FASEB J. Uncover Your Biological Pathway
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Supplementary Figures
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thon, L.
Right arrow Articles by Adam, D.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Thon, L.
Right arrow Articles by Adam, D.
(The FASEB Journal. 2005;19:1945-1956.)
© 2005 FASEB

Ceramide mediates caspase-independent programmed cell death

Lutz Thon*, Heike Möhlig*, Sabine Mathieu*, Arne Lange*, Elena Bulanova{dagger}, Supandi Winoto-Morbach*, Stefan Schütze*, Silvia Bulfone-Paus{dagger} and Dieter Adam*,1

* Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany; and
{dagger} Abteilung Immunologie und Zellbiologie, Forschungszentrum Borstel, Germany

1Correspondence: Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany. E-mail: dadam{at}email.uni-kiel.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.—Thon, L., Möhlig, H., Mathieu, S., Lange, R., Bulanova, E., Winoto-Morbach, S., Schütze, S., Bulfone-Paus, S., Adam, D. Ceramide mediates caspase-independent programmed cell death.


Key Words: tumor necrosis factor • RIP1 • mitochondria


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INCREASING EVIDENCE supports the existence of caspase-independent cell death pathways that induce the cell’s demise equally well controlled and programmed as in caspase-dependent PCD (apoptosis) but in the complete absence of caspase activation. Although caspase-dependent apoptosis appears to be the principal suicide program in many physiological and developmental settings, caspase-independent PCD can provide a backup suicide mechanism under conditions where the classical apoptosis machinery fails (1) . In addition, caspase-independent PCD has been implicated in the homeostasis of an organism by, for instance, contributing to the negative selection of lymphocytes, embryonic removal of interdigital webs, or the death of chondrocytes controlling the longitudinal growth of bones (1) . In vivo, relevance for caspase-independent PCD has been demonstrated directly for TNF-induced hyperacute shock (2) and is discussed for the deletion of T cells (1 , 3) . At the clinical level, characterization of caspase-independent death pathways promises new options to prevent death processes in neurodegenerative disease or to kill tumor cells that have developed strategies to evade apoptotic death signals (1 , 4) .

The sphingolipid ceramide has been described as one of the first mediators of TNF-induced cell death. However, with regard to apoptosis, the relevance of ceramide—either being an essential inducer or a mere by-product generated in consequence of cell death—has been the topic of intense discussions (e.g., refs 5 , 6 ). With regard to caspase-independent PCD, it has been demonstrated that the killing of NB16 neuroblastoma cells by exogenous C2-ceramide is not inhibited by broad-spectrum caspase inhibitors (7) . Likewise, addition of exogenous ceramide analogs or induction of intracellular ceramide accumulation has been observed in conjunction with caspase-independent PCD in several cell types (8 9 10 11 12 13) . Except for these studies, however, the role of ceramide has remained largely uninvestigated. Here, we have focused on the function of ceramide in caspase-independent PCD and establish the relevance of ceramide as a general mediator of this form of PCD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Highly purified human recombinant TNF was provided by G. Adolf (Bender Research Institute, Vienna, Austria). (2S, 3S)-Trans-epoxyscuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d) and fumonisin B1 were ordered from Calbiochem (San Diego, CA, USA). Benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk), benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone(zDEVD-fmk), and benzyloxycarbonyl-Ile-Glu(OMe)-Thr-DL-Asp(OMe)-fluoromethylketone (zIETD-fmk) were obtained from Bachem (Bubendorf, Switzerland), anti-FLAG M5 monoclonal antibody, desipramine, cycloheximide (CHX), benzyloxycarbonyl-Phe-Ala-fluoromethylketone (zFA-fmk), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), geldanamycin, and radicicol were purchased from Sigma (St. Louis, MO, USA). Tricyclodecan-9-yl (D609) was obtained from MoBiTec (Göttingen, Germany), N-[L-3-trans-(propylcarbamoyl)-oxirane-2-carbonyl]-L-Ile-L-Pro methyl ester (CA-074 Me) from Biomol (Plymouth Meeting, PA, USA), and tert-butyl hydroperoxide (BuOOH) from Fluka (St. Louis, MO, USA). Scyphostatin was a kind gift from T. Ogita (Sankyo, Tokyo, Japan). Human recombinant FLAG-tagged FasL was purchased from Alexis (Grünberg, Germany).

