Effects Of Recombinant Adenovirus AdE-SH2-Caspase8on The Proliferation And Apoptosis Of K562and K562/G01Cell Lines

Objective To study the effects of SH2-Caspase8fusion proteinexpressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP on theproliferation and apoptosis of K562and imatinib-resistant K562/G01celllines.Methods (1) AdE-SH2-Caspase8-HA-GFP adenovirus expressing theSH2-Caspase8fusion protein had been purified formerly. The BCR/ABLpositive cell lines K562and imatinib-resistant K562/G01cell lines werethen treated with AdE-SH2-Caspase8-HA-GFP adenovirus（SC）. The AdE-SH2m-Caspase8-HA-GFP（SmC），AdE-GFP（CMV）; And PBS were usedas control simutanously. Infection efficiency and optimal MOI ofadenovirus in K562and K562/G01cell by GFP expression was analyzedusing fluorescence microscopy and flow cytometry (FCM). The expressionof fusion protein was measured by Western blot.(2) The cell growth was detected by MTT test and cell countting. Thecell cycle was determined by FCM. The proliferation of cells was observedby colony formation test. (3) Cell morphology was detected by Wright’s staining. The apoptosisof the cells were detected by FCM and DNA ladder. The expression ofCaspase3and PARP were checked by Western blotting.Results The high infection efficiency of SC adenovirus vector andoptimal MOI in K562and K562/G01cells was verified by fluorescencemicroscopy and FCM. Both SH2-Caspase8-HA and SH2m-Caspase8-HAfusion protein were expressed correctly in K562and K562/G01cells．MTT and cell counts test were performed following the K562andK562/G01cells infection SC adenovirus inhibited cell proliferation. FCManalysis showed increased cell countting in G1phase, while cell counttingin S phase and G2phase decreased. Colony formation test displayed thatSC adenovirus reduced clone formation ability of cells.After treatment with SC adenovirus vector the morphology ofapoptotic K562and K562/G01cells was visible, such as cytoplasmappeared empty bubble and nuclear gather. The results of FCM showed thatearly apoptosis of the K562and K562/G01cells increased, DNA ladderelectrophoresis indicated classic DNA ladder pattern of apoptotic cellappeared in K562and K562/G01cells. Western blotting revealed activatedfragments of caspase3and PARP proteins significantly increased in K562and K562/G01cells.．Conclusions Recombinant adenovirus AdE-SH2-Caspase8-HA-GFPexpressing SH2-Caspase8fusion protein can significantly inhibit the proliferation and induced the apoptosis of K562and imatinib-resistantK562/G01cells.