| Research Abstract |
There are several lines of evidence indicating that tumor-nectosis factor (TNF)-induced apoptosis involves various signaling phospholipases, e.g. phospholipase A_2, phospatidylinositol-specific phospholipase C (PI-PLC), PC-specific PLC (PC-PLC), phospholipase D (PLD), sphingomyelinase (SMase). However, the regulatory mechanisms of PC-PLC,PLD,SMase except PI-PLC and PLA_2 have not yet been disclosed. Especially, the activation mechanisms of SMase participated in apoptosis has been poorly understood. In this research project, we have first investigated the downstream signaling of ceramide, the immediate haydrolytic product of SM via SMase, on the expressed PLD's mRNA levels by Northen blotting and also the activity of interleukin 1beta-converting enzyme (ICE) and CPP32 during ceramide-induced apoptosis in rat basophilic leuchemia RBL cells, glioma C6 cells and pheochromcytoma PC12 cells.
C6 cells were found to undergo the apoptotic process by exposure to membrane-permeable ceramide (C2-ce
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ramide) for 6-12 h, as inferred by nuclear DNA fragmentation and laddering. During the progress of apoptotic process, the PLD activity measured by the in vitro assay was considerably decreased to less than 10%. The levels of mRNA of PLD isozymes (rPLDa, b, c) were examined in the course of the apoptotic process induced by C2-ceramide. The mRNA levels of PLDa, b were greatly decreased whereas the level of PLDc was in contrast increased.
In PC12 cells, C2-ceramide induced DNA fragmentation due to apoptosis. There was a trandient elevation of CPP32 activity after the 12h-treatment with C2-ceramide. However, no canges were observed in cell morphology and CPP32 activity in BCL-2 overexpressed PC12 cells.
It has also demonstrated in basophilic leukemia RBL cells that the pretreatment of cells with C2-ceramide inhibited the IgE receptor-mediated PLD activation. This and those data by other groups have provided evidence that ceramide generated from SM via SMase may partake in induction of apoptosis. Less
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