The Subcellular Localization of cyclin A Regulates Apoptosis in Cardiomyocytes

阿霉素（Dox）是一种具有心脏毒性副作用的抗癌药物。然而，阿霉素诱导心肌细胞凋亡的确切分子机制尚不清楚。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，因此我们验证了细胞周期蛋白A的亚细胞定位决定细胞凋亡命运的假设。采用TUNEL法观察阿霉素对原代大鼠心肌细胞凋亡的影响，观察细胞凋亡呈剂量依赖性（10-5 ~ 10-7M）和活细胞数的增加。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治疗6小时后积累，达到最大反应。此外，通过组蛋白h1激酶测定，阿霉素增加了细胞周期蛋白a和cdk2相关激酶的活性。免疫组化结果显示，细胞周期蛋白A在心肌细胞中为细胞质，而在成纤维细胞增殖状态下为细胞核。为了检测细胞周期蛋白a相关激酶是否为细胞凋亡的效应因子，我们用显性阴性cdk2腺病毒（dncdk2）感染心肌细胞。阿霉素（10-6M）可显著降低细胞凋亡（对照值为4.8±1.8%，对照值为Dox. 46.2±4.0%，对照值为Dox+dncdk2 25.8±6.0%），提示细胞周期蛋白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，这些发现证实了细胞质中细胞周期蛋白A的激活介导了阿霉素诱导的心肌细胞凋亡。

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.