viable cells. The cyclin A protein level assessed by immunoblot analysis accumulated with a maximum response after 6 hours treatment in cardiomyocytes. Also, doxorubicin increased in the activity of cyclin A-and cdk2-associated kinase by histone-H1 kinase assay. By immnohistochemistry, cyclin A was cytoplasm in cardiomyocytes, however, cyclin A was nuclear in proliferating condition of fibroblasts. To test whether the cyclin A-associated kinase was the effector for apoptosis, cardiomyocytes were infected by dominant-negative cdk2 adenovirus (dncdk2). The apoptosis by doxorubicin(10-6M) was significantly reduced (cont. 4.8± 1.8%, Dox. 46.2±4.0%, Dox+dncdk2 25.8±6.0%), suggesting that cyclin A/cdk2 kinase activity play significant roles in the doxorubicin induced apoptosis. In conclusion, these findings confirm that the activation of cyclin A in cytoplasm mediates doxorubicin-induced apoptosis in cardiomyocytes.

阿霉素（Dox）是一种抗癌药，具有心脏毒性的副作用。然而，阿霉素诱导心肌细胞凋亡的确切分子机制仍不清楚。我们之前报道过细胞周期调节因子之一的细胞周期蛋白 A/cdk2 激酶活性介导缺氧诱导的心肌细胞凋亡。由于细胞凋亡通常始于增殖细胞的细胞质，因此我们测试了细胞周期蛋白 A 的亚细胞定位决定细胞凋亡命运的假设。原代大鼠心肌细胞暴露于阿霉素，通过TUNEL方法和活细胞数量评估，细胞凋亡呈剂量依赖性增加（10-5至10-7M）。通过免疫印迹分析评估的细胞周期蛋白 A 蛋白水平在心肌细胞中处理 6 小时后积累了最大反应。此外，通过组蛋白 H1 激酶测定，阿霉素可增加细胞周期蛋白 A 和 cdk2 相关激酶的活性。免疫组织化学显示，心肌细胞中cyclin A位于细胞质，而成纤维细胞增殖状态时，cyclin A位于细胞核。为了测试细胞周期蛋白 A 相关激酶是否是细胞凋亡的效应子，心肌细胞被显性失活 cdk2 腺病毒 (dncdk2) 感染。阿霉素(10-6M)引起的细胞凋亡显着减少(cont. 4.8± 1.8%, Dox. 46.2±4.0%, Dox+dncdk2 25.8±6.0%)，表明cyclin A/cdk2激酶活性在阿霉素诱导的细胞凋亡中起重要作用。总之，这些发现证实细胞质中细胞周期蛋白 A 的激活介导阿霉素诱导的心肌细胞凋亡。