Construction Of Cell Lines For Screening Anti-cancer Drugs Targeted On Death Receptor-effector Complex Of DISC

Cancer results from complex,progressive and random processes that cause normal cells towards malignant biotransformation triggered by genetic and acquired abnormalities.Currently cancer is still one of the biggest killer diseases worldwide.Cancer is characterized by high mortality,no conspicuous effect,which are the consequence of the complex mechanism and without specific targets.The drawbacks and disadvantages of traditional cancer therapies have made biologically targeted therapies attractive.Rationals:TRAIL(TNF-related apoptosis-inducing ligand)induce apoptosis only in virus-infected tumor cells and transformed cells without killing normal cells.This feature makes TRAIL attractive as anti-cancer drugs.But TRAIL is a biological protein,consequently,the high production costs,inconvenient to save and transport,difficult to administer etc.If some substance similar to TRAIL is discovered and used to replace TRAIL,it will excel TRAIL and make the cost far lower.According to the ligand-receptor-effector fluorescence imaging(REFI)technique,we construct Procaspase8-GFP fusion protein cell line,in theory,TRAIL treatment induces a change in fluorescence distribution in cells that can be visualized under fluorescence microscopy before and after TRAIL treatment.The redistribution of fluorescence in cells can be visiulized,analyzed and quantified.This cell line can be used to screen TRAIL mimics or DR agonists in natural materials.Results:The first step:prc8DED1-DED2-GFP plasmid was constructed and transfected into U20S cells.Cells expressing green fluorescent proteins were found dead after 24 hours later under the observation of fluorescence microscopy;Step two:pCMV6-procaspase8WT-GFP,and plasmids of fusing proteins with mutants at cysteine and aspartates were then constructed.Again,U20S cells transfected and expressed these fusing proteins were found dead or dying after 24 hours.The third step:GFP Plasmids containing only DED1(prc8DED1)or DED2(prc8DED2)were then constructed and transfected into U20S cells again.As observed under fluorescence microscopy,green fluorescence evenly distributed cells were obtained.The forth step:Cells expressing prc8DED1 and prc8DED2 were cloned with 400ug/ml of G418.Finally,these cells of prc8DED1 but not prc8DED2 treated with TRAIL resulted in changes in green fluorescence distribution,forming fluorescence aggregated in a time and TRAIL concentration-dependent manner,Conclusions:1)Procaspase8 can bind to FADD by prc8DED1 recruitment;2)Expression of prc8DED1-DED2-GFP,procaspase8WT-GFP and mutants at cysteine and aspartates procaspase8mutants-GFP of procaspase8 in U2OS cells all lead to apoptosis;3)Expression of prc8DED1-GFP of procaspase8 does not affect cell growth and survival;4)Cells expressing prc8DED1-GFP respond to TRAIL in term of fluorescence redistribution,and can be used for screening for TRAIL mimics,and death receptor5(DR5)agonists,antagonists and modulators in the absence or presence of TRAIL from compounds library and extracts of natural materials.