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(The FASEB Journal. 2000;14:36-47.)
© 2000 FASEB

Stress-induced apoptosis is not mediated by endolysosomal ceramide

BRUNO SÉGUI*,1, CHRISTINE BEZOMBES{dagger},1, EMMANUELLE URO-COSTE*,1, JEFFREY A. MEDIN{ddagger}, NATHALIE ANDRIEU-ABADIE*, NATHALIE AUGÉ*, ANNE BROUCHET*, GUY LAURENT{dagger}, ROBERT SALVAYRE*, JEAN-PIERRE JAFFRÉZOU{dagger} and THIERRY LEVADE*,2

* INSERM U466, Laboratoire de Biochimie, Maladies Métaboliques, Institut Louis Bugnard, C.H.U. Rangueil, 31403 Toulouse, France;
{dagger} INSERM E9910, Institut Claudius Régaud, 31052 Toulouse, France; and
{ddagger} Section of Hematology/Oncology, University of Illinois at Chicago, Chicago, Illinois, USA

2Correspondence: INSERM U. 466, Laboratoire de Biochimie, ‘Maladies Métaboliques’, Institut Louis Bugnard, Bât. L3, C.H.U. Rangueil, 1 Avenue Jean Poulhès, F-31403 Toulouse Cedex 4, France. E-mail: levade{at}rangueil.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major lipid-signaling pathway in mammalian cells implicates the generation of ceramide from the ubiquitous sphingolipid sphingomyelin (SM). Hydrolysis of SM by a sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, the apoptotic cell death triggered by stress-inducing agents. In the present study, we investigated whether the ceramide formed within or accumulated in lysosomes indeed triggers apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-{alpha} nor anti-CD95 induced the degradation to ceramide of a natural SM that had been first introduced selectively into acidic compartments. 3) Stimulation of SV40-transformed fibroblasts by TNF-{alpha} or CD40 ligand resulted in apoptosis equally well in cells derived from control individuals and from patients affected with Farber disease, having a genetic defect of acid ceramidase activity leading to lysosomal accumulation of ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-{alpha}, daunorubicin, and ionizing radiation was similar in control and Farber disease lymphoid cells. In all cases, apoptosis was preceded by a comparable increase of intracellular ceramide levels. 4) Retroviral-mediated gene transfer and overexpression of acid ceramidase in Farber fibroblasts, which led to complete metabolic correction of the ceramide catabolic defect, did not affect the cell response to TNF-{alpha} and CD40 ligand.— Ségui, B., Bezombes, C., Uro-Coste, E., Medin, J. A., Andrieu-Abadie, N., Augé, N., Brouchet, A., Laurent, G., Salvayre, R., Jaffrézou, J.-P., Levade, T. Stress-induced apoptosis is not mediated by endolysosomal ceramide.


Key Words: sphingomyelin • sphingomyelinase • Farber disease • ceramidase • lysosome • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RECENT EVIDENCE HAS accumulated that, besides their structural role, sphingolipids play important functions in cell regulation and signal transduction. Among this class of lipids is ceramide (N-acylsphingosine), which serves as a precursor for sphingolipid biosynthesis (1 , 2) . Ceramide also derives from the stepwise degradation of all sphingolipids through the action of specific hydrolases, the best documented of which are located in the lysosomes. In particular, ceramide can originate from the breakdown of the major sphingolipid, sphingomyelin (SM) by an acid sphingomyelinase. Then, in the lysosomal compartment, acid ceramidase degrades ceramide to liberate sphingosine and a free fatty acid (2 , 3) .

A wide array of biological responses to extracellular stimuli have been described to be mediated by the intracellular generation of ceramide (for reviews, see refs 4 5 6 7 8 9 10 ). Among these effects is the induction, in a variety of cell types, of apoptotic cell death triggered by various stress agents, including ligation of the 55 kDa tumor necrosis factor (TNF) and CD95 (Fas/APO1) receptors, ionizing and UV radiation, anticancer drugs, heat shock, and exposure to hydrogen peroxide (5 , 11 12 13 14 15) . Ceramide has been proposed to mediate apoptosis induced by these agents because 1) the stimuli prompt the production of ceramide from SM hydrolysis, 2) the stimuli activate a sphingomyelinase, and 3) exogenously added ceramide (usually, synthetic short-chain, cell-permeant analogs of ceramide) mimics the cytotoxic effect.

Whereas different sphingomyelinases having distinct subcellular locations may implement different biological effects of ceramide (7 , 9) , an apoptotic function has been attributed by some authors to the ceramide produced in acidic organelles through the action of an acid sphingomyelinase. An increase in sphingomyelinase activity measured at acidic pH, which preceded the onset of apoptosis, has been reported in extracts of cells treated with TNF-{alpha} or anti-CD95 antibodies (16 , 17) . In addition, lymphoid cells derived from patients affected with Niemann-Pick disease, an inborn disorder characterized by the deficient activity of lysosomal sphingomyelinase (18 , 19) , failed to generate ceramide and to undergo apoptosis on irradiation (20) or CD95 ligation (21) . Similar observations were described on endothelial cells from acid sphingomyelinase knock-out mice that were irradiated (20) or exposed to lipopolysaccharide (22) . Taken together, these findings suggested that diverse stress agents can mediate their cytotoxic action through the generation of ceramide in endosomes/lysosomes. However, conflicting data have been published, including studies using the same cell lines and the same agonist. Indeed, not only has activation of a nonacidic sphingomyelinase frequently been reported subsequent to cell injuries (see Discussion), but also significant ceramide production and a normal apoptotic response have been observed in cells genetically lacking functional acid sphingomyelinase (23 , 24) .

The present study was designed to elucidate the role of the natural ceramide present or formed in acidic compartments in the transduction of apoptotic signals. We speculated that if the ceramide liberated by acid lysosomal sphingomyelinase is involved in apoptosis signaling, its accumulation in acidic compartments should result in cytotoxicity and/or sensitivity to apoptotic stress agents. To test this hypothesis, we first examined the response of cells loaded with ceramide under conditions that allow lysosomal targeting of the exogenous lipid. Secondly, we examined the response to apoptotic signals of cells derived from patients affected with Farber disease. Farber disease (lipogranulomatosis; McKusick 22800) is a rare, autosomal recessive, lysosomal storage disorder, which is characterized by the intracellular accumulation of ceramide as a result of a deficiency in the activity of acid lysosomal ceramidase (or N-acylsphingosine deacylase, E.C. 3.5.1.23) (25 , 26) . We postulated that in these cultured Farber cells the levels of lysosomal ceramide would be augmented, and, consequently, if this ceramide plays a role in apoptosis, these cells might have an altered sensitivity to stress-induced apoptosis. This study demonstrates that endosomal/lysosomal ceramide does not mediate the apoptotic cell death induced by diverse stress agents and that acid ceramidase is not essential in this signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Lipids and reagents
[ceramide-3H]Sphingomyelin ([ceramide-3H]SM, 400 mCi/mmol), prepared by catalytic tritiation of bovine brain SM, was obtained from C.E.A. (Gif-sur-Yvette, France) (27) . Radiolabeled ceramide was obtained by hydrolysis of [ceramide-3H]SM using B. cereus sphingomyelinase; N-palmitoyl-D-sphingosine, [palmitoyl-1-14C] (55 mCi/mmol) was purchased from A.R.C. (St Louis, Missouri). [{gamma}-32P]ATP (7000 Ci/mmol) was purchased from ICN (Orsay, France), and [methyl-3H]thymidine from Amersham (Les Ulis, France). Sphingomyelinase (B. cereus), C2-ceramide (N-acetyl-sphingosine), and dioleoylphosphatidylglycerol were supplied from Sigma (Lisle d’Abeau, France). Recombinant E. coli diacylglycerol kinase, octyl-ß-glucoside (Ultrol grade) and concanamycin A were from Calbiochem (Meudon, France); silica gel 60 TLC plates (Art. 5721) were from Merck (Darmstadt, Germany). Recombinant human TNF-{alpha} was purchased from PeproTech (Tebu, France). Antibodies to human CD95 (clone CH-11) and CD40 (clone M3) were from Upstate Biotechnology-Euromedex (Strasbourg, France) and Genzyme (Cergy, France), respectively. Soluble recombinant human CD40 ligand was kindly provided by Immunex (Seattle, Wash.). Daunorubicin was obtained from the National Cancer Institute Drug Repository. All solvents and other reagents obtained from Merck or SDS (Peypin, France) were of analytical grade. DMEM Glutamax, RPMI 1640 Glutamax, penicillin, streptomycin, and trypsin-EDTA were from Gibco BRL (Cergy-Pontoise, France); fetal calf serum was from Boehringer Ingelheim (Gagny, France).

