Effect And Mechanism Of PUMA On The Apoptosis Of Mouse Liver Cell In Vitro

PUMA (p53 up - regulated modulator of apoptosis) was discovered in colon carcinoma cells in 2001. PUMA expression causes rapid p53-dependent apoptosis and growth inhibition. It belongs to Bcl-2 family and consists of a BH3 domain. It is a member of BH3-only subgroups. BH3-only proteins sharing only the short BH3 interaction domain, by which they can bind to the pro-survival relatives to induce apoptosis. BH3-only proteins play a critical role in regulating apoptosis initiation through the mitochondria. BH3-only proteins function through multidomain Bcl-2 family members to induce apoptosis, either by antagonizing antiapoptotic proteins or by directly activating proapoptotic proteins.The basal protein expression level of PUMA is relatively low according to past researches. However, in response to a range of stress signals,(Such as the DNA-damage, hypoxia and serum deprivation). PUMA could be induced in a p53 dependent or independent manner, and shows its potent pro-apoptotic activity. PUMA, an essential initiator of programmed cell death, can transduct signals of distinct apoptotic stimuli such as above factors. PUMA is a downstream target of the p53 tumor suppressor gene.Previous studies in colon cancer cells showed that PUMA interacts with several antiapoptotic members of Bcl-2 family through its BH3 domain to induce apoptosis。PUMA initiates apoptosis partly by promoting Bax multimerization and mitochondrial translocation via dissociating Bax and Bcl-XL, thereby promoting Bax multimerization and mitochondrial translocation. Disrupting Bax Multimerization or Mitochondrial Localization could block PUMA-induced apoptosis. Therefore, it may be an important pathway of apoptosis.In our previous works, we found that the PUMA expression in the early phase of liver regeneration was down-regulated transcriptionally. What does this finding suggest? Whether or not there is a close relationship between puma gene and liver regeneration? What is its the regulatory mechanism?We are very interasted in these problems and hope to solve these problems by experiment in vitro and in vivo. This paper mainly investigates its function in PUMA-mediated apoptosis and its mechanisms in mouse liver cells in vitro. It may help to explain functions and molecular mechanisms of PUMA in hepatocyte and provide envidence for exploring its role in liver regeneration. The next, respecting PUMA induce apoptosis in most mammalian cells, many studies indicated that PUMA adenovirus have broad applied prospect in gene therapy. Therefore, an experimental study investigat- ing the biological effects,toxicity and adverse reactions about adenovirus and adenovirus vector would be necessary.This study consists of the following three parts:1. The packaging and preparation of adenoviruses expressing PUMA (Ad-PUMA) and Adenoviruses expressing GFP (Ad-GFP).2. The cellular biological function of BNL CL.2 cells after infected by Ad-PUMA.3. Molecular mechanisms of the effection of PUMA on mouse liver cells.The results indicated that the packaging and preparation of Adenoviruses expressing PUMA (Ad-PUMA) was available and effective expression in mouse liver cell BNL CL.2. PUMA could induce apoptosis in these cells and the number of cells at S stage displayed increasing tendency with the infection time. The caspase activation also seems to be dependent on these changes. Taking into account above factors, we then analyzed that PUMA induction affects the interactions between Bax and Bcl-XL, between PUMA and Bcl-XL, as well as the interactions between Bax and PUMA by immunoprecipitation.