The Cloning, Expression Of C-terminal Truncated Human AIF Molecules And Their Apoptosis Induction On HeLa Cells

Apoptosis is programmed cell death triggered by various physiological or pathological stimuli. Apotosis inducing factor (AIF) is a caspase-independent death effector, which was found in 1996. In heathlty cells, AIF protein is normally confined to mitochondrial intermembrane space. However, after apoptosis is induced, AIF protein can be released from mitochondria and translocates into nucleus leading by its nuclear localization signal(NLS). Once in the nucleus, AIF causes chromatin condensation and large-scale DNA fragmentation to fragments of ~50kb.The pro-apoptotic activity of AIF is not affected by over expression 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 with caspases and other apoptosis effectors, AIF could directly induce apoptotic cell death, beyond the inhibitory regulation 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.The research done by Susin A indicates that the molecular weight of human AIF precursor protein is 67kD. It contains several structure domains that is (1) mitochondria location sequence (MLS); (2) Spacer sequence; (3) nuclear location sequence(NLS); (4) C-terminal. The mature AIF consists of three parts: FAD binding domain(122-262aa and 400-477aa), NADH binding domain and C-terminal.The mature AIF was cloned successfully by Dr Yu Cui-juan, at the same time, she demonstrated its pro-apoptotic activity. As followed Master Liu Xiang-li has successfully cloned four truncated human AIF genes named as AIFΔ1-300(301-613aa), AIFΔ1-352(353-613aa), AIFΔ1-400(401-613aa) AIFΔ1-480(481-613aa) respectively, and she also demomstrated the four truncated AIF genes can induced tumor cell aopotosis. However, the molecular weight of mature AIF is too high, which limits its application. So, our aim is to clone C-terminal truncated AIF genes so that we can obtain one kind of truncated AIF gene that is shorter, easier to use and its pro-apoptotic function remains unaffected. In this paper, we cloned two C-terminal truncated AIF as experimental group named AIFΔ1-500(501-613aa) and AIFΔ1-530 (531 -613aa), we also clone the AIF360-480 as a negative control. Then, the three C-terminal truncated AIF genes were inserted into pcDNA3. The sequence of genes was completely right as determined by DNA sequencing. After the three C-terminal truncated AIF genes were transiently transfected into HeLa cells, the expression of C-terminal truncated AIF genes were demonstrated by immunohistochemical staining assay. Using fluorescence microscope, the C-terminal truncated AIF genes were found in cytosol and the cells transfected with experimental group genes become round and shrink compared to the HeLa cells and the negative control.Because of the molecular weight of truncated AIF is too smallto be detected by Western blot. So we insert the C-terminal truncated AIF genes into the vector pFlag-CMV4, which has a Flag labelling on its N terminal. Then we transient transfected these fusion vector into HeLa cells and detected the expression protein by Western blot, meantime confocal microscopy was conducted to confirm these AIF positioned in cytoplasm. Through immunofluorescent, MTT and AnnexinV analysis, expression of the experimental group C-terminal truncated AIF genes can induce Cyt c release from mitochondria, and they also can inhibite the proliferation of HeLa cells.In summary, although the N-terminal amino acid 1-500 or 1-530 of AIF gene was deleted, the C-terminal truncated AIF AIF gene remains the pro-apoptotic activity. Comparing with mature AIF, the C-terminal truncated AIF genes are easier to apply in clinic. Our research provides the foundation of using them into cancer gene therapy and clinical research.