The Effects And Mechanisms Of IASPP Inhibiting The Apoptotic Pathway Of The P53Family

BackgroundThe p53protein is one of the best-known tumor suppressors. The p53protein familyconsists of three transcription factors: p53, p63, and p73. These proteins share significantstructural and functional similarities and each has unique biological functions as well.Compared to p53, the regulation of p73and p63activities is less understood but is likely to beas complex. Many years of research have uncovered an impressive number of p53-interactingproteins, but much less is known about protein interactions of p63and p73.The discovery of the apoptosis stimulating protein of p53(ASPP) family of proteinsrevealed a new level of p53regulation. iASPP belongs to the ASPP family consisting of threeproteins(ASPP1, ASPP2and iASPP), which are unified by their protein structure. ASPP1andASPP2selectively stimulate the transactivation function of all three p53family members p53,p63, and p73, increasing the transactivation of proapoptotic genes. In contrast, iASPP is anoncoprotein that specifically inhibits p53,and the most important tumor suppression functionof p53is its ability to induce apoptosis. The mechanism of iASPP suppressing the cellapoptotosis is through inhibiting the transactivation function of p53on the promoters ofproapoptotic genes. Then, how iASPP influences cell apoptotosis in the tumor cells withp53-deficiency remains unclear? To answer this question, we investigated the transactivationfunction of iASPP, iASPP RNAi, p63, p73on the promoters of Bax and Puma in the p53wide,null and mutational cells:U2OS, H1299and SW480. This study showed the functionof iASPP in the p53/p63/p73apoptosis pathway and provided a new thinking for the treatmentof cancers with p53loss or mutation.Methods:1. The iASPP RNAi plasmid was transfected into p53wild,null, mutational cell line,U2OS,H1299,SW480, respectively.Apoptosis was determined by cytofluorimetry using theAnnexin V-FITC Apoptosis detection kit and DNA fragmentation was detectable by DNA ladder extraction kit.2. The proliferation effects of cancer cells trandfected iASPP plasmid and iASPP RNAiplasmid were observed by CCK8kit.3. To detect the expression of iASPP in the cell before and after transfected usingWestern-blot analysis.4. The target fragments of Bax/Puma promoters were amplified by RT-PCR method fromHepG2cells and the fragments were inserted into the pGL3-basic luciferase reporter vector.The acquired Bax-Luc plasmid and Puma-Luc plasmid were transfected into U2OS,H1299,SW480cells, respectively and their activity was detected using the Luciferase Assay.5. Various combinations of plasmid DNA including reporters, p63and p73weretransfected into U2OS,H1299, SW480cells, respectively.The cells were assayed using theLuciferase Assay kit48hours after transfection.6. The protein products of iASPP, p53, p63and p73were produced in vitro using TheTNT T7Quick coupled Transcription/Translation System. The interaction of iASPP withp53/p63/p73in vitro was determined by immunoprecipitation and Western blot analysis.7. The interaction of iASPP with p53/p63/p73in vivo and was determined by celltransfection, immunoprecipitation and Western blot analysis.8. iASPP can inhibit the DNA Binding Function of p63and p73on the promoter ofproapoptotic genes. We studied the DNA binding activity of p63/p73on the promoters of Baxand PUMA in vivo using the chromatin immunoprecipitation technique.9. Apoptosis in tumor mass from BALB/c-nu detected by TUNEL assay. For in situdetection of DNA fragmentation in paraffin-embedded tissue sections, the TUNEL methodwas performed using the ApopTag1peroxidase kit,10. Expression of iASPP in tumor mass was detected by Real-Time quantitative PCRResult:1. iASPP can inhibit apoptosis independently of p53in tumor cells. In our previous study,we noticed that inhibiting the expression of endogenous iASPP can increase cell apoptosis notonly in cells with wild-type p53, but also in the p53null and mutational cells, suggestingthat iASPP may inhibit apoptosis independently of p53.2. iASPP can interact with the DNA binding domain of p53and inhibit its apoptoticfunction. As the most homologous region among all p53family members is their DNA binding domain, iASPP could also interact with p63and p73and influence their apoptoticfunction.3. iASPP suppressing the cell apoptotosis is through inhibiting the transactivationfunction of p53on the promoters of proapoptotic genes. The results indicated that iASPPinhibited apoptosis independently of p53in tumor cells mainly by inhibiting thetranscriptional activity of p63/p73on the promoters of proapoptotic genes.4. The transactivation function of proapoptotic genes Bax and Puma was inhibited afteriASPP plasmid was transfected into H1299,U2OS,SW480cells and the transactivationfunction of Bax and Puma was enhanced after iASPP RNAi plasmid.5. The transactivation function of proapoptotic genes Bax and Puma was enhanced afterp53/p63/p73was transfected into H1299,U2OS,SW480cells.6. iASPP can interact with p53/p63/p73in vitro and in vivo. When the expression ofiASPP was interfered,does not affect the protein expression of p53,p63and p73.7. The number of apoptosis cells increased obverously after transfected iASPP RNAiplasmid in tumors of animal models.8. The expression levels of ASPP mRNA after RNAi are reduced in tumors.Conclusion:1. Inhibiting the expression of endogenous iASPP can increase cell apoptosis not only incells with wild-type p53, but also in the p53null cells, suggesting that there is an apoptosispathway independent of p53in the p53null and mutation cells.2. iASPP can inhibit the transactivation function of proapoptotic genes such as Bax andPuma in the p53cells.3. p53,p63and p73can enhance the transactivation function of proapoptotic genes suchas Bax and Puma in the p53cells.4. iASPP can interact with p53,p63and p73in vitro and in vivo and iASPP does notaffect the protein expression of p53,p63,p73.5. iASPP maybe through inhibiting the transactivation function of p53/p63/p73on thepromoters of proapoptotic genes and induced cell apoptosis.6. iASPP inhibited apoptosis independently of p53in tumor cells mainly by inhibitingthe transcriptional activity of p63/p73on the promoters of proapoptotic genes.7. iASPP can promote on the development of tumor in nude mice model 8. There is also an important functional relationship between the p53family members: inthe absence of p53, p63/p73dependent apoptosis will take over the fuction of p53.9．iASPP can promote on the development of tumor in nude mice model...