| Gene therapy has been widely 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 newly found apoptosis effector, its accumulation in the extramitochondrial compartment and in particular in nuclei, will be both sufficient and required to induce apoptosis at least in some cases, resulting in peripheral chromatin condensation, large-scale DNA fragmentation (~50kb) and cell death. The pro-apoptosis activity of AIF is not affected by overexpression of Bcl-2, and is in caspases-independent fashion. Moreover, AIF is involved in apoptosis process throughout eukaryotic kingdom and is essential for the first wave of caspase-independent programmed cell death during mammalian development. Compared to caspases and other apoptosis effectors, AIF could directly induce apoptotic cell death, beyond the inhibitory regulation-6-from anti-apoptotic factor (eg.Bcl-2) in cytosol. Thus, it is significant and promising for AIF to be applied to induce tumor cell apoptosis in gene therapy.In this paper, we firstly constructed the truncated AIF (AIFA1-120) gene. After transiently transfected into human cervical carcinoma HeLa cells, AIFA1-120, with the N-terminal mitochrondrial localization sequence deleted, was observed to express in the cytosol and then translocate into nuclei to induce apoptotic cell death directly as demonstrated by fluorescent microscope, immonohisto-chemistry staining, HE staining and electron microscope observation.Then two recombinant fusion genes were constructed by combining the truncated AIFA1-120 and part of PE transmembrane domain (PE280-358aa and PE280-364aa). The recombinant genes were transiently transfected into HeLa cells, still it was demonstrated through confocal microscopy observation that the recombinant proteins were expressed in the plasma and then translocated into nuclei to induce apoptosis. The transfected cells displayed morphological characteristics of apoptosis, such as plasma membrane blebbing and multilobed nuclei under fluorescent microscope. Following with culture time, the number of transfected cells and the fluorescent of GFP in transfected cells reduced, suggesting many transfected cells died. The results of cell counting and MTT assay showed that the number of survival cells in transfected experiment group was much reduced than that of transfected void vector as control at the same time, which indicated that expression of AIFA1-120 and its fusion genes could inhibit the growth of tumor cells. Fluorescence staining of cell framework and nuclei were conducted, the result of which revealed that the transfected cells displayed disorder in cell skeleton, shrinkage and cleavage in nuclei. It was illuminated that chromosome DNA occurred cleavage and fragment since the result of TUNEL detection was positive. The transfected cells also displayed more typical apoptotic character, including exposure of phosphatidylserine in the plasma membrane by Annexin V staining detection, shrinkage nuclei, chromatin condensation, plasma membrane blebbing and chromatin breakage fragments by-7-electron microscopy analyses. Diffuse staining of Cyt c emerged in some transfected cells by indirect immunofluorescence staining, which suggested overexpression of AIF could trigger the release of cytochrome c from mitochondria. Thus, expression of recombinant human AIF could induce the transfected HeLa cells apoptosis. The part of PE peptide fused in N-terminal of AIFA1-120 did not affect its pro-apoptosis activity, while, the fusion molecule, PEII(280-364)-AIFAl-120, showed stronger apoptogenic activity.In our research, novel "ImmunoAIF" genes were further generated, coding fusion proteins of leading peptide, single-chain anti-TSA (tumor specific antigen) antibody, translocation domain of PE, and truncated AIFA1-120. The ImmunoA...
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