Cell lines and cell culture
Human SV40-transformed skin fibroblasts were derived from normal individuals, from a patient affected with Niemann-Pick disease type A (line Ber), or from a patient affected with Farber disease, who died at 3 days of age (line Moh) (28) . The cells were routinely grown in a humidified 5% CO2 atmosphere at 37°C in DMEM medium containing Glutamax (2 mmol/l), penicillin (100 U/ml), streptomycin (100 µg/ml), and heat-inactivated fetal calf serum (10%), as previously reported (28 , 29) . Fibroblasts were transduced using a recombinant retroviral vector carrying the full-length human acid ceramidase cDNA as described (30) . Cultured skin fibroblasts were used after reaching confluency. Human Epstein-Barr virus-transformed lymphoid cell lines were derived from control subjects, from a patient affected with Niemann-Pick disease type A (line Tre), or from patients with Farber disease (lines GM5748, obtained from the Human Genetic Mutant Cell Repository, Camden, N.J.; or line Moz). These cell lines have previously been characterized with respect to SM and ceramide metabolism (27 , 31) . They were routinely grown in a humidified 5% CO2 atmosphere at 37°C in RPMI 1640 medium containing Glutamax (2 mmol/l), penicillin (100 U/ml), streptomycin (100 µg/ml), and heat-inactivated fetal calf serum (10%). Jurkat T cells were grown under the same conditions as the lymphoid cells. Irradiations were performed using a 60Co source (1.25 MeV, Alcyon, General Electric) at a dose rate of ~1 Gy/min.

Incubation of intact cells with sphingomyelin
Cells were incubated at 37°C for the indicated periods of time with medium containing 10% fetal calf serum and [ceramide-3H]SM (~106 dpm/ml, which was added as an ethanolic solution) (27) . Next, cells were briefly washed with fresh medium and further incubated in the presence or absence of TNF-{alpha} or anti-CD95. At the end of incubation, cells were washed three times with phosphate-buffered saline (PBS) containing bovine serum albumin (2 mg/ml) and twice with PBS alone, and harvested using a rubber policeman. The cell pellets were stored at -20°C.

Lipid extraction and analyses
Cell pellets were suspended in 0.6 ml distilled water and sonicated for 2 x 15 s (Soniprep MSE sonicator). After an aliquot was taken for protein determination (32) , the lipids were extracted (33) .

For analysis of the intracellular distribution of [ceramide-3H]SM metabolites, lipids were resolved by analytical TLC developed in chloroform/methanol/water (100:42:6, by vol.) up to two-thirds of the plate and then in chloroform/methanol/acetic acid (94:1:5, by vol.). The distribution of the radioactivity on the plate was analyzed using a Berthold LB2832 radiochromatoscan. Unlabeled or radioactive lipid standards were used to identify the various metabolic products, which were scraped and quantified by liquid scintillation.

Total intracellular SM levels were determined on cells metabolically labeled for 48 h with [3H]choline (1 µCi/ml) using the previously described procedure (34) . Ceramide levels were quantitated in the lipid extracts essentially as described (35) , using E. coli diacylglycerol kinase and [{gamma}-32P]ATP. Radioactive ceramide-1-phosphate was isolated by TLC using chloroform/acetone/methanol/acetic acid/water (50:20:15:10:5, by vol.) as the developing solvent. Alternatively, ceramide was quantitated after metabolic labeling of the cells for 48 h with 1 µCi/ml [9, 10-3H]palmitic acid (53.0 Ci/mmol; Amersham) (14 , 36) .

Acid ceramidase assay
Fibroblasts were harvested and washed in cold 250 mM sucrose/50 mM NaCl/10 mM Tris/HCl, pH 7.5. Cell pellets were suspended and disrupted in 250 mM sucrose/1 mM EDTA by brief sonication. Ceramidase activity was assayed as described previously (37) using [palmitoyl-1-14C]sphingosine (105 dpm/assay) as the substrate along with detergents in the presence of 125 mM citrate-phosphate buffer, pH 4. After a 2 h incubation at 37°C, the liberated radioactive fatty acid was isolated by phase partition, followed by TLC separation in chloroform/methanol/acetic acid (94:1:5, by vol.). After exposure to iodine vapors and/or radiochromato-scanning to locate the fatty acid on the plate, the radioactive spots were scraped and counted by liquid scintillation.

Sphingomyelinase assay
Neutral and acid sphingomyelinase activities were determined on freshly isolated cell pellets essentially as described (14 , 38) , using [choline-methyl-14C]SM (NEN; 100,000 dpm/assay) as substrate.

Morphological studies, cell viability, and DNA fragmentation assays
Lymphoid cells were centrifuged at low speed on glass slides and examined under the light microscope after May-Grünwald-Giemsa (MGG) staining. Cell membrane permeability was assessed by the trypan blue dye exclusion test. In some experiments, [3H]thymidine incorporation was monitored as previously reported (34) . Fibroblast viability was estimated directly on the culture flasks by coincubation with propidium iodide (6 µM) and Syto-13 (1 µM; Molecular Probes, Leiden, The Netherlands). The percentage of apoptotic cells (having a condensed and fragmented nucleus) was evaluated by counting cells under a Leica fluorescence-equipped inverted microscope.

Quantitative DNA fragmentation was determined by the spectrofluorometric DAPI procedure as described previously (39 , 40) .

