The Apoptosis Induced By SfaMNPV In SL-1 Cell And The Detection Of EndonucleaseG

Our laboratory have established two stable apoptosis systems,one is the apoptosis induced by Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in Spodoptera litura cell line(SL-l),the other one is that induced by the Syngrapha falcifera multiple nuclear polyhedrosis virus(SfaMNPV).In this study we used the second system to investigate the changes in SL-1 cell during the apotosis process,and detected the release of Endonuclease G. The main results were as follows:Tn-5B1 cell line was very sensitive to this virus,the titers in the culture system is 2.41×10⁷ PFU/ml, Microscopic examination of SL-1 cell deal with SfaMNPV revealed progressive cell blebbing at 8-16h of pi and all cells were destructued at 24h. The fragmentation of the induced cell nuclei was showed with DAPI. Agarose gel electrophoresis analysis of DNA extracted from deal cell showed typical DNA ladder. Using the flow cytometry, the effect of SfaMNPV on mitochondrial membrane potential(Δψm) was investigated by Rh-123 staining of cells. The results showed that the Rh-123 fluorescent intensity in SL-1 cells was reduced after treatment for 2 hr,and the decrease of fluorescent intensity typical in time-dependent manners.As the research in mammalian, endoG, which belongs to an important family of Mg²⁺ dependent nucleases,is encoded by a nuclear gene,translated in the cytosol. After mitochondrial import, the N-terminal region of 48 amino acids is removed yielding the mature form. As a mitochondrion-specific nuclease, upon apoptotic stimulus, once released from mitochondria,endoG cleaves chromatin DNA into nucleaosomal fragements independently of caspase.We investigated whether there was EndoG in SL-1 cell, the release of EndoG and the changes of EndoG in mitochondria and cytosol by western blotting. The results showed that EndoG couldn't be detected in mitochondrial and cytosol,suggesting that the events of EndoG release from mitochondria to cytosol and then cleaving chromatin DNA during the SfaMNPV-induced apoptosis in SL-1 cells possibly didn't occur. These data suggested that apoptosis induced by SfaMNPV in SL-1 cells was dependent on caspases.