Although both ceramide and interleukin-ll] convert-apoptosis [15][16][17][18]. However, ICE itself might not be a true mammalian Ced-3 counterpart because apoptosis proceeds aling enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report most normally in ICE-deficient mice [19,20]. So far at least here that cell-permeable ceramide induced cleavage and activaseven mammalian Ced-3 homologues have been identified, tion of CPP32, a Ced-3/ICE-like protease, but not ICE. , especially the death domain, but not Apoptosis is a well-regulated process of cell death that is TNFRII(p75), raising the possibility that ceramide and ICEimportant in events such as embryogenesis, cell growth conlike proteases exist in the same signaling pathway. We theretrol and lymphocyte repertoire formation. Recent investigafore addressed this question in the present study and demontions have revealed that there are several key pathways in strated that CPP32 acts downstream of ceramide and its actiapoptotic signal transduction, including the sphingomyelinvation is required for ceramide-induced apoptosis. ceramide pathway [1,2] and the pathway involving interleukin-l~ converting enzyme (ICE)/Ced-3 family proteases [3].2. Materials and methods Ceramide is a newly identified lipid second messenger that is generated through the hydrolysis of sphingomyelin by 2.1. Reagents and cell lines sphingomyelinases (SMase) [1,2]. Signals from cell surface re-N-Acetyl ceramide (C2-ceramide) was purchased from Wako (Osaceptors such as Fas (CD95/Apol) [4][5][6][7] and tumor necrosis ka), C2-dihydroceramide from Calbiochem and dioctanoylglycerol factor receptor (TNFR) [8][9][10][11] activate SMase and generate from Sigma, and dissolved in ethanol. The final ethanol concentration in the culture medium was always less than 0.1%. Mouse IgM anticeramide, even in the cell-free system [12]. Synthetic cellhuman Fas monoclonal antibody (CH-11) was purchased from Medpermeable ceramide can mimic such apoptosis. This cytotoxic ical Biological Laboratories (Nagoya) and anti-CPP32 monoclonal effect is specific because structural analogues such as dihydroantibody from Transduction Laboratories. Tetrapeptide inhibitors ceramide do not induce apoptosis [13,14]. These data suggest for CPP32 (Ac-DEVD-CHO) and ICE (Ac-YVAD-CHO), and tetrathat ceramide is a physiological mediator of Fas-and TNFRpeptide substrates for CPP32 (Ac-DEVD-AMC) and ICE (Ac-YVAD-AMC) [22,27] were purchased from Peptide Industries (Osamediated apoptosis, ka) and dissolved in DMSO. The human T cell line, Jurkat, was ICE family proteases, which are mammalian Ced-3 homomaintained in RPMI 1640 medium containing 10% fetal calf serum logues, have also been implicated in Fas-and TNF-mediated (FCS). An anti-Fas-resistant (FR) subline of Jurkat was established by culturing wild-type Jurkat cells in the presence of 100 ng/ml antiFas (CH-11) for 1 month. *Corresponding author. Fax: (81) (3) 5803-0131.2.2. Immunoblotting of CPP32 **Research fellow o...

We have studied the glycosphingolipid composition in an F-11 neuroblastoma cell line originated from hybridization of a mouse neuroblastoma cell line (N18TG-2) with rat dorsal root ganglion cells. The total lipid-bound glucose of F-11 cells was estimated to be 0.28 micrograms/mg of protein and the total lipid-bound sialic acid was 0.82 micrograms/mg of protein. The major neutral glycosphingolipids were Gb4 (37% of the total neutral glycosphingolipids), Gb3 (15%), LacCer (21%), and GlcCer (15%). The major gangliosides were found to be GM3 (37% of the total gangliosides), GD3 (27%), O-acetylated GD3 (18%), and GD1a (4%), with trace amounts of GD2. The unusually high concentration of O-acetylated GD3 is consistent with its putative role as a tumor marker. Immunocytochemical localization studies of GD3 and O-acetylated GD3, examined by mouse monoclonal antibodies R24 and D1.1, respectively, revealed that the cell bodies and processes were all positively stained. To elucidate the role of O-acetylated GD3 in tumorigenesis, we transfected F-11 cells with the O-acetylesterase gene from influenza C virus. Compared with the original cell line, the transfected cells showed a dramatic increase in the level of GD3 (150% of that in the control cells) and a significant decrease of the concentration of O-acetylated GD3 (27% of control cells). In addition, the transfected F-11 cells exhibited a morphology different from the parental cells with enlarged cell bodies and elongated neurites. We conclude that alteration of ganglioside composition, particularly the expression of GD3 and O-acetylated GD3, may be associated with the morphological changes observed in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Seventeen steviol derivatives, i.e., 2-18, and 19 isosteviol derivatives, i.e., 19-37, were prepared from a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, nine steviol derivatives, i.e., 5-9 and 11-14, and five isosteviol derivatives, i.e., 28-32, exhibited activities with single-digit micromolar IC(50) values against one or more cell lines. All of these active compounds possess C(19)-O-acyl group, and among which, ent-kaur-16-ene-13,19-diol 19-O-4',4',4'-trifluorocrotonate (14) exhibited potent cytotoxicities against four cell lines with IC(50) values in the range of 1.2-4.1 μM. Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis-inducing activity by flow-cytometric analysis. These results suggested that acylation of the 19-OH group of kaurane- and beyerane-type diterpenoids might be useful for enhancement of their cytotoxicities with apoptosis-inducing activity.

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20–26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It is known that the proportion of VLC species in GSLs is higher than that in SM. To address the mechanism of the VLC-preference of GSLs, we used genome editing to establish three HeLa cell mutants that expressed different amounts of CERS2 and compared the acyl chain lengths of SM and GSLs by metabolic labeling experiments. VLC-sphingolipid expression was increased along with that of CERS2, and the proportion of VLC species in glucosylceramide (GlcCer) was higher than that in SM for all expression levels of CERS2. This higher proportion was still maintained even when the proportion of C16-Cer to the total ceramides was increased by disrupting the ceramide transport protein (CERT)-dependent C16-Cer delivery pathway for SM synthesis. On the other hand, merging the Golgi apparatus and the endoplasmic reticulum (ER) by Brefeldin A decreased the proportion of VLC species in GlcCer probably due to higher accessibility of UDP-glucose ceramide glucosyltransferase (UGCG) to C16-rich ceramides. These results suggest the existence of a yet-to-be-identified mechanism rendering VLC-Cer more accessible than C16-Cer to UGCG, which is independent of CERT.

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