Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-XL.
Prolonged (>24 h) exposure to anti-IgM (an antigen surrogate that induces membrane cross-linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by 32P labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [3H]palmitate in a B-lymphocyte cell line, WEHI 231. This correlated with the induction of apoptosis. Shorter exposure times to anti-IgM (up to 8 h) failed to elicit apoptosis and did not elicit increased ceramide formation. After 8 h, apoptosis occurs concomitantly with ceramide formation over the next 40 h. Further, we showed that exogenous ceramide mimicked anti-IgM-induced apoptosis and that apoptosis was potentiated in serum-free media. Treatment of cells with an inhibitor of ceramide catabolism, N-oleoylethanolamine, increased both ceramide formation and apoptosis and accelerated apoptosis induced by anti-IgM. To examine further how ceramide metabolism is involved in apoptosis, we derived cell lines from a small population of cells resistant to N-oleoylethanolamine. These cell lines were selected based on an altered ceramide metabolic pathway, were resistant to apoptosis induced by anti-IgM, and showed no significant increase in ceramide when challenged with anti-IgM. The basis of this resistance was shown to be the failure to activate neutral sphingomyelinase activity following 24-h treatment with anti-IgM, in contrast to the 2-fold increase in neutral sphingomyelinase activity observed in wild type cells. We have shown previously that transfection of WEHI cells with bcl-xL conferred resistance to anti-IgM-induced apoptosis, whereas transfection with bcl-2 did not (Gottschalk, A., Boise, L., Thompson, C., and Quintans, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7350-7354). In this study, these bcl-xL transfectants also displayed increased resistance to exogenous N-acetylsphingosine (C2-ceramide) or N-hexanoylsphingosine (C6-ceramide). However, when challenged with anti-IgM the bcl-xL transfectants produced levels of ceramide similar to wild type cells, suggesting that ceramide formation is upstream of bcl-xL and that it is a major determinant of B-cell death.
Wiesner
D A
DA
Department of Pediatrics, The University of Chicago, Chicago, Illinois 60637, USA.
Kilkus
J P
JP
Gottschalk
A R
AR
Quintáns
J
J
Dawson
G
G
D000581:Amidohydrolases / Q000037:antagonists & inhibitors; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology*; D017209:Apoptosis; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects*; D002264:Carboxylic Acids / Q000494:pharmacology; D002460:Cell Line; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism*; D063388:Endocannabinoids; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004983:Ethanolamines / Q000494:pharmacology; D037341:Fumonisins; D051379:Mice; D009183:Mycotoxins / Q000494:pharmacology; D009829:Oleic Acids; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D013050:Spectrometry, Fluorescence; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D013723:Teratogens / Q000494:pharmacology; D051020:bcl-X Protein
D000888:Antibodies, Anti-Idiotypic; C494812:Bcl2l1 protein, mouse; D002264:Carboxylic Acids; D002518:Ceramides; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D037341:Fumonisins; D009183:Mycotoxins; D009829:Oleic Acids; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013109:Sphingomyelins; D013723:Teratogens; C044203:anti-IgM; D051020:bcl-X Protein; C033595:N-oleoylethanolamine; C056933:fumonisin B1; D013108:Sphingomyelin Phosphodiesterase; D000581:Amidohydrolases; D055572:Ceramidases