Study Of The Role. Lipid Rafts In Trail-induced Apoptosis In The Early Events

TRAIL(TNF-related apoptosis-inducing ligand) is a novel member of the TNF super-family.Unlike other members of TNF family,whose expression is restricted to some cells and tissues such as activated T-cells,natural killer(NK) cells,and immtme-privileged sites,TRAIL and TRAIL receptors are widely expressed in many cell types and tissues,suggesting that most tissues and cell types are potential targets for TRAIL.TRAIL selectively trigges apoptosis of tumor cells and is not toxic to most normal cells,suggesting that this unique molecule is a promising anti-cancer agent.However,reports on the resistance to TRAIL have been increased in various tumor cell lines,which might limit the utility of this molecule in clinical application. In the present study,recombinant soluble TRAIL(rsTRAIL) was used as apoptosis-inducer,and two Jurkat T lymphocyte cell lines(JKR and JKS) with different sensitivity to rsTRAIL were used to explore the molecular mechanism of apoptosis regulation,especially in the early stage of apoptosis induction.Lipid rafts,as defined by Simoms and Ikonen are unique membrane micro-domains,which contain large amount of cholesterol and sphingomyelin. Cholesterol and spingomyelin are the most stable lipid in the membrane,and thus made lipid rafts separated from other phospholipids bilayer and a platform for membrane proteins signal transduction or virus to come in or out of the cell.We speculated that different sensitivity to rsTRAIL stimuli might be layed under the relationship between the pro-apoptosis molecules and the recruitment ability of lipid rafts.In our experiments,two different phenotype cell lines of Jurkat T lymphocytes showed different sensitivity to rsTRAIL stimuli,JKS cells were more sensitive than JKR cells.At cytosolic level,JKS cells showed a more rapid response to the rsTRAIL stimulation than JKR cells.The procaspase-8 and RIP were cleaved upon rsTRAIL stimuli for half an hour.The background concentrations of procaspase-8 were different in the two cell lines,JKS cells containd more procaspase-8 than JKR cells. At plasma-membrane level,JKS cells recruit more FADD,procaspase-8 molecules than JKR cells when stimulated by rsTRAIL for half an hour.The background concentration difference of procaspase-8 in the two cell lines did not fully explain the sensitivity difference.The caspase-8 inhibitor,Z-IETD-FMK,could block the early stage apoptosis of JKR cells,but the blocking ratio dropped to about 50%in the JKS cells.It might be some factors upstream of caspase-8 also affect the sensitivity.Then, we detected the changes happened to the two cell lines upon rsTRAIL treatment at the lipid rafts level.DR5 was a pre-located molecule in lipid rafts in both of the two cell lines.During a 0 to 20 min time course,JKS cells recruited more DR5,FADD, procaspase-8 into lipid rafts,PI3K also was increased in lipid rafts,while RIP was decreased;DR5 and RIP were unaffected in the lipid rafts of JKR cells.FADD, procaspase-8 were not detectable and PI3K had a slightly elevation.2DE image showed that about 33%dots emerging or were increased in the rafts sample of JKS cells after treated with rsTRAIL for half an hour.Most importantly,ASMase,which enable rafts come into being and expanding,was different in concentration,enzyme activity and co-location relationship with lipid rafts.JKS cells contained more ASMase than JKR cells.The enzyme activity of ASMase in JKS cells was higher than that of the JKR cells,with time went on,the enzyme activity of ASMase was even higher than that of the JKR cells.In JKR cells ASMase was gradually co-located with GM1,the symbol of lipid rafts,while in JKS cells ASMase was consecutively co-located with GM1.The results above indicated that concentration,activity and localization of ASMase may contribute to the different sensitivity to TRAIL cytotoxicity of the two cell lines.This study may through light on the therapy and prediction of the outcome of T lymphocyte leukemia.