Cell culture
L929, NIH3T3, and Jurkat wild-type cells were originally obtained from the American Type Culture Collection (Manassas, VA, USA). Fas-associated death domain protein (FADD)-, RIP1-deficient, and Bcl-2-overexpressing Jurkat cells were kindly provided by J. Blenis, B. Seed, and S. Korsmeyer (all Harvard Medical School, Boston, MA, USA). Cells were maintained in a mixture of Click’s/RPMI 1640 (50/50% v/v) supplemented with 10% v/v FCS, 10 mM glutamine, and 50 µg/mL each of streptomycin and penicillin in a humidified incubator containing 5% w/v CO2. L929 cells overexpressing the full-length acid ceramidase (AC) cDNA have been described (14) . Lung fibroblasts from acid sphingomyelinase (A-SMase) -deficient and wild-type mice (15) kindly provided by E. Gulbins (Essen, Germany) were generated essentially as described (16) except cells were cultivated in DME supplemented with 20% v/v FCS. Lung fibroblasts were immortalized by stable transfection with the SV40 large T antigen expression vector pMSSVLT (17) . For reconstitution of A-SMase deficiency, 293 cells were transfected with the A-SMase expression construct pRK5-ASM by the calcium phosphate method; 48 h later the culture supernatant was collected, filtered, diluted with fresh medium (1:2), and added to lung fibroblasts for 16 h before stimulation.

Ceramide quantitation
Ceramide was quantitated in duplicate by the diacylglycerol kinase assay essentially as described by Dressler and Kolesnick (18) following recommendations of Perry and Hannun (19) . Alternatively, ceramide was quantitated by the charring method following high-performance thin layer chromatography exactly as described (20) . Autoradiographs and thin layer chromatography plates were scanned and analyzed using the software package PCBAS (Raytest).

Cytotoxicity assays
For flow cytometric measurement of cell death, cells were seeded in 6-well plates at 5 x 105 cells/well. After treatment detached and adherent cells were collected, followed by centrifugation. The cells were resuspended in PBS/5 mM EDTA containing 2 µg/mL propidium iodide (PI), and the red fluorescence was measured on a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA). Immortalized lung fibroblasts were alternatively analyzed by staining with crystal violet as follows: 104 cells were seeded in flat-bottomed 96-well plates. After stimulation, adherent cells were washed twice with PBS and incubated for 10 min at 37°C in 50 µL of staining solution (0.5% w/v crystal violet, 4% w/v formaldehyde, 30% v/v ethanol, and 0.17% w/v NaCl). The staining solution was washed away with tap water and cells were dried for 1 h at 50°C. Stained cells were dissolved in acetic acid (33% v/v) and the intensity of the staining was colorimetrically determined at 570 nm in a microplate reader (Tecan, Medford, MA, USA).

RNA interference
The predesigned siRNAs Smpd1_1, Smpd1_2, and Smpd1_3 specific for murine A-SMase, the predesigned siRNAs Ripk1_1, Ripk1_2, Ripk1_3, and the custom-made siRNA Ripk1_4 (target sequence 5'-CCACUAGUCUGACUGAUG-3') specific for murine RIP1 as well as the negative control siRNA were obtained from Ambion (Austin, TX, USA). To target expression, 106 cells were transfected with 150 pmol of siRNA by Amaxa nucleofection, using solution V and program T-20 (A-SMase) or solution R and program A-24 (RIP1). The efficiency of the transfection procedure was monitored by nucleofection of pmaxGFP (Amaxa) and routinely yielded 70–90% of green fluorescent cells. Cells were subsequently cultured for 24 to 72 h before further analysis.

