The Regulatory Mechanisms Of Multifunctional P100 Protein In Cell Cycle

Objective:The cell cycle is the most essential mechanism by which all living things reproduce. It's basic function is to duplicate accurately the vast amount of DNA in the chromosomes and then segregate the copies precisely into two genetically identical daughter cells.p100 is a multifunctional protein. It can participated in the gene transcriptional regulation of many transcription factors as a coactivator, and increase their transcriptional activity. Furthermore, the TSN domain of p100 protein interacts with snRNPs complexes, accelerate splicosome assembly. Since cell cycle is the basic process in organism at cell level, whose characteristic is DNA replication and related protein orderly transcription; while p100 protein is the important regulator of gene transcription, the basic events at molecular level, it is naturally for us to assume that p100 protein probably participate in the regulation of cell cycle, in turn affect the biological process in cells. The object of this experiment is to figure out the impact of p100 protein on cell cycle, and to study the molecular mechanism.Methods:The experiment is divided into three parts:in partⅠ, we isolate lymphocytes and granulocytes from the PHA, and get the different tissues from mouse, then detect the expression of p100 in different kinds of cell groups. In part II, we study the impact and mechanisms of p100 protein on cell cycle.First,we transfect cells with siRNA and pSG5-p100-FLAG plasmids,detect the distribution of cell cycle by flow cytometry assay.Then,synchronize the three groups of cells in G1/S phase or G2/M phase by thymidine double block or Nocodazole respectivly,the cell cycle distribution was detected by flow cytometry after the cells release from the arrest. In partⅢ, we observe the character of p100 in different cell cycle phases.Three tests were included in this part. After synchronize the cells in different phases, first we detect the expression of p100 in protein and mRNA level.Then we use the immunoprecipitation technique to investigate the modification of p100.Third, observe the loacation change through immunofluorescence.Result:①p100 is highly expressed in lymphocytes,while less frequently and intensely expressed in granulocytes.n the tumor cell lines which belong to the proliferating-cell population, p100 is highly expressed; In the liver,spleen,kidney tissues which break away from cell cycle temporarily,p100 also express intensely.In the end cell lines,such as granulocytes,muscle cells and neurocytes,p100 is rarely expressed.③Silencing p100 expression resulted in a decreased percentage of cells in S phase and an increased percentage of cells in G1 phase. On the other hand, overexpression of p100, led to an increased percentage of cells in S phase④Released cells from the G1/S phase or G2/M phase arrestment,silencing p100 expression led more cells stopping in the G1 phase,while overexpress it,resulted in little changes in cell cycle profile.⑤p100 can intensely expressed in every phase during the cell cycle,and only a little less in G1/S phase.⑥rrest cells in G1/S phases, the phosphoralation of p100 decreased dramatically compared to other phases。⑦The location of p100 changes dynamicly during the cell cycle progression,the same to the biological behavior of nucleolus.Conclusion:①p100 is highly expressed in cells that are in the cell cycle, while cells are out of cell cycle, p100 disappear. It indicates that p100 has functional link to the cell cycle.②p100 promotes the progress of cell cycle from G1 phase to S phase. Silencing p100 expression can result in the Gl phase arrest.③p100 can intensely expressed in every phase during the cell cycle, the phosphoralation of it decreased dramatically when cells were stopped in G1/S phase.④p100 may has functional link to the nucleolus, it's location changes dynamicly in different phases just like the behavior of the nucleolus.