Mechanism Of Pin1 Regulates P53 Stability And P53-mediated Apoptosis In Tumor Cells Response To DNA Damage

One of the most important strategies for cancer therapy including radiotherapy and chemotherapy is that using DNA damage induced by genotoxins insult kills tumor cells through apoptotic and non-apoptotic death program. Therefore, the mechanisms of DNA damage to induce cell apoptosis are a study hotspot in cancer research. wtp53 is a determinant factor for cell fate in reponse to DNA damage. DNA damage triggers p53 multiple modifications,such as phosphorylation including on the Ser-/Thr-pro motif, acethylation, and deubiquitination, then activates p53 functions like inducing cell arrest, DNA damage repair and inducing cell apoptosis.New research progresses have showed that those "post-phosphorylation protein" involved in gene transcription, cell cycle regulation and cell signal trancduction must be isomerized by Pin1(peptidyl-prolyl cis-transisomerase, PPIase, Pin1), then they obtain or lose the functional comformation. To date, Pin1 is the only cis-transisomerase which can specifically recognize and isomerize the phosphorylated Ser/Thr-Pro bonds in certain proteins. Response to DNA damage, Kinases activted by stresses catalyze multiple Ser-/Thr sites in Ser-/Thr-Pro motif to be phosphorylated, and promote Pin1 to binding p53. Pin1 leads to a prolyl isomerization in p53 , then catalytically induces its conformational changes , enhances p53 stabilization and promotes its function activation.However, in the apoptotic responses induced by DNA damage, Pin1 shows either pro-apoptotic or anti-apoptotic functions. Zheng et al. show that Pin1 is required for maintaining the DNA-damage cell cycle checkpoint by inducing the cell cycle inhibitor p21^WAF/cip1, which will protect cells from DNA damage-induced cell death. In contrast, Zacchi et al. claim that Pin1 accelerates apoptosis by enhancing pro-apoptotic genes downstream of p53, such as Bax and DR2/Killer.Especially Pin1 is overexpressed in numerous tumor cells promoting cell proliferation and transformation, is a 'catalyst' for tumorgenesis and development. This function of Pin1 is opposite to tumor suppression function of p53. Moreover, in tumor cells p53 is phosphorylated by those kinases activated by oncogenic signal, maybe has the phosphorylated Ser-/Thr-Pro motif which is essential for Pin1 recognizing and binding. Thus, it is possible that Pin1 cooperates with oncogenes to regulte p53 function negatively in tumor cells. Studies have shown that Mdm2 is main negative regulator for p53. As a specific ubiquitination E3 ligase, Mdm2 mediates p53 ubiquitination and degradation, maintaining p53 at a low expression level in unstressed situation. So, our question is whether Pin1 accelerates p53 degradation by Mdm2 mediated ubiquitination? Do the Irradiation and Chemotherapy drugs like Cisplatin, Ariamycin have any effect on this negative regulation of Pin1? How to regulate p53 function by Pin1 in various apoptotic response induced by different DNA damage agents?How to regulate the interaction between p53 and Mdm2 by Pin1 response to DNA dmage? To answer these questions is important for learning the roles and mechanisms of Pin1 in regulating p53 functions in unstressd and stressed situations, also in cell apoptotic responses induced by different DNA damage agents. This study focused on the questions above, and makes some progresses.1. The cell model stably suppresseing Pin1 expression by RNA interference technique was constructed successfully.2. It was found that under unstressed conditions Pin1 may involve in accelerating p53 degradation through regulating Mdm2 -mediated ubiquitination degradation in tumor cells.This impact of Pin1 is demonstrated here by the finding that knocking down Pin1 leads to an increase in p53 protein level and a decrease in p53-Mdm2 complex. Moreover, by CoIP tests using a specific p53 phosphorylated Ser315 antibody, it is found that p53 Ser315(-Pro316)was phosphorylated in nonstressed MCF-7 cells , and Pin1 and p53 formed an immunoprecipitated complex.3. On Cisplatin treatment Pin1 protected p53 from MDM2 mediated degradation, ia a important positive regulator for p53 stability. Pin1 knockdown led p53 atability and accumulation is severely impaired after Cisplatin treatment. CoIP test showed that after Cisplatin treatment 12h, the interaction between p53 and Mdm2 was inhibited significantly,it led to p53accumulation, but in Pin1 RNAi cells ,the amounts of Mdm2 combined by p53 was much more than control cells, p53 accumulation was impaired. This results indicate that DNA damage inhibits the negatively regulating p53 stability by Pin1,and enhances positive regulation by Pin1. Similar results was obtained from Irridiation.4. It was found that Pin1 RNAi directly impaired p53 transcriptional activity. It is demonstrated by using dual luciferase reporter system,. When exposure to Cisplatin and Irriadiation, endogenous p53 downstream genes, p21^WAF/cip1 and Bax expression decreased significantly in Pin1 RNAi cells. These results demonstrate that depletion of Pin1 impaired the transcriptional activation of p53 and reduced its downstream genes p21^WAF/cip1 and Bax expression after DNA damage.5. A DNA replication checkpoint impairment was observed in Pin1 RNAi cells after exposing to Cisplatin. Furthermore, a striking impairment in the apoptotic response was also observed following Cisplatin treatment. In Pin1 RNAi cells the number of apoptotic nuclei was reduced by about 50% as compared to the counterpart. Those results suggest that knockingdown Pin1 significantly suppresses apoptosis induced by Cisplatin in MCF-7.6. p53 regulation depends on the integration of various damage-sensing pathways, the final outcome will be influenced by the type and the intensity of DNA damage, as well as by cell type. we found that on Ariamycin treatment p53 accumulation was not impaired but enhanced, and apoptosis also increased about 10% in Pin1 RNAi cells.7. P53-Survivin pathway is important for Adriamycin induced apoptosis, and inhibitor of apoptosis protein survivin expression is transriptional repressed by p53. We found that in Pin1 RNAi cells survivin expression level significantly decreased, but survivin mRNA had no detectable change. Meanwhile, we found survivin half-lift was shorter in Pin1 RNAi cells. This indicates that Pin1 can enhance p53 stability in tumor cells, maybe this is a mechanism for Pin1 to play an anti-apoptotic function, and for sensitizing Adriamycin-induced apoptosis in Pin1 RNAi cells.In sum, our results revealed that Pin1 is an important regulator of p53 stability under unstressed or stressed conditions in tumor cells. On one hand, Pin1 accelerates apoptosis by enhancing p53 stability and transcriptional activity; on the other hand, under unstressed conditions Pin1 invovles in accelerating Mdm-mediated p53 degradation. In addition, Pin1 can enhance inhibitor of apoptosis protein survivin stability. These results presented a mechanism for Pin1 to regulate p53 stability and transcriptional activty in response to DNA damage in tumor cells, it will be helpful in Learnning the functionsof Pin1 in tumor cells and valuable in directing the cancer therapy.