Measurement of A-SMase activity
The micellar SMase assay using exogenous radiolabeled sphingomyelin was performed as described (21) . Briefly, cells were homogenized in 100 µL 0.2% v/v Triton X-100. Radioactive phosphocholine produced from [N-methyl-14C]-sphingomyelin (labeled in the choline moiety, Amersham CFA566) was identified by TLC and routinely determined in the aqueous phase by scintillation counting.

Immunoblots
Cells were collected and lysed in TNE buffer (50 mM Tris pH 8.0, 1% v/v NP-40, 2 mM EDTA) containing 10 µg/mL pepstatin/aprotinin/leupeptin, 1 mM sodium orthovanadate, and 5 mM NaF. 25 µg of cell protein per lane were resolved by electrophoresis on SDS polyacrylamide gels. After electrophoretic transfer to nitrocellulose, reactive proteins were detected using antisera specific for RIP1 (BD Biosciences) and the ECL detection kit (Amersham Biosciences, Arlington Heights, IL, USA). Expression of RIP1 protein was quantified after scanning of the autoradiographs using the software package PCBAS (Raytest, Wilmington, NC, USA).

Measurement of ROS
Cells were seeded at a density of 2 x 105 cells per well and stimulated as indicated, with addition of 1 µM dihydrorhodamine 123 for a total of 1 h. Subsequently, the cells were harvested, washed once with cold PBS, resuspended in PBS, and analyzed by flow cytometry.

Flow cytometric analysis of {Delta}{Psi}m
Cells were treated as indicated with the addition of 150 nM CMTMRos (Molecular Probes, Eugene, OR, USA) for the last 30 min of stimulation. After harvesting, the cells were washed once in cold PBS, fixed by incubation in paraformaldehyde (4% w/v) for 15 min at room temperature in the dark and analyzed on a FACSCalibur flow cytometer (BD Biosciences).

Analysis of cytochrome crelease
After treatment, cells were harvested at 4°C and resuspended in 1 mL of a buffer containing 20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride and Complete protease inhibitor mixture (Roche, Nutley, NJ, USA) as recommended by the manufacturer. Cells were lysed by passaging through a 27-gauge needle and homogenates were centrifuged at 1000 x g for 5 min at 4°C. Supernatants were further centrifuged at 10000 x g for 15 min at 4°C. The resulting mitochondrial pellets were resuspended in 40 µL of cold cell lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% v/v Triton X-100, 1 mM phenylmethylsulfonyl fluoride, Complete protease inhibitor mixture). The supernatants from the final centrifugation were centrifuged at 6000 x g for 65 min at 4°C using Centricon YM-10 centrifugal filter devices (Millipore, Bedford, MA, USA) to prepare concentrated cytosolic extracts. For detection of mitochondrial release of cytochrome c in immunoblots, 5–20 µg of cytosolic or mitochondrial extract (depending on the cell line used) was analyzed using cytochrome c antibody (BD Biosciences).

Flow cytometric analysis of membrane integrity and phosphatidylserine externalization
106 cells were collected, washed with cold PBS, and resuspended in 100 µL of a buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing 2% v/v of annexin-V-FLUOS labeling reagent (Roche) and 20% v/v 7-AAD reagent (BD Biosciences). The cells were incubated for 20 min in the dark at room temperature and analyzed by flow cytometry using a FACSCalibur Analyzer (BD Biosciences).

