微管抑制及尾桥蛋白磷酸化促进骨肉瘤细胞U2OS微核形成

Microtubule Inhibition and Gephyrin Phosphorylation Promote Micronucleus Formation in Osteosarcoma Cell Line U2OS

目的探讨微管及微管相关蛋白尾桥蛋白在肿瘤细胞微核形成中的作用。方法利用DAPI染色检测不同类型细胞自发微核率;利用诺考达唑(nocodazole)抑制微管聚集后,DAPI染色和胞质分裂阻滞(cytokinesis-block,CB)微核实验观察人骨肉瘤细胞株U2OS中微核形成情况;免疫荧光多重染色检测诺考达唑处理不同时间对U2OS细胞中微管及尾桥蛋白的影响;在U2OS细胞中过表达尾桥蛋白或利用collybistin使尾桥蛋白磷酸化,再利用免疫荧光及DAPI染色检测其对微管及微核形成的影响。结果相对于人胚肾HEK293细胞和原代神经细胞,U2OS自发微核率更高;诺考达唑处理使U2OS细胞微核数量增加,且与释放时间相关;诺考达唑处理使尾桥蛋白分布改变,逐渐聚集于细胞核;U2OS细胞中磷酸化尾桥蛋白主要表达于分裂期细胞;过表达尾桥蛋白和(或)增加其磷酸化水平并不影响U2OS细胞中微管蛋白的表达,但过表达尾桥蛋白并增加其磷酸化水平可使微核数量增多。结论肿瘤细胞内微核形成与微管聚集能力关系密切,可形成于细胞周期不同时期,具有积累效应。尾桥蛋白磷酸化后细胞中微核数量增多,提示其可能参与调节肿瘤细胞微核形成。

Objective To investigate the role of microtubule(MT)and MT associated protein gephyrin in micronucleus formation in tumor cells.Methods DAPI staining was used to detect the spontaneous micronucleus formation rate of different cell types.DAPI staining and cytokinesis-block micronucleus(CBMN)assay were applied to explore the micronucleus formation of U2OS cells.Double labelling immunofluorescence was used to detect the expression of microtubules and gephyrin in U2OS cells at different time points after nocodazole treatment.In U2OS cells,in which gephyrin was overexpressed or gephyrin was phosphorylated by collybistin,microtubule formation and micronucleus formation were examined by immunofluorescence and DAPI staining.Results Compared with human embryonic kidney HEK293 cells and primary nerve cells,osteosarcoma cell line U2OS had the highest spontaneous micronucleus formation rate.Nocodazole treatment increased the number of micronuclei in U2OS cells,which was correlated with the release time of nocodazole.The endogenous expression of phosphorylated gephyrin was predominantly observed in dividing U2OS cells.Neither transfection of gephyrin plasmid nor co-transfection of gephyrin plasmid and collybistin plasmid affected the expression of microtubules in U2OS cells,yet increased the number of micronuclei.Conclusion The formation of micronuclei in tumor cell is closely related to the ability of MT assembly,which can be formed at different periods of cell cycle and has an accumulation effect.Gephyrin phosphorylation could increase the number of micronuclei,thus we speculated that gephyrin may be involved in regulation of micronucleus formation.