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）是一种抗癌剂，具有心脏毒性的副作用。然而，阿霉素诱导的心肌凋亡的精确分子机制仍然未知。我们以前已经报道了细胞周期调节剂之一的细胞周期蛋白A/CDK2激酶活性是介导的缺氧诱导的心肌细胞凋亡。由于凋亡通常在增殖细胞的细胞质中开始，因此我们测试了细胞周期蛋白A的亚细胞定位确定凋亡命运的假设。将原发性大鼠心肌细胞暴露于阿霉素中，并通过TUNEL方法和可行细胞的数量评估凋亡剂量依赖性剂量依赖性剂量（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6小时治疗后积累了最大反应。同样，阿霉素通过组蛋白-H1激酶测定法中细胞周期蛋白A和CDK2相关激酶的活性增加。通过IMMNO组织化学，Cyclin a在心肌细胞中是细胞质，但是，在成纤维细胞增殖状态下，细胞周期蛋白A是核的。为了测试细胞周期蛋白A相关激酶是否是凋亡的效应子，心肌细胞被显性阴性CDK2腺病毒感染（DNCDK2）。阿霉素（10-6M）的凋亡显着降低（续4.8±1.8％，dox。46.2±4.0％，dox+dncdk2 25.8±6.0％），表明环氧蛋白A/CDK2激酶活性在doxorubiubicin staus opopopopopopopopopopsiss中起着显着的疾病。总之，这些发现证实了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中的凋亡。