PARP cleavage assay
Analysis of poly(ADP-ribose)polymerase (PARP) proteolysis was assessed by resuspending cells in sample buffer (62.5 mM Tris, pH 6.8, 4 M urea, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.04% bromophenol blue). Samples were boiled for 5 min, loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. PARP and its cleaved fragment were detected by using a rabbit polyclonal antiserum (Boerhinger-Mannheim, Meylan, France) and a donkey anti-rabbit secondary antibody (Immunotech, Marseille, France). The signal was visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, U.K.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ceramide loading of acidic compartments does not affect cell viability
To analyze the purported proapoptotic role of ceramide present in acidic organelles, cultured cells were loaded with natural ceramide. This ceramide was targeted to endosomes/lysosomes via receptor-mediated endocytosis (41) by incubating the living cells with human LDL that were pretreated with a bacterial sphingomyelinase to degrade virtually all the lipoprotein SM to ceramide (Fig. 1C ). As shown in Fig. 1A , although an agonistic anti-CD95 antibody induced cell death, loading Jurkat T cells with LDL-ceramide (see Fig. 1D ) did not significantly alter their viability. In contrast to the natural compound delivered to acidic compartments, synthetic short-chain ceramides (i.e., N-acetyl- or N-hexanoyl-sphingosines), which readily enter cells, were cytotoxic (Fig. 1A ).



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  		Figure 1. Ceramide loading of acidic compartments does not affect cell viability. A, B) Effect of ceramide loading on the viability of Jurkat cells. Jurkat cells (106 dpm/ml) were incubated in RPMI medium without fetal calf serum in the presence or absence of anti-CD95 ({alpha}CD95, 100 ng/ml), N-acetyl-sphingosine (C2-ceramide, 10 µM), or native (LDL) or bacterial sphingomyelinase-treated (SMase-LDL) low density lipoproteins (90 or 180 µg apoB/ml). In panel B, the cells were incubated with LDL (180 µg apoB/ml) or anti-CD95 and with the indicated concentration of concanamycin A. After 24 h incubation, trypan blue cells were counted. The data correspond to the mean ± SD of at least three separate determinations. C) SM and ceramide content in native and bacterial sphingomyelinase (SMase)-treated LDL. D) Ceramide content in cells incubated for 24 h in the presence of 90 µg apoB/ml of native and sphingomyelinase (SMase)-treated LDL. Ceramide was quantified using the diacylglycerol method (mean ± SE, n=3).

To test whether accumulation of ceramide in endosomes could affect cell viability, cells were coincubated with LDL-ceramide and concanamycin A, an antibiotic that blocks the transport from endosomes to lysosomes as a result of inhibition of vacuolar proton-ATPase and neutralization of the pH of acidic organelles. Fig. 1B shows that inhibition of ceramide traffic to lysosomes did not alter the viability of Jurkat cells. Identical results were obtained on EBV-transformed lymphoid cells and on the leukemic U937 cell line (data not shown). These findings strongly suggest that the natural ceramide introduced into endosomes and lysosomes does not signal cell death in cellular models where ceramide has been described to serve as an apoptotic mediator.

TNF-{alpha} or CD95 stimulation does not result in degradation of lysosomal sphingomyelin
Because ceramide has been proposed to be generated in acidic organelles through TNF-{alpha} or CD95 receptor-mediated activation of an acid sphingomyelinase (12 , 42) , and because we and others have found that acid sphingomyelinase-deficient (Niemann-Pick disease) cells do not exhibit defects in the apoptotic response nor in the activation of the ceramide pathway (see Discussion), we examined whether TNF-{alpha} or CD95 could stimulate the degradation of SM present in the endosomal/lysosomal compartment. Lymphoid cells and SV40-transformed fibroblasts derived from patients with Niemann-Pick disease type A, which accumulate SM intracellularly, were loaded with radioactive SM under conditions that target this SM to acidic organelles (27 , 31) , and then treated with TNF-{alpha} or with an agonistic anti-CD95 antibody (CH-11). As seen in Fig. 2 , the radiolabeled SM accumulated in endosomes/lysosomes because of the defect in acid sphingomyelinase. Notably, neither stimulus induced a detectable, time-dependent hydrolysis of the SM present in acidic compartments (compare with Figs. 3 , 4 , and 7 ), indicating that lysosomal SM is not accessible to the sphingomyelinase activated (even in Niemann-Pick cells) by apoptotic agents.



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  		Figure 2. TNF-{alpha} and CD95 ligation does not induce the degradation of sphingomyelin present in acidic compartments. EBV-transformed lymphoid cells (A) and SV40-transformed fibroblasts (B) from patients with type A Niemann-Pick disease were incubated with [ceramide-3H]SM (~106 dpm/ml) in the presence of lipoproteins. After 24 h incubation, cells were chased for 2 h in fresh medium and then treated for the indicated times in the absence or presence of anti-CD95 ({alpha}CD95, 200 ng/ml) or TNF-{alpha} (3 nM). Cellular lipids were extracted and separated by TLC. Radiochromato-scannings from a typical experiment (out of two with different cell lines) are shown.



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  		Figure 3. Ceramide accumulation in Farber cells. A, B) Intracellular ceramide concentration in EBV-transformed lymphoid cells (A) and SV40-transformed fibroblasts (B) from control subjects and patients with Farber disease. Ceramide concentration (mean ± SE, n=3) was determined using the diacylglycerol method and is expressed as pmole/mg of cell protein. C–F) Intracellular [ceramide-3H]SM-derived ceramide turnover in EBV-transformed lymphoid cells (C, E) and SV40-transformed fibroblasts (D, F) from control subjects (E, F) and patients with Farber disease (C, D). Cells were incubated for 24 h with [ceramide-3H]SM (~106 dpm/ml) in the presence of lipoproteins. Cellular lipids were then extracted and separated by TLC. Radiochromato-scannings from a typical experiment (out of more than three) are shown; the position of SM and ceramide (CER) is indicated.



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  		Figure 4. Activation of the sphingomyelin-ceramide pathway in Farber lymphoid cells by stress agents. A) Kinetics of anti-CD95–induced ceramide formation in control and Farber lymphoid cells. Cells were metabolically labeled with [3H]palmitic acid and then treated with anti-CD95 ({alpha}CD95, 500 ng/ml) or with an irrelevant antibody (-{alpha}CD95) for the indicated times. Cellular lipids were extracted and separated by TLC and radiolabeled ceramide was scraped and counted. The ceramide contents are expressed as percentage of the values at time 0 (mean ± SE, n=3–5). B) CD40 ligation-induced hydrolysis of SM in lymphoid cells. SM was quantified on cells that were metabolically labeled with [3H]choline and then treated with anti-CD40 ({alpha}CD40, 5 µg/ml) for the indicated times (mean ± SE, n=3). C) Production of ceramide in lymphoid cells after treatment with daunorubicin (DNR, 1 µM), TNF-{alpha} (3 nM) + cycloheximide (25 µg/ml), or ionizing radiation (IR, 12 Gy). [3H]Palmitic acid-labeled ceramide was quantified (values represent peak ceramide production obtained between 4 and 10 min); data are expressed at percentage of the values in untreated cells (mean ± SE,n=3). D) Neutral sphingomyelinase activation by stress agents in lymphoid Farber cells. Sphingomyelinase activity was determined on lymphoid cell extracts (values represent peak enzyme stimulation observed between 4 and 10 min) (mean ± SE, n=3). All changes were found to be significant (P<0.05) as compared with controls.



