METABOLISM AND APOPTOTIC PROPERTIES OF ELEVATED CERAMIDE IN HT29(REV)CELLS

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 C-2-Ce r, a short-chain analogue of the sphingolipid. bSMase treatment did no t induce apoptosis in these cells, as revealed by a lack of both DNA f ragmentation and cleavage of poly(ADP-ribose)polymerase. In contrast, apoptosis did occur upon addition of C-2-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 differen ce in apoptosis-inducing capacity. C-2-Cer was rapidly taken up by HT2 9(rev) cells and accumulated due to the absence of substantial metabol ic conversion. Upon addition of bSMase, hydrolysis of sphingomyelin re sulted in a reduction of that pool to 20% compared with control values , accompanied by a multifold increase in Cer level. In spite of the co ntinuous presence of active bSMase, the Cer increase turned out to be transient. Cer levels reached their maximum 1-2 h after addition of bS Mase, followed by a significant decrease. Excessive Cer was mainly tur ned over via cerebrosides into complex glycolipids, including ganglios ides. In the presence of glucosylceramide synthase- and/or ceramidase inhibitors, this conversion was significantly blocked and bSMase-gener ated Cer accumulated in the cells. However, even under these condition s apoptosis did not occur. in conclusion, the inability of bSMase to i nduce 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 C-2-Cer, we therefore propose that the intracellular targ et involved in the propagation of the apoptotic signal is reached by C -2-Cer, but not by bSMase-generated Cer.