Proapoptotic Activity And Related Molecular Mechanism Of AIF C-terminal Domain

The malignant tumor has become one of the principal killers of human life. Gene therapy has been used to cure cancers in the past decades and provides us great hopes and chances for conquering this serious disease.The thought of inducing tumor cell apoptosis will lead to the emergence of novel antitumor gene therapy strategies with high efficacy and low toxicity.Apotosis inducing factor(AIF) is a mitochondria-assoeiated protein,whose accumulation in the cytoplasm and nucleus will be sufficient to induce programed cell death at least in some cases,resulting in peripheral chromatin condensation, large-scale DNA fragmentation(～50 kb) and cell death.The pro-apoptosis activity of AIF is not affected by either overexpression of Bcl-2,or inhibition of caspases.Moreover,AIF is involved in apoptotic process of almost all eukaryotic cells and is essential for the first wave of caspase-independent programmed cell death during embryo development.Thus,AIF holds out great promises to be applied to induce tumor cell apoptosis in gene therapy. In this study,we firstly constructed the FLAG-tagged C-terminal domain of AIF(AIFΔ1-480).After transiently transfected into human cervical carcinoma HeLa cells,AIFΔ1-480,was observed in the cytosol to induce apoptotic cell death directly as demonstrated by fluorescent microscope,colony forming assay, TUNEL staining and MTT assay.Indirect immunofluorescence staining revealed that the expression of AIFΔ1-480 in HeLa cells induced cytochrome c release from the mitochondria, which is a process also observed in AIFΔ1-120-transfected cells but not in the parent cells or cells transfected withΔAIF360-480 constructs.In cells undergoing typical apoptosis,the release of cytochrome c from the mitochondria leads to the activation of caspase 9,and then,caspase 3.This involves the formation of an apoptosome,which is a multiprotein complex comprising cytochrome c,Apaf-1,pro-caspase 9,and ATP.To test whether caspase-9 participates in the apoptotic pathway mediated by AIFΔ1-480,RNAi was used to knock down the expression of caspase-9 in HeLa cells.The stable expression of caspase-9-targeted siRNA from a pSUPER.puro vector resulted in a decrease of approximately 70%in caspase-9 expression.Cell viability assay demonstrated the concurrent inhibition of AIFΔ1-120- or AIFΔ1-480-induced apoptotic cell death by the knockdown of caspase-9.These results suggest that the proapoptotic activity of AIFΔ1-480 is mediated by caspase-9-dependent cytoplasmic signaling events.In our research,novel "ImmunoAIF" genes were further generated,coding fusion proteins of a leading peptide,single-chain anti-TSA(tumor specific antigen) antibody,translocation domain of Pseudomonas exotoxin A(PE),and truncated AIFΔ1-120 or AIFΔ1-480.The ImmunoAIF genes were stably tranfected into Jurkat T-lymphocytes,followed by G418 selection.RT-PCR was used to detect the expression of the aim genes in RNA level.Western Blot of the condensate of culture medium of the transduced cells confirmed that the transduced Jurkat cells could express and secret ImmunoAIF protein stably. The normal cell growth curve of the transduced lymphocytes indicated that these cells could survive for long periods.The secreted immunoAIF protein thus specifically recognized HER-2-overexpressed tumor cells,and translocate into cytoplasma.ImmunoAIF exhibited targeted pro-apoptotic activity on HER-2 possitive tumor cells,while had no significant influence on HER-2 negative tumor and normal cells in cell co-culture assays.In summary,our data suggest that the C-terminus of AIF is sufficient to mediate apoptosis,a process dependent upon the release of cytochrome c from the mitochondrion.When fused to anti-HER-2 scFv and PE domainⅡ,the AIF C-terminal domain can recognize and kill HER-2 over-expressing tumor cells. Therefore,the present study probably provides a novel strategy of gene therapy for HER-2 positive tumors.