Clonogenic survival assays
To analyze the ability of cells to form colonies in soft agarose, after treatment 1000 viable cells (as determined by Trypan blue staining; 10,000 cells for Jurkat wild-type) were mixed with 2 mL of 0.4% Sea Plaque agarose (Cambrex) in complete medium, transferred into 6-well plates containing 3 mL of a 1% SeaKem LE agarose underlayer (Cambrex) in complete medium, and incubated at 37°C. Colony formation (>20 cells) was scored after 14 days of incubation on an inverted Zeiss Axiovert 100 microscope. For this purpose, viable cells were stained by adding 1 mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO, USA; 2.5 mg/mL in PBS) and incubation for 2 h at 37°C to allow metabolization of MTT to blue MTT-formazan. For visual representation, NIH3T3 cells were plated in complete medium without agarose as above and stained with crystal violet after 7 days as described under "Cytotoxicity assays," except that all steps subsequent to washing with tap water were omitted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ceramide generated by A-SMase mediates TNF/zVAD-induced caspase-independent PCD of L929 cells
We previously demonstrated that ceramide plays a pivotal role in the TNF-induced caspase-independent PCD of murine L929 fibrosarcoma cells (14) . In contrast to L929 cells, where caspases are not activated by TNF (Supplementary Fig. 1A), analysis of TNF-induced caspase-independent PCD in most other cell systems requires active inhibition of interfering caspase-dependent responses. Therefore, we initially used the established L929 system to investigate whether ceramide is still crucial for TNF-induced death of these cells in the presence of caspase inhibitors. TNF in combination with the broad-spectrum caspase inhibitor zVAD-fmk induced a much more pronounced increase of intracellular ceramide than TNF alone (Fig. 1 A), corresponding to the much more rapid killing described for TNF/zVAD-treated L929 cells (ref 22 ; Fig. 1B ). No caspase activity was detected under these conditions (Supplementary Fig. 1A). Analysis of nuclear morphology (Supplementary Fig. 2A) suggested that PCD induced by TNF/zVAD was "necrosis-like" (1) .



View larger version (34K):
[in this window]
[in a new window]
  		Figure 1. Ceramide mediates TNF/zVAD-induced caspase-independent PCD of L929 cells. A) L929 cells were stimulated with 100 ng/mL hTNF and/or 20 µM zVAD-fmk for 3 h before intracellular ceramide levels were quantified by the diacylglycerol kinase assay. B) Left panel: L929 cells were incubated with increasing concentrations of hTNF alone or with hTNF and 20 µM zVAD-fmk for 14 h before cell death was analyzed by flow cytometric counting of PI-positive cells. Right panel: incubation with 20 µM zVAD-fmk alone for 24 or 46 h was not toxic. C) Left panel (dose-response): parental (L929) and AC-transfected L929 cells (AC23, AC52) were left untreated or incubated with 10 ng/mL hTNF in combination with increasing concentrations of zVAD-fmk for 14 h before cell death was determined. Right panel (time course): cells were incubated with 100 ng/mL hTNF and 20 µM zVAD-fmk. D) L929 cells were preincubated with the indicated concentrations of D609, desipramine, scyphostatin, or fumonisin B1 for 2 h before 100 ng/mL hTNF and 20 µM zVAD-fmk were added. After 5 h, cell death was analyzed. For all figures, values of cytotoxicity assays represent the means from 3 independent determinations, error bars indicate the respective SDs.

In line with a causative role of ceramide in TNF/zVAD-induced PCD, two independent AC-overexpressing L929 clones (AC23 and AC52) displayed a higher resistance to TNF/zVAD than parental L929 cells. AC degrades ceramide generated by A-SMase in response to TNF, thereby protecting L929 cells against the cytotoxic action of TNF (14) . Enhanced survival was consistently seen for different concentrations of TNF/zVAD as well as for different incubation times (Fig. 1C ).

Consistent with the above results, the pharmacological agents D609 and desipramine also enhanced resistance against TNF/zVAD in parental L929 cells (Fig. 1D ). D609 indirectly inhibits the TNF-dependent activation of A-SMase and thereby ceramide generation by this enzyme in the lysosomal compartment by impairing the activity of phosphatidylcholine-specific phospholipase C, an enzyme upstream of A-SMase (21 , 23) . Desipramine causes a rapid and irreversible reduction of A-SMase activity by inducing proteolytic degradation of the enzyme (24) . In contrast, neither scyphostatin nor fumonisin B1 could protect the cells (Fig. 1D ). Scyphostatin inhibits neutral sphingomyelinase (25) , an enzyme distinct from A-SMase generating ceramide at the plasma membrane, whereas fumonisin B1 is an inhibitor of ceramide synthase that generates ceramide by de novo synthesis (26) . With regard to interpreting results obtained with D609, however, a certain caution should be taken since murine and human forms of phosphatidylcholine-specific phospholipase C have not yet been cloned; therefore, evidence that D609 inhibits its target in vitro (i.e., directly) is still missing.