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  		Figure 7. Acid ceramidase activity is dispensable for stress-induced apoptosis of SV40-transformed fibroblasts. A) Acid ceramidase activity in SV40-transformed fibroblasts from a control individual and from a patient with Farber disease before (Farber mock) and after retrovirally mediated gene correction (Farber corr) (mean ± SE, n=4). B) TNF-{alpha}-induced activation of SM hydrolysis in control, Farber, and acid ceramidase-transduced Farber fibroblasts. SM was quantified on cells that were metabolically labeled with [3H]choline and then treated with TNF-{alpha} (3 nM) for the indicated times (mean ± SE, n=3). (C–E) Effect of TNF-{alpha} and CD40 ligation on the viability of control, Farber, and acid ceramidase-transduced Farber SV40-transformed fibroblasts. Cells were incubated for 24 h with cycloheximide (CHX, 50 µg/ml) in the presence or absence of TNF-{alpha} (3 nM) or soluble recombinant CD40 ligand (sCD40L, 5 µg/ml), and [3H]thymidine incorporation (C) was monitored during the last 16 h (mean ± SE, n=3). Apoptotic cells were also counted after 24 h incubation using MGG (D) or Syto13/propidium iodide (E) staining.

Accumulation of ceramide in Farber disease cell lines does not affect cell growth
To further evaluate the role of lysosomal ceramide and acid ceramidase in apoptosis, we used both SV40-transformed skin fibroblasts and EBV-transformed lymphoid cell lines derived from patients affected with Farber disease. As illustrated in Fig. 3 , these mutant cells exhibited a 3- to 10-fold increase in intracellular ceramide concentration (A, B) and failed to degrade the ceramide produced intra-lysosomally from the hydrolysis of a radiolabeled SM (C–F) that was targeted to lysosomes through lipoprotein receptor-mediated endocytosis (27 , 31 , 41) . These results are in accordance with the previously reported defect in acid ceramidase activity measured in cell extracts (28 , 43) .

Most notably, even though these cells accumulate large amounts of ceramide, their growth rate in either 1 or 10% fetal calf serum-containing medium was similar to that of their normal counterparts (data not shown), as was the basal number of apoptotic cells (see Fig. 5 ).



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  		Figure 5. Effect of CD95 ligation on the viability of control and Farber lymphoid cells. EBV-transformed lymphoid cells (106/ml) were treated with anti-CD95 ({alpha}CD95, 500 ng/ml) for 5 h in the absence of fetal calf serum. Then, cells were spun on glass slides for MGG staining (A, B), lysed for quantifying DNA fragmentation (C), or analyzed by Western blotting using an anti-PARP antibody (D). CD95 ligation induced cell size reduction and nuclear condensation and fragmentation of both control and Farber lymphoid cells (A); the presence of these features was considered as typical for apoptosis in counting apoptotic cells (B).

The sphingomyelin-ceramide pathway can be activated in Farber lymphoid cells
Because ceramide has been proposed to be produced on treatment with, and to transduce the apoptotic effect of, diverse stress agents, we sought to determine whether the apoptosis of Farber cells induced by these stimuli was accompanied by the activation of the ceramide pathway. This was studied either by measuring the sphingomyelinase activity or by following the SM breakdown and ceramide generation. Fig. 4 demonstrates that all stress stimuli used here activated the ceramide pathway equally well in Farber and control lymphoid cells. Indeed, CD95 stimulation (Fig. 4A ), as well as daunorubicin, TNF-{alpha}, and ionizing radiation treatment (Fig. 4C ), resulted in an increase of cellular ceramide concentration and a concomitant increase in neutral sphingomyelinase activity (Fig. 4D ). SM hydrolysis was also stimulated similarly in normal and Farber cells by anti-CD40 (Fig. 4B ). Acid sphingomyelinase activity was not significantly affected after treatment with daunorubicin or anti-CD95 (data not shown). Of note, despite their deficient acid ceramidase activity the Farber cells did not exhibit a higher amount of agonist-induced ceramide than their normal counterparts, suggesting little contribution of the lysosomal pathway in the production of this ceramide.

Consistent with these findings, when radiolabeled SM loading experiments (as in Fig. 2 ) were performed on Farber fibroblasts or lymphoid cells, neither TNF-{alpha} nor anti-CD95 promoted an enhanced catabolism of lysosomal ceramide (data not shown).

Control and Farber lymphoid cells are equally sensitive to CD95-mediated apoptosis
Because CD95 ligation has been reported to activate the SM-ceramide pathway and to promote apoptosis in EBV-transformed lymphoid cell lines (21 , 24) , we investigated the effect of an agonistic anti-CD95 (CH-11) antibody on lymphoid cells derived from Farber disease patients. As shown in Fig. 5 , triggering of the CD95 receptor led to apoptotic cell death of both control and Farber cells. Indeed, the characteristic features of apoptosis were observed morphologically (i.e., cell size reduction, chromatin condensation, and membrane blebbing; Fig. 5A ), by monitoring the DNA fragmentation (Fig. 5C ), and by examining the cleavage of PARP (Fig. 5D ). These alterations were followed by modifications of plasma membrane permeability as measured by trypan blue dye uptake (data not shown), indicative of postapoptotic necrotic features. Quantification of these phenomena indicated that Farber lymphoid cells underwent apoptosis in a similar fashion and with a similar time course (data not shown) as their normal counterparts (Fig. 5B ).

Control and Farber lymphoid cells are equally sensitive to TNF-{alpha}, CD40, daunorubicin, and ionizing radiation-induced apoptosis
The sensitivity of Farber lymphoid cells to apoptotic agents other than anti-CD95 was evaluated. Control and Farber lymphoid cells were exposed to TNF-{alpha}, daunorubicin, ionizing radiation, and anti-CD40, all stimuli that have been shown to induce apoptosis. Fig. 6 demonstrates that the cytotoxic effect of these agents was similar in control and Farber cells. Indeed, not only was the growth of control and mutant cells similarly inhibited (Fig. 6A, B ), but also PARP was cleaved (Fig. 6C ) on treatment by the stress agents. Furthermore, morphological studies using MGG, DAPI, or TUNEL staining indicated characteristic features of apoptosis (data not shown). Finally, control and Farber cells were equally sensitive to cell-permeant ceramides (data not shown), indicating that these synthetic short-chain ceramides are cytotoxic irrespective of the degree of accumulation of natural ceramide in acidic compartments.



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  		Figure 6. Effect of daunorubicin, TNF-{alpha}, ionizing radiation and CD40 ligation on the viability of control and Farber lymphoid cells. A) Lymphoid cells (4x105/ml) were exposed to daunorubicin (DNR, 1 µM) for 1 h and then placed in fresh medium, or treated with TNF-{alpha} (3 nM) + cycloheximide (25 µg/ml), or exposed to ionizing radiation (IR, 12 Gy). After 24 h, viable (trypan blue-negative) cells were counted (mean ± SE, n=3). B) Effect of CD40 ligation on thymidine incorporation in lymphoid cells. Cells were treated with anti-CD40 ({alpha}CD40, 5 µg/ml) for 72 h; [3H]thymidine was added to the medium during the last 16 h (mean ± SE, n=3). C) Effect of stress agents on PARP cleavage. Control (N) and Farber (F) cells were treated as indicated above, and cell lysates were immunoblotted using an anti-PARP antibody.

