B-Myb Participates In Ionizing Radiation-induced Apoptosis And Cell Cycle Arrest In Human Glioma Cells

Objective:The purpose of this study is to preliminarily investigate the role of B-Myb in apoptosis and cell cycle arrest induced by ionizing radiation in human glioma cells and its regulatory mechanism.Methods:U87 and U251 cells were treated with ionizing radiation.The expression levels of transcription factor B-Myb and its phosphorylation form were detected by Western blot and immunofluorescence.The distribution of B-Myb was confirmed by nuclear-plasmid separation experiments.Flow cytometry was used to detect cell cycle and apoptosis,as well as the expression of cell cycle-related proteins such as cyclin B1,cyclin A and CDK 2 in both cells.After decreasing the level of B-Myb protein by small interfering RNA transfection,the expression of cell cycle-related proteins,cell cycle distribution and apoptosis were determined.Two different p53 mutant plasmids were used to transfect H1299 cells.The alternations of B-Myb and p-B-Myb were detected after ionizing radiation.Immunoprecipitation determined the interaction between p53 and B-Myb.Chromatin immunoprecipitation was used to investigate the binding of SP1 on B-Myb promoter.Western blot and immunohistochemistry were used to confim the relationship between p53 status and B-Myb expression in clinical human glioma samples.Results:In this study 1)Ionizing radiation could induce the elevated-expression of B-Myb and p-B-Myb in U251 cells.2)B-Myb played a role in ionizing radiation-induced G2/M arrest and the expression of cell cycle-related proteins in U251 cells.Reducing B-Myb protein can increase the sensitivity of U251 cells to ionizing radiation,but have no significant effect on U87.3)The increase of B-Myb after ionizing radiation may relate to mutant p53.4)p53 and SP1 regulate the expression of B-Myb gene through different pathways in U251 cells.5)Analysis of clinical human glioma samples revealed that B-Myb,p-B-Myb and cell cycle-related proteins were highly expressed in the samples with mutant p53.Conclusion:Cells with different p53 states responded distinctly to ionizing radiation and transcription factor B-Myb played an important role in it.p53 and SP1 were involved in regulating the expression of B-Myb.