Tepper A D, Cock J G, de Vries E, Borst J, van Blitterswijk W J
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
J Biol Chem. 1997 Sep 26;272(39):24308-12. doi: 10.1074/jbc.272.39.24308.
The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (hours) often parallels apoptosis onset. We investigated CD95-induced ceramide formation in Jurkat cells, using two methods (radiolabeling/thin layer chromatography and benzoylation/high performance liquid chromatography), which, unlike the commonly used diglyceride kinase assay, discriminate between ceramide species and de novo formed dihydroceramide. We demonstrate that ceramide accumulates after several hours, reaching a 7-fold increase after 8 h, kinetics closely paralleling apoptosis induction. No fast response was observed, not even in the presence of inhibitors of ceramide metabolism. The majority ( approximately 70%) of the ceramide response remained unaffected when apoptosis was completely inhibited at the level of caspase-3/CPP32 processing by the inhibitor peptide DEVD-CHO. Exogenous cell-permeable C2-ceramide induced the proteolytic processing of caspase-3, albeit with somewhat slower kinetics than with CD95. DEVD-CHO dose-dependently inhibited C2-ceramide- or exogenous sphingomyelinase-induced apoptosis. The results support the idea that ceramide acts in conjunction with the caspase cascade in CD95-induced apoptosis.
目前关于内源性神经酰胺在介导CD95/Fas诱导的细胞凋亡中的相关性存在混淆，主要基于以下两点：（i）使用相同凋亡刺激的不同研究中神经酰胺反应动力学存在差异；（ii）观察到晚期神经酰胺形成（数小时）通常与细胞凋亡开始平行。我们使用两种方法（放射性标记/薄层色谱法和苯甲酰化/高效液相色谱法）研究了Jurkat细胞中CD95诱导的神经酰胺形成，这两种方法与常用的甘油二酯激酶测定法不同，能够区分神经酰胺种类和新形成的二氢神经酰胺。我们证明神经酰胺在数小时后积累，8小时后增加7倍，其动力学与细胞凋亡诱导密切平行。未观察到快速反应，即使在存在神经酰胺代谢抑制剂的情况下也是如此。当凋亡在caspase-3/CPP32加工水平被抑制剂肽DEVD-CHO完全抑制时，大部分（约70%）的神经酰胺反应不受影响。外源性可渗透细胞的C2-神经酰胺诱导了caspase-3的蛋白水解加工，尽管其动力学比CD95诱导的稍慢。DEVD-CHO剂量依赖性地抑制C2-神经酰胺或外源性鞘磷脂酶诱导的细胞凋亡。这些结果支持了神经酰胺在CD95诱导的细胞凋亡中与caspase级联协同作用的观点。