Untransduced and genetically corrected Farber fibroblasts are equally sensitive to TNF-{alpha} and CD40-induced apoptosis
To investigate whether the sensitivity of Farber cells to apoptotic agents was restricted to lymphoid cells, we examined the effect of stress inducers on another cell type (i.e., skin fibroblasts). SV40-transformed fibroblasts have been reported to undergo apoptotic cell death on stimulation of members of the TNF receptor superfamily (e.g., TNF, CD95, and CD40 receptors) (44) . In addition, this response in SV40-transformed fibroblasts is accompanied by the activation of the ceramide pathway (Ségui, B., and Levade, T., unpublished results). As illustrated in Fig. 7 (C–E), exposure of SV40-transformed fibroblasts from a patient with Farber disease to TNF-{alpha} or CD40 ligand led to a reduction in thymidine incorporation, which was linked to apoptosis as evidenced by morphological alterations. No difference was found in cytotoxicity between control and Farber fibroblast cell lines. In addition, TNF and CD40 receptor stimulation resulted in SM hydrolysis in both mutant and control fibroblasts (Fig. 7B ).

To further examine the importance of acid ceramidase in this apoptotic signaling pathway, we tested the ability of SV40-transformed fibroblasts that overexpress acid ceramidase to respond to TNF-{alpha} or CD40 ligation. Toward this aim, we used Farber fibroblasts that had been infected with a recombinant retroviral vector encoding acid ceramidase (30) . These transduced cells not only exhibit a fully corrected catabolism of ceramide but also overexpress acid ceramidase (~5x above the activity found in normal control cells; Fig. 7A ). Despite their high acid ceramidase activity, these transduced cells responded to TNF-{alpha} and CD40 ligation exactly as mock-transduced cells, by activating the ceramide pathway (Fig. 7B ) and by undergoing apoptosis (Fig. 7C-E ).

Similar observations were made on Farber lymphoid cells that were challenged with anti-CD95, TNF-{alpha}, daunorubicin, or ionizing radiation after cross-correction of their metabolic deficiency (data not shown) (i.e., after having been incubated with the culture medium of genetically corrected Farber fibroblasts to normalize the acid ceramidase activity) (30) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The sphingolipid ceramide has recently emerged as an important second messenger molecule, mediating a number of cellular responses to various exogenous stimuli and specially stress agents (4 , 5 , 7 8 9 10 , 45) . In fact, the intracellular concentration of ceramide has been shown to increase after cell stimulation with cytokines (TNF-{alpha}), antibodies (anti-CD95, anti-IgM), UV and ionizing radiation, anticancer drugs (e.g., daunorubicin, vincristine), serum withdrawal, heat, and hypoxia. The generation of ceramide was not merely the consequence of general cell dysfunction and death, because inhibition of apoptosis by certain caspase synthetic or viral protein inhibitors did not abrogate the ceramide increase (5 , 10 , 46) . Rather, ceramide is believed to belong to the pathways of the signaling/commitment phase of apoptosis (5 , 9 , 10) . Indeed, exogenously supplied ceramide or the ceramide formed at the cell surface after treatment with bacterial sphingomyelinase can activate multiple pathways that play a critical role in the apoptotic process (e.g., the JNK/SAPK pathway, mitochondrial alterations, caspase cascade, and NF-{kappa}B nuclear translocation).

While the targets of ceramide begin to be deciphered, controversy exists as to the mechanisms of its generation, in particular the nature and subcellular localization of the enzyme sphingomyelinase. Various sphingomyelinases are present in mammalian cells that can be distinguished by their subcellular localization and pH optimum (1 , 2 , 18) . Of these, the best characterized enzyme is the acid sphingomyelinase. This enzyme is present in acidic organelles, and its deficiency gives rise to the lipid storage disorder Niemann-Pick disease (18 , 19) . The implication of an acid sphingomyelinase in signal transduction was first reported by Krönke et al. as an intermediate for TNF-induced nuclear translocation of NF-{kappa}B in leukemic cells (42) . Subsequent studies by these authors indicated that only the ceramide liberated by the acid sphingomyelinase in endosomes/lysosomes could mediate the TNF-induced activation of NF-{kappa}B and the induction of cell death (38) . In this model, TNF-dependent activation of an acid sphingomyelinase is believed to be signaled by the death domain region of the TNF p55 receptor and involves the recruitment of the TRADD and FADD proteins (9 , 47 , 48) . Other observations, still based on in vitro measurements of enzyme activity, have suggested a signaling function for acid sphingomyelinase in apoptosis triggered by anti-CD95 (12 , 16) and anti-IgM (49) . Moreover, cultured cells derived from patients with Niemann-Pick disease or certain cell types from acid sphingomyelinase-deficient mice were reported to be resistant to ionizing radiation (20) , doxorubicin (23) , lipopolysaccharide (22) , or CD95 stimulation (21) . Overall, these findings gave support to the idea that the ceramide formed in acidic compartments serves as a second messenger for diverse stress stimuli.

However, this concept is challenged by several observations that strongly argue against this view (50) . First, activation of a neutral, but not acid, sphingomyelinase has been associated with induction of apoptosis by a number of stress agents, including TNF-{alpha} (17 , 38 , 51 , 52) , anti-CD95 (53) , anti-IgM (54) , anti-CD40 (Ségui, B., Andrieu-Abadie, N., and Levade, T., unpublished results), daunorubicin (14) , Ara-C, cis-platinum, taxol, mitoxantrone (55 , 56) , serum withdrawal (57) , ionizing radiation (58) , and hypoxia (59) . Second, cells having a genetic deficiency of acid sphingomyelinase (i.e., Niemann-Pick cells) were found to generate ceramide and to undergo apoptosis after CD95 stimulation (24) or exposure to anti-class I antibodies (60) . We recently extended these findings to mutant cells exposed to a variety of stress stimuli (Bezombes, C., Ségui, B., Bruno, A.P., Uro-Coste, E., Andrieu-Abadie, N., Laurent, G., Jaffrézou, J.P., Levade, T., unpublished results). Moreover, hydrolysis of SM was observed in TNF-stimulated Niemann-Pick cells (34) , and diverse biological responses supposed to be mediated by ceramide were noted in these deficient cells, ranging from NF-{kappa}B (61 62 63) , ERK or JNK activation (23 , 64) to cytokine production (65 , 66) . Even when apoptosis was monitored in acid sphingomyelinase-deficient mice, only some cell types resisted the stress agent (20) . Consistent with these findings is the marked absence of similitude between the Niemann-Pick condition and the phenotypes of CD95 or CD95 ligand-defective animals (50 , 67) . Third, a series of experiments have indicated that the signal-induced hydrolysis of SM occurs in (or very close to) the inner leaflet of the plasma membrane but not in endosomes (36 , 52 , 68 , 69) . Finally, natural ceramide formed or introduced into the acidic compartments appears to be unable to escape these organelles (41) , which makes it difficult to understand how a ceramide present in the lumen of endosomes/lysosomes could activate protein targets present in other subcellular compartments.

