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 32 P labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [ 3 H]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, Noleoylethanolamine, 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-x L conferred resistance to anti-IgMinduced 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-x L transfectants also displayed increased resistance to exogenous N-acetylsphingosine (C 2 -ceramide) or N-hexanoylsphingosine (C 6 -ceramide). However, when challenged with anti-IgM the bcl-x L transfectants produced levels of ceramide similar to wild type cells, suggesting that ceramide formation is upstream of bcl-x L and that it is a major determinant of B-cell death.Apoptosis (1) can be induced readily in lymphocytes by means of irradiation, corticosteroids, or cross-linking of their antigen receptors. The apoptotic response of the murine Blymphoma WEHI 231 cell line to the cross-linking of surface IgM receptors provides a model to study the induction of physiological cell death (apoptosis) in lymphocytes (2-4). In WEHI 231, the response is specifically triggered via surface IgM since cross-linking other surface proteins such as IgD, Fc receptor, or major histocompatibility complex class II have no effect (5). Apoptosis is reversed by both phorbol esters and by bacterial lipopolysaccharide (6). Apoptosis can also be reversed by the removal of the antigen prior to 12 h of exposure, emphasizing its slow acting nature compared with necrosis and some types of apoptosis. We showed recently that the time course and ...

We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1-1 .0 pM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N-Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [ 3H]palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 pM), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death. Key Words: Programmed cell death-Apoptosis-Neurons---Ceramide-Sphingomyelin-Staurosporine.

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 microM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.

Gangliosides were isolated from the sera of recently diagnosed breast-cancer patients and from individuals who were apparently free of disease. Quantificative and qualitative analyses were carried out by 2-dimensional high-performance thinlayer chromatography and gas chromatography. The locations of isolated gangliosides on thin-layer chromatograms were determined by visualization with resorcinol, and each spot was quantified by digital image densitometry. The ganglioside profiles of cancer patients were compared to those of the control group, revealing a significant increase in total lipid-bound sialic acid and a specific increase in polysialogangliosides in the patients with breast cancer. Furthermore, an increase was noted in the ratio of gangliosides of the b-series biosynthetic pathway over those of the a-series in the cancer sera, as compared to the controls. Gas chromatographic analysis of the peracetylated methanolysis mixtures derived from the total ganglioside fraction of cancer patients supported the HPTLC data, with an increase in total sialic acid, galactose, and sphingosine residues. No unusual gangliosides were found in the mixture from breast-cancer patients.o 1995 Wilq-Liss, Inc. Many studies have been made both of neutral glycosphingolipids and of gangliosides associated with cancers. It is clear that an alteration in the composition of glycosphingolipids, of both the neutral type and gangliosides, accompanies oncogenic transformation, and both direct and indirect roles of gangliosides in tumorigenesis have been proposed (reviewed by Hakomori, 1981). With the development of monoclonal antibodies against tumor-associated antigens, unusual, more highly glycosylated or sialylated ganglioside tumor markers have been identified. Magnani et a/. (1981) identified the first monoclonal antibody-defined ganglioside of colon cancer and a large number of antibodies to tumor-associated ganglioside antigens have subsequently been found (reviewed by Hakomori, 1981). Furthermore, gangliosides are shed from these cancer cells into the local environment and eventually into the bloodstream (Li and Ladisch, 1991) and attempts have been made to identi@ unusual or increased levels of gangliosides in the plasma of cancer patients. Circulating tumor-associated antigens as well as tumor-associated tissue gangliosides have been considered as targets for use in antibody therapy (Portoukalian et al., 1991). A circulating tumor marker of this type may be equally useful in the diagnosis and monitoring of breast cancer and may serve as a target molecule in the treatment of cancer. In this study, we report the ganglioside profiles of several plasma and serum samples obtained from women suffering from ductal-cell carcinoma of the breast and an appropriate control group to ascertain whether an unusual profile or unique lipid may be present, as well as to describe the ganglioside profiles in serum of breast-cancer patients. MATERIAL AND METHODS MaterialAll solvents were of reagent grade or better, while chloroform and methan...

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