Ceramide accumulation elicits caspase-independent PCD in NIH3T3 fibroblasts and in Jurkat leukemic T cells
After confirming that ceramide (generated by A-SMase) is responsible for the TNF-induced dying of L929 cells not only in the absence but also in the presence of caspase inhibitors, we extended our investigations to other systems of caspase-independent PCD. In both murine NIH3T3 fibroblasts and human leukemic Jurkat T cells, TNF can trigger an alternative route to PCD provided caspase activity is actively inhibited or absent (27 , 28) . While zVAD-fmk or TNF alone did not alter ceramide levels, corresponding to the inability of either substance to kill NIH3T3 cells when applied individually (27) , treatment of NIH3T3 cells with TNF/zVAD led to a pronounced increase of intracellular ceramide comparable to results obtained in L929 cells (Fig. 2 A). Concomitantly, the viability of the cells was substantially reduced but could be restored by addition of D609. As in L929 cells, scyphostatin and fumonisin B1 did not protect the cells (Fig. 2B ). Although less pronounced than in L929 cells, changes in nuclear and cellular morphology in response to TNF/zVAD argued for a mode of caspase-independent death resembling necrosis-like PCD (Supplementary Fig. 2B).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 2. Ceramide accumulation elicits caspase-independent PCD in NIH3T3 fibroblasts and Jurkat leukemic T cells. A) NIH3T3 cells were left untreated or stimulated with 100 ng/mL hTNF and/or 20 µM zVAD-fmk for 18 h before intracellular ceramide levels were determined by the charring method. B) NIH3T3 cells were preincubated with the indicated concentrations of D609, scyphostatin, or fumonisin B1 for 2 h before 100 ng/mL hTNF and 20 µM zVAD-fmk were added. After 16 h, cell death was analyzed by flow cytometric counting of PI-positive cells. C) Ceramide levels in Jurkat wild-type (WT) cells were quantified by the diacylglycerol kinase assay after preincubation with 50 µM zVAD-fmk for 4 h followed by incubation for 0–12 h with a combination of 100 ng/mL hTNF, 50 µM zVAD-fmk, and 2 µg/mL CHX. D) Cell death was measured in parallel. E) Jurkat wild-type cells were treated essentially as in panel B except that cells were incubated for 20 h with 100 ng/mL hTNF, 50 µM zVAD-fmk and 2 µg/mL CHX before cell death was determined. F) FADD-deficient Jurkat cells were stimulated with 100 ng/mL hTNF for the indicated times before intracellular ceramide was quantified as in panel C. G) In parallel, cell death was measured.

In Jurkat wild-type T cells, Holler and co-workers have demonstrated that caspase-independent necrosis-like PCD in response to both TNF (see Supplementary Fig. 2C) and Fas ligand is substantially enhanced by addition of the protein biosynthesis inhibitor CHX, and have suggested this is due to the degradation of a short-lived inhibitor that selectively controls caspase-independent PCD in these cells (28) . As we observed the same increase in TNF/zVAD-induced caspase-independent PCD after addition of CHX (Supplementary Fig. 3A), Jurkat wild-type cells were sensitized for TNF by addition of CHX in subsequent experiments. We verified that any caspase activity was completely suppressed in TNF/CHX/zVAD treated cells, confirming that death indeed occurred by caspase-independent PCD (Supplementary Fig. 1B). As shown in Fig. 2C , TNF/CHX/zVAD-induced caspase-independent PCD in Jurkat wild-type cells was again accompanied by a pronounced accumulation of intracellular ceramide with a linear increase over time. Ceramide accumulation started well before the onset of cell death (Fig. 2D ), suggesting that ceramide represents a cause rather than a consequence of caspase-independent PCD. Cell death was once more reduced by the addition of D609, but not by fumonisin B1 or scyphostatin (Fig. 2E ).