In addition to the above observations, the present study strongly argues against an essential role for endosomal/lysosomal ceramide in stress-induced apoptosis. Two experimental approaches have been used to investigate directly the possible signaling function of endosomal/lysosomal ceramide. One approach was to selectively introduce natural ceramide in acidic organelles of cells (like Jurkat) that are very sensitive to ceramide-induced cell death. We demonstrate that ceramide loading of acidic organelles failed to result in apoptosis, even when the transport between endosomes and lysosomes was blocked. In addition, we show that apoptotic agents known to trigger the SM-ceramide pathway did not stimulate the breakdown of a SM initially targeted to the endosomal/lysosomal compartment of Niemann-Pick cells where it accumulated owing to the deficient activity of acid sphingomyelinase. In the same cells, those agents induced an apoptotic cell death that was preceded by neutral sphingomyelinase activation and hydrolysis of SM (as followed by labeling of all cellular SM pools) (Bezombes, C., Ségui, B., Bruno, A.P., Uro-Coste, E., Andrieu-Abadie, N., Laurent, G., Jaffrézou, J.P., Levade, T., unpublished results).

To further analyze the role of endosomal/lysosomal ceramide, we used a genetic approach. Cultured cells having a genetic defect in acid ceramidase activity (Farber disease) were tested for their ability to respond to various stress stimuli. These cells, which were of the fibroblast or lymphoid type, accumulated ceramide as a result of the block in its lysosomal degradation. However, despite the ceramide storage in acidic compartments, they did not manifest any cell viability abnormality, either in the absence or presence of a stress agent. When stimulated with TNF-{alpha}, anti-CD95 or CD40 antibodies, anthracycline, or ionizing radiation, as well as cell-permeant ceramides, Farber cells underwent apoptosis just as control cells, both quantitatively and with the same dose-dependency and kinetics. In addition, Farber fibroblasts were equally sensitive to oxidized LDL (data not shown), which has been previously shown to activate the ceramide pathway (70) and to induce apoptosis (71) . These results agree with the notion that Farber disease is characterized clinically by symptoms that do not evoke any anomaly of apoptotic processes (26) and that cultured cells derived from patients affected with this inborn disorder do not exhibit particular growth difficulties.

While the above observations provide evidence for the absence of signaling functions of endosomal/lysosomal ceramide, other findings support the idea that functional acid ceramidase is not required for and does not influence transduction of apoptotic signals. Indeed, cells having a genetic deficiency of acid ceramidase are shown here to activate the SM pathway and to generate ceramide in response to various stress agents in a similar way as control cells. Furthermore, stable overexpression of acid ceramidase (up to 500% of the activity in normal cells) was not accompanied by any alteration in cell viability or in stress-induced apoptotic response and hydrolysis of SM. These results are in agreement with those of Boesen de Cock et al. (24) , showing that acid sphingomyelinase is fully dispensable for CD95-induced cell death. They also demonstrate that metabolic transformation of lysosomal ceramide (e.g., production of sphingosine) is not necessary for apoptosis signaling, suggesting that if sphingosine formation is an important step for mediating apoptosis (45) , this sphingosine is not generated in acidic organelles.

Taken together, these findings strongly argue against endosomal/lysosomal ceramide playing a role in stress-induced apoptosis and further strengthen the notion that the ceramide produced in cells stimulated by apoptotic agents does not result from an endosomal/lysosomal sphingomyelinase activity. Whereas some studies have described the implication of an acid sphingomyelinase based on in vitro measurements of enzyme activity or on observations on genetically acid sphingomyelinase-deficient cells, this investigation presents topological arguments that exclude the endosomal/lysosomal compartment as a site for generation of apoptotic ceramide. Whether the signaling ceramide can be formed in another acidic compartment by a yet undefined sphingomyelinase remains to be clarified, as does the role of the secretory form of acid sphingomyelinase, which is also encoded by the gene mutated in Niemann-Pick disease (72) .


   ACKNOWLEDGMENTS
 
Note added in proof: In agreement with the present study, Tohyama et al. (1999), J. Inher. Metab. Dis. 22, 649–662, have recently reported the lack of increase in apoptosis in Farber fibroblasts.

The authors thank Dr. K. Harzer for providing the primary culture of Farber fibroblasts (line Moh), and S. Carpentier, J. P. Basile, and J. C. Thiers for acid ceramidase assays and iconography. Financial support by INSERM, Faculté de Médecine-Rangueil Université Paul Sabatier Toulouse, Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, Vaincre les Maladies Lysosomales, and Centre National d’Etudes Spatiales is gratefully acknowledged.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Abbreviations: SM, sphingomyelin; TNF, tumor necrosis factor; EBV, Epstein-Barr virus; LDL, low-density lipoprotein; MGG, May-Grünwald-Giemsa; PARP, poly(ADP-ribose)polymerase; TLC, thin-layer chromatography.

