Microtubule Inhibition and Gephyrin Phosphorylation Promote Micronucleus Formation in Osteosarcoma Cell Line U2OS
Objective To investigate the role of microtubule(MT)and MT associated protein gephyrin in micronucleus for-mation 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 phos-phorylated 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 pre-dominantly 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 micronu-clei.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.
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