We analyzed Jurkat cells genetically deficient for FADD (29) . The absence of FADD prevents caspase activation by TNF (Supplementary Fig. 1C) but simultaneously renders these cells more sensitive to TNF (Supplementary Fig. 3B; ref 28 ). Therefore, TNF kills FADD-deficient Jurkat cells exclusively by caspase-independent necrosis-like PCD, similar to L929 cells (Supplementary Fig. 2C). Confirming our results from Jurkat wild-type cells, FADD-deficient Jurkat cells displayed a pronounced linear accumulation of intracellular ceramide in response to TNF alone, again well before the onset of cell death (Fig. 2F, G ). In summary, these data corroborate the concept that TNF-induced intracellular accumulation of ceramide elicits caspase-independent PCD in cells of fibroblastoid as well as T cell origin.

Lung fibroblasts genetically deficient for A-SMase are protected from caspase-independent PCD
Since results obtained with pharmacological inhibitors such as D609 must be interpreted with a certain caution due to potential nonspecific effects, we sought to further substantiate the function of ceramide in caspase-independent PCD by selectively targeting A-SMase expression using RNA interference (RNAi). As shown in Fig. 3 A, 48 h after transfection A-SMase activity in L929 cells was reduced to a minimum of 30%, which was not further diminished by variation in the transfection conditions (e.g., time, Fig. 3A or amount of siRNA, not shown). However, we did not detect a corresponding inhibition of TNF- or TNF/zVAD-induced caspase-independent PCD (Fig. 3B ). Parallel transfection of NIH3T3 cells likewise did not confer protection (not shown). As one possible explanation for this result, the achieved reduction of A-SMase activity might not yet be sufficient to inhibit the death response. Alternatively, this result might indicate lack of a role for A-SMase and leave the possibility that ceramide is generated by sources other than A-SMase.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. Genetic deficiency for A-SMase protects from caspase-independent PCD. A) L929 cells were transfected with a mixture of 3 siRNAs specific for murine A-SMase (Smpd_1-3) and the activity of the enzyme was measured at the indicated times as described in Materials and Methods. A-SMase activity is displayed relative to cells transfected with a negative control siRNA that does not elicit an RNAi response. B) L929 cells were left untransfected or were transfected as in panel A. 48 h after transfection, cells were treated with 100 ng/mL hTNF for 20 h or with 100 ng/mL hTNF and 20 µM zVAD-fmk for 3 h before cell death was determined by flow cytometric counting of PI-positive cells. C) Enzymatic activity of A-SMase in immortalized lung fibroblasts from wild-type and A-SMase-deficient mice was measured as described in Materials and Methods. D) Immortalized lung fibroblasts from wild-type and A-SMase-deficient mice were left untreated or stimulated for 8 h with a combination of 100 ng/mL hTNF and 20 µM zVAD-fmk before ceramide levels were quantified by the charring method. E) Immortalized lung fibroblasts from wild-type and A-SMase-deficient mice were treated with 100 ng/mL hTNF and 20 µM zVAD-fmk for 48 h before viability was measured by staining with crystal violet. F) Parallel analysis of cell viability after 33 h in cells cultured in regular medium (0) or medium containing exogenous A-SMase (ASM) to restore A-SMase deficiency. Values represent the means from 5 independent determinations, error bars indicate the respective SDs.

We therefore immortalized and analyzed lung fibroblasts from A-SMase-deficient mice in a direct genetic approach. As shown in Fig. 3C , these cells are completely devoid of any residual A-SMase activity. In consequence, treatment with TNF/zVAD induced ceramide accumulation in wild-type, but not in A-SMase-deficient fibroblasts (Fig. 3D ). In viability assays, A-SMase-deficient cells displayed a pronouncedly higher resistance to TNF/zVAD than wild-type cells (Fig. 3E ). Supernatant from cells transfected with an A-SMase expression construct contains secreted A-SMase and can restore A-SMase deficiency when added to cells (30) . Reconstitution of the A-SMase defect in lung fibroblasts by this method did restore their sensitivity to TNF/zVAD to the level of wild-type cells (Fig. 3F ). In time course experiments, ceramide levels in wild-type cells increased well before the onset of cell death (not shown), again arguing for ceramide being an inducer rather than a consequence of caspase-independent PCD. Taken together, the results obtained in lung fibroblasts provide strong support for a causative role of ceramide in TNF-induced caspase-independent PCD in yet another cell type.