Received for publication April 22, 1999. Revised for publication September 10, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Merrill, A. H., Jr, Jones, D. D. (1990) An update of the enzymology and regulation of sphingomyelin metabolism. Biochim. Biophys. Acta 1044,1-12[Medline]
  2. Kolesnick, R. N. (1991) Sphingomyelin and derivatives as cellular signals. Prog. Lipid Res. 30,1-38[Medline]
  3. Sandhoff, K., Kolter, T. (1996) Topology of glycosphingolipid degradation. Trends Cell Biol 6,98-103[Medline]
  4. Ballou, L. R., Laulederkind, S. J. F., Rosloniec, E. F., Raghow, R. (1996) Ceramide signaling and the immune response. Biochim. Biophys. Acta 1301,273-287[Medline]
  5. Hannun, Y. A. (1996) Functions of ceramide in coordinating cellular responses to stress. Science 274,1855-1859[Abstract/Free Full Text]
  6. Spiegel, S., Foster, D., Kolesnick, R. N. (1996) Signal transduction through lipid second messengers. Current Opin. Cell Biol. 8,159-167[Medline]
  7. Testi, R. (1996) Sphingomyelin breakdown and cell fate. Trends Biol. Sci. 21,468-471
  8. Riboni, L., Viani, P., Bassi, R., Prinetti, A., Tettamanti, G. (1997) The role of sphingolipids in the process of signal transduction. Prog. Lipid Res. 36,153-195[Medline]
  9. Kolesnick, R. N., Krönke, M. (1998) Regulation of ceramide production and apoptosis. Annu. Rev. Physiol. 60,643-665[Medline]
  10. Levade, T., Jaffrézou, J. P. (1999) Signalling sphingomyelinases: which, where, how and why?. Biochim. Biophys. Acta 1438,1-17[Medline]
  11. Obeid, L. M., Linardic, C. M., Karolak, L. A., Hannun, Y. A. (1993) Programmed cell death induced by ceramide. Science 259,1769-1771[Abstract/Free Full Text]
  12. Cifone, M. G., De Maria, R., Roncaioli, P., Rippo, M. R., Azuma, M., Lanier, L. L., Santoni, A., Testi, R. (1994) Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J. Exp. Med. 180,1547-1552[Abstract/Free Full Text]
  13. Haimovitz-Friedman, A., Kan, C. C., Ehleiter, D., Persaud, R. S., McLoughlin, M., Fuks, Z., Kolesnick, R. M. (1994) Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J. Exp. Med. 180,525-535[Abstract/Free Full Text]
  14. Jaffrézou, J. P., Levade, T., Bettaieb, A., Andrieu, N., Bezombes, C., Maestre, N., Vermeersch, S., Rousse, A., Laurent, G. (1996) Daunorubicin-induced apoptosis: triggering of ceramide generation through sphingomyelin hydrolysis. EMBO J 15,2417-2424[Medline]
  15. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., Kolesnick, R. N. (1996) Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature (London) 380,75-79[Medline]
  16. Cifone, M. G., Roncaioli, P., De Maria, R., Camarda, G., Santoni, A., Ruberti, G., Testi, R. (1995) Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J 14,5859-5868[Medline]
  17. Wright, S. C., Zheng, H., Zhong, J. (1996) Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease (AP24). FASEB J 10,325-332[Abstract]
  18. Levade, T., Salvayre, R., Douste-Blazy, L. (1986) Sphingomyelinases and Niemann-Pick disease. J. Clin. Chem. Clin. Biochem. 24,205-220[Medline]
  19. Schuchman, E. H., Desnick, R. J. (1995) Niemann-Pick disease types A and B: acid sphingomyelinase deficiencies. Scriver, C. R. Beaudet, A. L. Sly, W. S. Valle, D. eds. The Metabolic and Molecular Bases of Inherited Disease 7th Ed ,2601-2624 McGraw-Hill New York.
  20. Santana, P., Pena, L. A., Haimovitz-Friedman, A., Martin, S., Green, D., McLoughlin, M., Cordon-Cardo, C., Schuchman, E. H., Fuks, Z., Kolesnick, R. (1996) Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 86,189-199[Medline]
  21. De Maria, R., Rippo, M. R., Schuchman, E. H., Testi, R. (1998) Acidic sphingomyelinase (ASM) is necessary for Fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells. J. Exp. Med. 187,897-902[Abstract/Free Full Text]
  22. Haimovitz-Friedman, A., Cordon-Cardo, C., Bayoumy, S., McLoughlin, M., Gallily, R., Edwards, C. K., Schuchman, E. H., Fuks, Z., Kolesnick, R. (1997) Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation. J. Exp. Med. 186,1831-1841[Abstract/Free Full Text]
  23. Herr, I., Wilhelm, D., Böhler, T., Angel, P., Debatin, K. M. (1997) Activation of CD95 (Apo-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis. EMBO J 16,6200-6208[Medline]
  24. Boesen de Cock, J. G. R., Tepper, A. D., de Vries, E., van Blitterswijk, W. J., Borst, J. (1998) CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase. J. Biol. Chem. 273,7560-7565[Abstract/Free Full Text]
  25. Sugita, M., Dulaney, J. T., Moser, H. W. (1972) Ceramidase deficiency in Farber’s disease (lipogranulomatosis). Science 178,1100-1102[Abstract/Free Full Text]
  26. Moser, H. W. (1995) Ceramidase deficiency: Farber lipogranulomatosis. Scriver, C. R. Beaudet, A. L. Sly, W. S. Valle, D. eds. The Metabolic and Molecular Bases of Inherited Disease 7th Ed ,2589-2599 McGraw-Hill New York.
  27. Graber, D., Salvayre, R., Levade, T. (1994) Accurate differentiation of neuronopathic and nonneuronopathic forms of Niemann-Pick disease by evaluation of effective residual lysosomal sphingomyelinase activity in intact cells. J. Neurochem. 63,1060-1068[Medline]
  28. Chatelut, M., Harzer, K., Christomanou, H., Feunteun, J., Pieraggi, M. T., Paton, B., Kishimoto, Y., O’Brien, J., Basile, J. P., Thiers, J. C., Salvayre, R., Levade, T. (1997) Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies. Clin. Chim. Acta 262,61-76[Medline]
  29. Chatelut, M., Feunteun, J., Harzer, K., Fensom, A. H., Basile, J. P., Salvayre, R., Levade, T. (1996) A simple method for screening for Farber disease on cultured skin fibroblasts. Clin. Chim. Acta 245,61-71[Medline]
  30. Medin, J. A., Takenaka, T., Carpentier, S., Garcia, S., Basile, J. P., Ségui, B., Andrieu-Abadie, N., Augé, N., Salvayre, R., Levade, T. (1999) Retrovirus-mediated correction of the metabolic defect in cultured Farber disease cells. Hum. Gene Ther. 10,1321-1329[Medline]
  31. Levade, T., Leruth, M., Graber, D., Moisand, A., Vermeersch, S., Salvayre, R., Courtoy, P. (1996) In situ assay of acid sphingomyelinase and ceramidase based on LDL-mediated lysosomal targeting of ceramide-labeled sphingomyelin. J. Lipid Res. 37,2525-2538[Abstract]
  32. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, M. N., Olson, B. J., Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150,76-85[Medline]
  33. Folch, J., Lees, M., Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497-509[Free Full Text]
  34. Andrieu, N., Salvayre, R., Levade, T. (1994) Evidence against involvement of the acid lysosomal sphingomyelinase in the tumour-necrosis-factor- and interleukin-1-induced sphingomyelin cycle and cell proliferation in human fibroblasts. Biochem. J. 303,341-345
  35. van Veldhoven, P. P., Matthews, T. J., Bolognesi, D. P., Bell, R. M. (1992) Changes in bioactive lipids, alkylacylglycerol and ceramide, occur in HIV-infected cells. Biochem. Biophys. Res. Commun. 187,209-216[Medline]
  36. Andrieu, N., Salvayre, R., Jaffrézou, J. P., Levade, T. (1995) Low temperatures and hypertonicity do not block cytokine-induced stimulation of the sphingomyelin pathway but inhibit nuclear factor-{kappa}B activation. J. Biol. Chem. 270,24518-24524[Abstract/Free Full Text]
  37. Dulaney, J. T., Moser, H. W. (1977) Farber disease (lipogranulomatosis). Glew, R. H. Peters, S. P. eds. Practical Enzymology of the Sphingolipidoses ,283-296 Alan R. Liss New York.
