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pubmed:abstractText |
Ceramide (Cer) has been implicated in the regulation of apoptosis. In this study, we elevated cellular Cer levels in human colon-carcinoma (HT29(rev)) cells by incubating the cells in the presence of bacterial sphingomyelinase (bSMase) or, alternatively, in the presence of C2-Cer, a short-chain analogue of the sphingolipid. bSMase treatment did not induce apoptosis in these cells, as revealed by a lack of both DNA fragmentation and cleavage of poly(ADP-ribose)polymerase. In contrast, apoptosis did occur upon addition of C2-Cer. These findings led us to study whether differences in the metabolic fate of the excess of Cer, as generated by both treatments, contributed to the observed difference in apoptosis-inducing capacity. C2-Cer was rapidly taken up by HT29(rev) cells and accumulated due to the absence of substantial metabolic conversion. Upon addition of bSMase, hydrolysis of sphingomyelin resulted in a reduction of that pool to 20% compared with control values, accompanied by a multi-fold increase in Cer level. In spite of the continuous presence of active bSMase, the Cer increase turned out to be transient. Cer levels reached their maximum 1-2 h after addition of bSMase, followed by a significant decrease. Excessive Cer was mainly turned over via cerebrosides into complex glycolipids, including gangliosides. In the presence of glucosylceramide synthase- and/or ceramidase inhibitors, this conversion was significantly blocked and bSMase-generated Cer accumulated in the cells. However, even under these conditions apoptosis did not occur. In conclusion, the inability of bSMase to induce apoptosis of HT29(rev) cells does not appear to be due to rapid metabolic conversion of excessive Cer. Since apoptosis is induced upon addition of C2-Cer, we therefore propose that the intracellular target involved in the propagation of the apoptotic signal is reached by C2-Cer, but not by bSMase-generated Cer.
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pubmed:affiliation |
Department of Physiological Chemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
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