Mechanistic Studies Of P53 Mitochondrial Translocation In Transcriptional-Independent Apoptotic Pathway

P53 protein is a core protein of cellular homeostatic mechanism. P53 controls the cellular processes and the start of the cell cycle. P53 detect cellular damage signal to raise repair factors for DNA damage. P53 also can induce apoptosis to avoid immortalized cells into cancer. Accordingly, p53 is an important tumor suppressor gene. From the perspective of apoptosis, cancer immortalize precisely because the apoptosis mechanism is suppressed, leading to the cells which should be initiated apoptotic program not be cleared. As an important tumor suppressor gene, p53 was found mutation in more than half of the tumor cell lines. This mutation p53 can not travel its important pro-apoptotic as a tumor suppressor gene. Studies have shown that p53-induced apoptotic pathway include transcriptional-dependent apoptotic pathway and transcriptional-independent apoptotic pathway. P53 transcriptional-dependent apoptotic pathway has been studied more thorough. In recent years, studies have shown that p53-induced apoptosis is not entirely dependent on the transcriptional regulation. At present, p53 transcriptional-independent apoptotic pathway has become a hot topic of p53 pathway. It is important for p53 that may be a new method of cancer therapy.Purpose: Explore the possible mechanistic studies of p53 mitochondrial translocation in transcriptional-independent apoptotic pathway.Research content: By exogenous stimulation, p53 migrate to the mitochondria whether or not. How long after exogenous stimulation, p53 can be transferred to the mitochondria. And which part of the p53 domain regulates the migration to the mitochondria. And verify that the direct effect of purified p53 protein on mitochondria in vitro.Research methods: Firstly, construct plasmid of p53-related. Secondly, induce p53 protein expression and finish the purification of p53 protein. Thirdly, observe the direct effect of p53 protein on the mitochondria by cell-free system in vitro. In vivo,HCT116 wide type cell lines were treated with 5-Fu, and detect the migration of endogenous p53 to the mitochondria by western blot. In HCT116 null cell lines, aftertransient transfect of p53-related plasmid, cells were treated with 5-Fu, and detect the migration of exogenous p53 to the mitochondria by western blot.Result: Successfully constructed a series of p53-related plasmid. Successfully induced the expression of p53 protein and purified the p53 prokaryotic protein. The cell-free results showed that after p53 protein was added to the mitochondria suspension, the outer mitochondrial membrane was rupture. Western blot results showed that after 5-Fu treated HCT116 wide type cells, the endogenous p53 gradually accumulate in the mitochondrial, and the amount of protein increases with time. After5-Fu treated HCT116 null cells which transient transfect of p53-related plasmid, the exogenous p53 gradually accumulate in the mitochondrial. After 5-Fu treated HCT116 null cells which transient transfect of p53 segment plasmids, some of them may migrate to the mitochondria.Conclusion: P53 can induce mitochondria rupture in vitro. In vivo, when subjected to exogenous stimulation, endogenous p53 rapid accumulate in the whole cell, and migrate to the mitochondria and accumulation in 6 hours. Exogenous p53 can migrate to the mitochondria without exogenous stimulation. And N-terminal domain and DBD domain on p53 might regulate the migration of p53 to the mitochondria.