  38. Wiegmann, K., Schütze, S., Machleidt, T., Witte, D., Krönke, M. (1994) Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78,1005-1015[Medline]
  39. McConkey, D. J., Aguilar-Santelises, M., Harztell, P., Eriksson, I., Mellstedt, H., Orrenius, S., Jondal, M. (1991) Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells. J. Immunol. 146,1072-1076[Abstract]
  40. Kapuscinski, J., Skooczylas, B. (1977) Simple and rapid fluorimetric method for DNA microassay. Anal. Biochem. 83,252-257[Medline]
  41. Chatelut, M., Leruth, M., Harzer, K., Dagan, A., Marchesini, S., Gatt, S., Salvayre, R., Courtoy, P., Levade, T. (1998) Natural ceramide is unable to escape the lysosome, in contrast to a fluorescent analogue. FEBS Lett 426,102-106[Medline]
  42. Schütze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., Krönke, M. (1992) TNF activates NF-{kappa}B by phosphatidylcholine-specific phospholipase C-induced ‘acidic’ sphingomyelin breakdown. Cell 71,765-776[Medline]
  43. Antonarakis, S., Valle, D., Moser, H., Zinkham, W., Qualman, S. (1983) Farber’s lipogranulomatosis: variability of expression and clinical overlap with histiocytosis. Pediatr. Res. 17,206A
  44. Hess, S., Engelmann, H. (1996) A novel function of CD40: induction of cell death in transformed cells. J. Exp. Med. 183,159-167[Abstract/Free Full Text]
  45. Spiegel, S., Merrill, A. H., Jr (1996) Sphingolipid metabolism and cell growth regulation. FASEB J 10,1388-1397[Abstract]
  46. Dbaibo, G. S., Perry, D. K., Gamard, C. J., Platt, R., Poirier, G. G., Obeid, L. M., Hannun, Y. A. (1997) Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-{alpha}: CrmA and Bcl-2 target distinct components in the apoptotic pathway. J. Exp. Med. 185,481-490[Abstract/Free Full Text]
  47. Schwandner, R., Wiegmann, K., Bernardo, K., Kreder, D., Krönke, M. (1998) TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J. Biol. Chem. 273,5916-5922[Abstract/Free Full Text]
  48. Wiegmann, K., Schwandner, R., Krut, O., Yeh, W. C., Mak, T. W., Krönke, M. (1999) Requirement of FADD for tumor necrosis factor-induced activation of acid sphingomyelinase. J. Biol. Chem. 274,5267-5270[Abstract/Free Full Text]
  49. Chen, L., Kim, T. J., Pillai, S. (1998) Inhibition of caspase activity prevents anti-IgM induced apoptosis but not ceramide generation in WEHI 231 B cells. Mol. Immunol. 35,195-205[Medline]
  50. Hofmann, K., Dixit, V. M. (1998) Ceramide in apoptosis: does it really matter?. Trends Biochem. Sci. 23,374-377[Medline]
  51. Chatterjee, S. (1994) Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-{alpha} in the synthesis of cholesteryl esters in human fibroblasts. J. Biol. Chem. 269,879-882[Abstract/Free Full Text]
  52. Bezombes, C., Maestre, N., Laurent, G., Levade, T., Bettaïeb, A., Jaffrézou, J. P. (1998) Restoration of TNF-{alpha}-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833. FASEB J 12,101-109[Abstract/Free Full Text]
  53. Tepper, C. G., Jayadev, S., Liu, B., Bielawska, A., Wolff, R., Yonehara, S., Hannun, Y. A., Seldin, M. F. (1995) Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. Proc. Natl. Acad. Sci. USA 92,8443-8447[Abstract/Free Full Text]
  54. Wiesner, D. A., Kilkus, J. P., Gottschalk, A. R., Quintans, J., Dawson, G. (1997) Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-xL. J. Biol. Chem. 272,9868-9876[Abstract/Free Full Text]
  55. Jaffrézou, J. P., Bettaïeb, A., Levade, T., Laurent, G. (1998) Antitumor agent-induced apoptosis in myeloid leukemia cells: a controlled suicide. Leuk. Lymph. 29,453-463[Medline]
  56. Bettaïeb, A., Plo, I., Mansat-de Mas, V., Quillet-Mary, A., Levade, T., Laurent, G., Jaffrézou, J. P. (1999) Daunorubicin- and mitoxantrone-triggered phosphatidylcholine hydrolysis: implication in drug-induced ceramide generation and apoptosis. Mol. Pharmacol. 55,118-125[Abstract/Free Full Text]
  57. Jayadev, S., Liu, B., Bielawska, A. E., Lee, J. Y., Nazaire, F., Pushkareva, M. Y., Obeid, L. M., Hannun, Y. A. (1995) Role for ceramide in cell cycle arrest. J. Biol. Chem. 270,2047-2052[Abstract/Free Full Text]
  58. Bruno, A. P., Laurent, G., Averbeck, D., Demur, C., Bonnet, J., Bettaïeb, A., Levade, T., Jaffrézou, J. P. (1998) Lack of ceramide generation in TF-1 human myeloid leukemic cells resistant to ionizing radiation. Cell Death Diff 5,172-182[Medline]
  59. Yoshimura, S. I., Banno, Y., Nakashima, S., Takenaka, K., Sakai, H., Nishimura, Y., Sakai, N., Shimizu, S., Eguchi, Y., Tsujimoto, Y., Nozawa, Y. (1998) Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. J. Biol. Chem. 273,6921-6927[Abstract/Free Full Text]
  60. Woodle, E. S., Smith, D. M., Bluestone, J. A., Kirkman, W. M., Green, D. R., Skowronski, E. W. (1997) Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway. J. Immunol. 158,2156-2164[Abstract]
  61. Kuno, K., Sukegawa, K., Ishikawa, Y., Orii, T., Matsushima, K. (1994) Acid sphingomyelinase is not essential for the IL-1 and tumor necrosis factor receptor signaling pathway leading to NF-{kappa}B activation. Int. Immunol. 6,1269-1272[Abstract/Free Full Text]
  62. Gamard, C. J., Dbaibo, G. S., Liu, B., Obeid, L. M., Hannun, Y. A. (1997) Selective involvement of ceramide in cytokine-induced apoptosis. J. Biol. Chem. 272,16474-16481[Abstract/Free Full Text]
  63. Zumbansen, M., Stoffel, W. (1997) Tumor necrosis factor {alpha} activates NF-{kappa}B in acid sphingomyelinase-deficient mouse embryonic fibroblasts. J. Biol. Chem. 272,10904-10909[Abstract/Free Full Text]
  64. Adam, D., Ruff, A., Strelow, A., Wiegmann, K., Krönke, M. (1998) Induction of stress-activated protein kinases/c-Jun N-terminal kinases by the p55 tumour necrosis factor receptor does not require sphingomyelinases. Biochem. J. 333,343-350
  65. Stoffel, B., Bauer, P., Nix, M., Deres, K., Stoffel, W. (1998) Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice. Eur. J. Immunol. 28,874-880[Medline]
  66. Manthey, C. L., Schuchman, E. H. (1998) Acid sphingomyelinase-derived ceramide is not required for inflammatory cytokine signalling in murine macrophages. Cytokine 10,654-661[Medline]
  67. Skowronski, E. W., Kolesnick, R. N., Green, D. R. (1996) Fas- mediated apoptosis and sphingomyelinase signal transduction: the role of ceramide as a second messenger for apoptosis. Cell Death Differ 3,171-176[Medline]
  68. Linardic, C. M., Hannun, Y. A. (1994) Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle. J. Biol. Chem. 269,23530-23537[Abstract/Free Full Text]
  69. Andrieu, N., Salvayre, R., Levade, T. (1996) Comparative study of the metabolic pools of sphingomyelin and phosphatidylcholine sensitive to tumor necrosis factor. Eur. J. Biochem. 236,738-745[Medline]
  70. Augé, N., Andrieu, N., Nègre-Salvayre, A., Thiers, J. C., Levade, T., Salvayre, R. (1996) The sphingomyelin-ceramide signaling pathway is involved in oxidized low density lipoprotein-induced cell proliferation. J. Biol. Chem. 271,19251-19255[Abstract/Free Full Text]
  71. Harada-Shiba, M., Kinoshita, M., Kamido, H., Shimokado, K. (1998) Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms. J. Biol. Chem. 273,9681-9687[Abstract/Free Full Text]
  72. Schissel, S. L., Schuchman, E. H., Williams, K. J., Tabas, I. (1996) Zn2+-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J. Biol. Chem. 271,18431-18436[Abstract/Free Full Text]



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