The Regulative Mechanism Of IASPP On The Tranactivation Function Of Proapoptotic Genes In The Apoptosis Pathways Independent Of P53

Objective:The p53 protein is one of the best-known tumor suppressors. The recently identified ASPP family (apoptosis-stimulating protein of p53) can interfere with the working of p53. Among them, iASPP can inhibit the normal function of p53 as a suppressor. The mechanism of iASPP suppressing the cell apoptotosis is through inhibiting the transactivation function of p53 on the promoters of proapoptotic genes. Then, how iASPP influences cell apoptotosis in the tumor cells with p53-deficiency? To understand the mechanism of iASPP suppressing the cell apoptotosis in the p53 null cells, we constructed the Bax and Puma reporter plasmids, investigated the transactivation function of iASPP, iASPP RNAi, p63, p73 on the promoters of Bax and Puma. This study showed the function of iASPP in the p63/p73 apoptosis pathway and provided a new thinking for the treatment of cancers with mutated p53.Methods:1. The iASPP RNAi plasmid was transfected into p53 null cell line, H1299. The stable transfected cells were obtained by G418 screening. Apoptosis was determined by cytofluorimetry using the Annexin V-FITC Apoptosis detection kit.2. The target fragments of Bax/Puma promoters were amplified by RT-PCR method from HepG2 cells and the fragments were inserted into the pGL3-basic luciferase reporter vector. The acquired Bax-Luc plasmid and Puma-Luc plasmid were transfected into H1299 cell line and their activity was detected.3. Various combinations of plasmid DNA including reporters, iASPP and iASPP RNAi were transfected into H1299 cells. The cells were assayed using the Luciferase Assay kit 48 hours after transfection. 4. Various combinations of plasmid DNA including reporters, p63 and p73 were transfected into H1299 cells. The cells were assayed using the Luciferase Assay kit 48 hours after transfection.5. The protein products of iASPP, p63 and p73 were produced in vitro using The TNT T7 Quick coupled Transcription/Translation System. The interaction of iASPP with p63/p73 in vitro was determined by immunoprecipitation and Western blot analysis.6. The interaction of iASPP with p63/p73 in vivo was determined by cell transfection, immunoprecipitation and Western blot analysis.Results:1. iASPP can decrease cell apoptotosis in the p53 null H1299 cells. This pathway of iASPP suppressing the cell apoptotosis is not p53 mediated apoptosis pathway and it maybe a p53-independent pathway.2. The cloning of human Bax/Puma gene promoters and the construction of their reporter vectors were successful. The biological activity of Bax/Puma reporter plasmids were verified by cell transfection and luciferase activity detection.3. The transactivation function of proapoptotic genes Bax and Puma was inhibited after iASPP plasmid was transfected into H1299 cells and the transactivation function of Bax and Puma was enhanced after iASPP RNAi plasmid was transfected into H1299 cells.4. The transactivation function of proapoptotic genes Bax and Puma was enhanced after p63/p73 was transfected into H1299 cells.5. iASPP can interact with p63 and p73 in vitro and in vivo.6. iASPP does not affect the protein expression of p63 and p73.Conclusion:1. There is an apoptosis pathway independent of p53 in the p53 null H1299 cells.2. iASPP can inhibit the transactivation function of proapoptotic genes such as Bax and Puma in the p53 null H1299 cells.3. p63 and p73 can enhance the transactivation function of proapoptotic genes such as Bax and Puma in the p53 null H1299 cells.4. iASPP can interact with p63 and p73 in vitro and in vivo. 5. iASPP does not affect the protein expression of p63 and p73.6. iASPP maybe through inhibiting the transactivation function of p63/p73 on the promoters of proapoptotic genes and induced cell apoptosis.