The Role Of Caspase Mediated IASPP Cleavage In Apoptosis

iASPP is a novel oncogene identified recently. The best studied iASPP functionis that iASPP can inhibit apoptosis by negative regulation of transcriptionalactivities of p53family members. Nevertheless iASPP was first reported to bindwith and inhibit NF-κB. However the effect of the interaction between iASPP andNF-κB has not been understood well. Furthermore, nuclear localization is aprerequisite for its well known functions dentified so far, however, the regulatorymechanisms of iASPP localization remains clarify. Our previous data showed thatactivated caspase could mediate iASPP cleavage at the early stage of apoptosis,suggesting that caspase mediated cleavage might play a role in regulating iASPPactivity and subsequent apoptosis progression. Based on our previous data, we usedthe NF-κB constitutively activated tumor cells as a model to investigate the effect ofcaspase-mediated iASPP cleavage on the cellular localization of iASPP and therelationship between iASPP cleavage and NF-κB regulated apoptosis progression.We first examined the effect of caspase mediated iASPP cleavage onapoptosis progression, cell cycle distribution by using Annexin V-FITC and PIstaining, PI staining followed by FACS assay. We also made a further step in themechcanism behind these phenomenon. We use the immunofluorescence cellstaining to investigate whether caspase mediated iASPP cleavage change iASPP’scelluler localization. Luciferase reporter assay was then used to understand theeffect of iASPP cleavage on the transcriptional activities of NF-κB. Finally, we usedRT-PCR to study the effect of iASPP cleavage on the NF-κB target genes whichregulates apoptosis and cell cycle arrest.Our data showed that WT-iASPP, cleaved iASPP fragements have no obviousimpact on cell cycle. Although iASPP-(295-828) significantly promotes apoptosis.NF-κB inhibitor BAY11-7082succesfully block iASPP-(295-828)’s impact onapoptosis suggesting that the pro-apoptotic effect of iASPP-(295-828) is dependenton NF-κB. Luciferase reporter data showed that both WT-iASPP and iASPPfragements inhibit the transcriptional activity of NF-κB. The inhibitory effect ofiASPP-(295-828) on NF-κB transcriptional activity was much stronger than others.Furthermore, the downstream target genes of NF-κB also change significantly aftertransfection with iASPP-(295-828) fragement. The mRNA level of Bax wassignificantly decreased, while the mRNA level of Bcl-xl and CIAP1has significantlyincreased.Therefore, our data revealed a novel regulatory mechanism of iASPP.Caspase medaited iASPP cleavege and then changes the intracellular localization of iASPP. After iASPP fragment enter the nuclei, its inhibitory effect of iASPP onNF-κB was increased, so that it efficiently promotes apoptosis by inhibiting thetranscriptional activity of NF-κB. These data provides new insights into the drugresistance mechanisms of NF-κB constitutive activated malignancies. Themechanisms of NF-κB-iASPP interaction might be able to provide a new target tosensitize cancer cells’s response to chemotheray.