The Potential Function Of 53BP2 In Cell Cycle And Apoptosis Regulation And The Preliminary Study Of HDIs-sensitivity Function By Radiosensitizer In Tumor Cells

Cell homeostasis involves in damage monitor,cycle regulation,damage repairation and apoptosis induction in long-term evolution.One hand ,damage is repaired by inducing,recognizing and starting complicated repair mechanism;in another hand,cell cycle is delayed or blocked by the activity mechanism of cell cycle check-point induced by DNA damage stress.The stressing reaction of DNA damage is basis of maintaining stability of genome,and is correlated with nosogenesis and development of many human diseases. Research into cell homeostasis has led to the possibility of new therapeutic approaches to human tumor.How to promote tumor cells apoptosis by finding suitable targets and methods is a key purpose in tumor therapy.The tumour suppressor protein p53 was first described in 1979, and ten years later, it was identified as a tumour suppressor. It is well known that p53 is not functional or functions incorrectly in most human cancers, and that it plays a crucial role in the prevention of tumour development. p53 is a sequence-specific nuclear transcription factor that binds to defined consensus sites within DNA as a tetramer and affects the transcription of its target genes, including more than up-regulated 100 genes and about down-regulated 60 genes. p53 enables the repair of damaged DNA by programmed cell death or cell cycle arrest and the damaged cells beyond repair are eliminated,thus preventing the fixation of DNA damage as mutations.All of above,p53 is a gatekeeper in cell homeostasis.What determines the choice of p53 between growth arrest and apoptosis is unknown.53BP2 was generally identified by its ability to interact with wild type (not mutation)p53 in a yeast two-hybrid assay.TP53BP2 gene is a single copy gene and has been mapped on the long arm of chromosome 1 at q42.1.The gene encoded two mRNA species by alternative splicing:53BP2S(Bbp) and 53BP2L(ASPP2). As a cofactor of p53, 53BP2 was paid close attention for its role in pro-apoptosis. It has been identified that 53BP2 can promote p53 only to transactivite pro-apoptosis genes,but not cycle-arrest like genes.53BP2 can interact with wtp53,p63,p73,Bcl-2,NFκB's p65 subunit,PP1,APCL,YAP,APP-BP1,Hapatitis C virus core protein and so on.In the first part of this thesis,we mainly discuss the role of 53BP2 in cell apoptosis and cell cycle.Histone deacetylase inhibitors (HDAC inhibitors, HDIs) have been tested as antitumor drugs recently.They can induce cell cycle arrest, stimulate differentiation, and provoke apoptosis in tumors as reported. The naturally occurring anti-fungal antibiotic TSA is one of the first HDAC inhibitors identified as an antiproliferative agent, and induces many cancer cells apoptosis and/or cycle arrest.It has been known that the transactivity of HDIs to p21WAF1/CIP1 depends on the kinase activity of ATM ( ataxia telanglectasia muted);PIKK inhibitor such as Wortmannin and caffeine can inhibit activity of ATM/ATR . We plan to use Wortmannin and caffeine as radiosensitizer with HDIs(TSA,SAHA)on tumour cells(Molt-4,HeLa)for the research of the meaning of DNA damage signalling net induced by ATM and ATR in tumour cell apoptosis induced by HDIs.In this thesis, efforts were made on two aspects: First,we established 53BP2 over-expressed and knock-down cell lines to detect the 53BP2 effect on the expression level of p53 and survivin, and to observe the change of cell cycle and apoptosis by Flow cytometry,Western blot,RT-PCR and Dual-luciferase Reporter Assay. Secondly,we need to confirm weather PIKK inhibitors can enhance tumour apoptosis induced by HDIs. The results obtained as follows:1) The plasmid including full-length 53BP2 was constructed and the 53BP2 stably knock-down cell line was established.2) The over-expression of 53BP2 can increase the apoptosis of Hela with wild-type p53,but cannot influence the apoptosis of H1299 with null p53.These results showed the important of p53 in the pro-apoptosis pathway of 53BP2 .3) The expression level of survivin increased in 53BP2 knock-down cells,but 53BP2 had no effect on transcription of survivin.4) The expression level of p53 decreased in 53BP2 knock-down cells afterγ-ray radiation for 12h, but it increases obviously in control cells. Meanwhile,the G2/M arrest in 53BP2 knock-down cells is longer than that in control cells.The results showed that 53BP2 in DNA damage signalling pathway maybe a key factor not only in cell apoptosis but also in cell cycle.5) PIKK inhibitor such as Wortmannin and caffeine enhanced HDIs(TSA,SAHA) induced-tumour cell apoptosis obviously but not influenced that in nomal cells,especially,the apoptosis happening before inhibition of PIKK inhibitor to G2/M.In short, the two most significant results obtained in this thesis exhibited that(1) 53BP2 had the effect on cell apoptosis and cell cycle in HeLa cells and had the effect on the expression of survivin,a member of inhibitor apoptosis protein family; (2) the combination administration of HDIs and PIKK inhibitor in cancer therapy exhibited strong potentiality.