STEREOSPECIFIC INDUCTION OF APOPTOSIS IN U937 CELLS BY N-OCTANOYL-SPHINGOSINE STEREOISOMERS AND N-OCTYL-SPHINGOSINE - THE CERAMIDE AMIDE GROUP IS NOT REQUIRED FOR APOPTOSIS

We investigated the ability of N-octanoyl-sphingosine (C-8-Cer) stereo isomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C-8-Cer amine), to induce apoptosis in U937 cells. We found the C-8-Cer stereo isomers to be stereospecific with the D- and L-threo stereoisomers bei ng severalfold more potent than the erythro in inducing nucleosomal fr agmentation. The order of potency was. D-t-C-8-Cer = L-t-C-8-Cer > L-e -C-8-Cer > D-e-C-8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivativ e, D-e-C-8-Ceramine, in which the carbonyl group was replaced by a met hylene group. The carbonyl group was not necessary for triggering apop tosis. In fact, replacement of the carbonyl group decreased substantia lly the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e C-8-Cer. To e xplore possible mechanisms by which these compounds trigger the apopto tic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activa ted protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C-8-Ceramine and L-t-C-8-Cer failed to increase the cel lular ceramide levels, D-e-C-8-Cer, D-t-C-8-Cer and D-e-C-8-Ceramine a ctivated the CAPK equally. These studies suggest that the DNA fragment ation-inducing ability of the three stereoisomers and D-e-C-8-Ceramine cannot be attributed either to an increase in the activity of CAPK, o r, as illustrated by D-e-C-8-Ceramine and L-t-C-8-Cer, to the differen tial elevation of endogenous ceramide. The phosphatase inhibitor okada ic acid failed to protect U937 cells from apoptosis induced by D-e-C-8 -Cer.