骨肉瘤细胞中KDM5C与有丝分裂期纺锤体组装检查点关系的研究

Association of KDM5C with SAC on mitosis of osteosarcoma

目的:探讨赖氨酸特异性去甲基化酶5C(lysine-specific demethylase 5C,KDM5C)在骨肉瘤细胞有丝分裂期的变化及其对有丝分裂期纺锤体组装检查点(spindle assembly checkpoint,SAC)的调控。方法:利用细胞周期同步化方法,收集不同骨肉瘤细胞系有丝分裂期各时间节点的细胞样品,利用Western blot检测各时间点KDM5C蛋白质含量的变化及其翻译后修饰与有丝分裂期进程的关系;并用特异性抑制剂CPI-455抑制KDM5C的去甲基化转移酶活性,利用Western blot检测有丝分裂期进程中抑制KDM5C的去甲基化酶活性对SAC相关标志蛋白细胞分裂周期蛋白-27(cell division cycle protein 27,cdc27)、周期蛋白B1(Cyclin B1)的影响。结果:在有丝分裂进程中,与SAC相关的标志蛋白cdc27的磷酸化滞后条带的位移变化和Cyclin B1的蛋白降解均与文献报道相似,但KDM5C蛋白含量无明显变化;KDM5C截短体表达未观察到明显的位移减慢或泛素化降解条带;抑制其去甲基化酶活性后,相较对照组,标志蛋白cdc27的去磷酸化曲线和Cyclin B1降解曲线未见明显变化。结论:在骨肉瘤细胞系有丝分裂期中,KDM5C的蛋白质含量不随有丝分裂期进程而改变,抑制其去甲基化酶活性不影响对SAC的灭活。

Objective:To investigate the alteration of Lysine-specific demethylase 5C(KDM5C) protein level and the regulatory effect of KDM5C on spindle assembly checkpoint(SAC),related mitotic progression of osteosarcoma cell lines. Methods:The various osteosarcoma cells in prometaphase were collected by synchronization. The alteration of KDM5C protein level and its potential posttranslational modification in mitosis were determined by Western blot. The effect of inhibitor CPI-455 on mitosis process was detected using Western blot. Results:In mitosis,the alterations of SAC related markers,cell division cycle protein 27(cdc27) and Cyclin B1,were similar to previous literatures,but KDM5C protein level had no obvious change. The shift and ubiquitination of KDM5C truncations were not observed by Western bolt. When demethylase activity of KDM5C was inhibited by CPI-455,SAC related markers,cdc27 and Cyclin B1,had no obvious change. Conclusion:Mitotic process of osteosarcoma cell and the SAC is unaffected by KDM5C protein level and its demethylase activity.