Construction Of Recombinantadeno Virus With Apoptosis Inducing Factor Gene Or Its Mutants And Their Effct Of Apoptosis On K562 Cells

Chronic Myeloid Leukemia (CML) is a hematopoietic malignancy origins from the translocation of chromosome 9 chromosome 22 with the formation of Bcr-Abl fusion protein which increases the strong activity of tyrosine kinase and accelerates cell proliferation and inhibits cell apoptosis through activating several downstream pathways. So understanding the molecular mechanism of cells apoptosis inhibited in CML is of great significance to CML pathogenesis and treatment.Apoptosis inducing factor (AIF) participates caspase-independent apoptosis pathway. AIF is a flavor-protein which located on mitochondrial membrane, involving in electron transfer in energy metabolism, when cells receive apoptosis signal, AIF can release from mitochondria to cytoplasm, relocates into nucleus subsequently and can cut DNA into large fragments of 50 kb, finally leading to cells apoptosis. Researchers have found that AIF release from mitochondria is related with mitochondrial membrane permeability, calpain and so on.Luigi et al. found that Heat Shock Protein 70(HSP70) could prevent AIF re-translocation into nuclear and rescue cells apoptosis in Apafl-/-HeLa. However, the role of AIF in cell apoptosis-inhibited of CML is unclear.To address this question, we proposed to construct recombinant adenovirus with wild apoptosis inducing factor (Ad-Apoptosis inducing factor, Ad-AIF) or apoptosis inducing factor mutant deleted mitochondria localization signal domain (Ad-DMLS-AIF) or apoptosis inducing factor mutant deleted HSP70 binding domain (Ad-DHBD-AIF) and detect the apoptotic effect of them on K562 cells after infection, in order to exclude the relationship between AIF and abnormal apoptosis of CML.The main contents and methods:1. Construct recombinant adenovirus Ad-AIF, Ad-DMLS-AIF and Ad-DHBD-AIF.AIF, DMLS-AIF and DHBD-AIF fragments were obtained by PCR (cDNA from K562 cells as a template) and were cloned to shuttle plasmids pAd-Track-CMV-HA. The recombinant shuttle plasmid were identified through double enzyme digestion、PCR and sequencing respectively. Subsequently the recombinant shuttle plasmid was recombined with the skeleton plasmid pAd-Easyl respectively. After lined by Pac I,the recombinant adenovirus plasmids were transfected into AD293 cells, wrapped and amplified. Recombinant adenovirus named Ad-AIF, Ad-DMLS-AIF and Ad-DHBD-AIF were obtained, pAd-Easy 1 plasmid was packaged and amplified at the same time for obtaining empty adenovirus as control. After the three recombinant adenovirus infected to K562 cells for 48h, Western blot was performed to detect their expression.2. Detect apoptotic effect of Ad-AIF, Ad-DMLS-AIF and Ad-DHBD-AIF on K562 cells.When empty adenovirus and the three recombinant adenovirus infected to K562 cells for 0h、24 h、48 h、72 h,CCK-8 was used to detect the cells proliferation-inhibited effect of these adenovirus on K562 cells. After 48 h, Hoechst33258 dye was applied to observe the changes of nucleus from K562 cells; Flow cytometry was used to assess apoptotic effect of K562 cells. After 24h, immunofluorescent staining was applied to assess the localizationthe of recombinant adenovirus expressing protein in K562 cells. Immune co-precipitation was used to detect whether AIF, DMLS-AIF or DHBD-AIF banded with HSP70.Through the above methods, we harvested the following results:1. The recombinant shuttle plasmid were correctly obtained through double enzyme, PCR and sequencing; the recombinant adenovirus plasmid were constructed successfully due to the special 4.5 kb DNA fragments obtained from recombinant adenovirus plasmid digested by Pac I; the expression of Ad-AIF, Ad-DMLS-AIF and Ad-DHBD-AIF were observed in K562 cells by western blot.2. Comparing with un-treatment group, empty adenovirus group, Ad-AIF group and Ad-DMLS-AIF group, the cells proliferation-inhibited effect of Ad-DHBD-AIF on K562 cells increased in a time-dependent manner through the result of CCK-8. The number of apoptotic K562 cells in Ad-DHBD-AIF group was significantly higher than that in un-treatment group, empty adenovirus group, Ad-AIF group and Ad-DMLS-AIF group assessed by flow cytometry; Ad-DHBD-AIF could lead to chromatin condense in K562 cells while the chromatin of K562 cells distributed uniformity in un-treatment group, empty adenovirus group, Ad-AIF group and Ad-DMLS-AIF group through the result of Hoechst33258. Immunofluorescent staining revealed that DHBD-AIF could re-translocate into nucleus in K562 cells, AIF and DMLS-AIF could not. Immune co-precipitation showed that AIF and DMLS-AIF could combine with HSP70 whereas DHBD-AIF could not.In summary, Ad-AIF, Ad-DMLS-AIF and Ad-DHBD-AIF was constructed successfully. After the three recombinant adenovirus infected to K562 cells, compared to un-treatment group, empty adenovirus group, Ad-AIF group and Ad-DMLS-AIF group, Ad-DHBD-AIF could induce obvious apoptosis in K562 cells, the protein encoded by which did not bind with HSP70 and could translocate into nucleus. It means that AIF banding with HSP70 could not enter into nucleus to execute the role of promoting apoptosis in K562 cells, finally the phenomenon of apoptosis-resistance was performed.