RIP1 function is indispensable for ceramide-induced caspase-independent PCD
RIP1 appears to be essential for TNF-induced caspase-independent PCD of Jurkat cells (28) . To gain further insight into the signaling pathways by which ceramide mediates caspase-independent PCD, we used Jurkat cells deficient for RIP1 expression (31) . We confirmed that, in the presence of zVAD-fmk, these cells are completely resistant against TNF/CHX-induced caspase-independent PCD (although they could still be killed by caspase-dependent mechanisms in the absence of zVAD-fmk; Supplementary Fig. 1D, 3C; ref 28 ). Correspondingly, these cells showed an exclusively apoptotic morphology (in contrast to the mixed apoptotic/necrosis-like morphology of dying Jurkat wild-type cells or to the solely necrosis-like morphology of dying FADD-deficient cells, Supplementary Fig. 2C). As shown in Fig. 4 A, ceramide accumulation was massively impaired in TNF/CHX/zVAD-treated, RIP1-deficient cells when compared with wild-type Jurkat cells, suggesting that, in Jurkat cells, RIP1 is essential for the induction of ceramide production by TNF and that RIP1 and ceramide mediate their death signals through the same pathway.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. RIP1 and ceramide mediate their death signals through the same pathway. A) Wild-type and RIP1-deficient Jurkat cells were left untreated or preincubated with 50 µM zVAD-fmk for 30 min, then stimulated with 100 ng/mL hTNF, 50 µM zVAD-fmk, and 2 µg/mL CHX for 20 h before intracellular ceramide levels were measured by the diacylglycerol kinase assay. B) L929, NIH3T3, and Jurkat wild-type cells were left untreated or preincubated with 1 µg/mL GA or 5 µg/mL RC for 24 h before 100 ng/mL hTNF with 20 µM zVAD-fmk (Jurkat wild-type cells: 100 ng/mL hTNF, 50 µM zVAD-fmk and 2 µg/mL CHX) were added. After 24 h of incubation, cell death was analyzed by flow cytometric counting of PI-positive cells. The fraction of surviving cells is displayed relative to cells that were treated as above, except that no hTNF/zVAD (hTNF/CHX/zVAD) was added. C) NIH3T3 cells were transfected with siRNAs specific for RIP1 (pooled siRNAs Ripk1_1, Ripk1_2, and Ripk1_3 or individual siRNA Ripk1_4) as described in Materials and Methods. For control, cells were left untransfected or transfected with an siRNA that does not elicit an RNAi response (negative control). 24 h after transfection, cells were treated with 100 ng/mL hTNF and 20 µM zVAD-fmk for another 18 h before cell death was analyzed by flow cytometric counting of PI-positive cells. Expression of RIP1 protein was visualized 24 h after transfection by Western blot analysis. Detection of ß-actin was used as a loading control (not shown).

To extend our analyses of RIP1 beyond the Jurkat T cell line, we neutralized RIP1 function by a pharmacological approach. The inhibitor geldanamycin (GA) has been used in Jurkat cells to limit the function of RIP1 (28) . GA disrupts the function of the heat-shock protein HSP90 by preventing the release of client proteins such as RIP1 undergoing refolding from HSP90. The stabilized complex is then ubiquitinated and degraded (32) . Both GA and the structurally unrelated HSP90 inhibitor radicicol (RC) pronouncedly decreased RIP1 protein levels in L929 cells (Supplementary Fig. 4A). Moreover, electrophoretic mobility shift assays revealed a complete block of RIP1-dependent nuclear factor-{kappa}B translocation in GA-pretreated cells in response to TNF, confirming the functional inhibition of RIP1 (Supplementary Fig. 4B). In consequence, GA- or RC-treated L929 cells proved completely resistant against TNF/zVAD-induced caspase-independent PCD (Fig. 4B ). Identical results were obtained in NIH3T3 cells or in Jurkat wild-type cells treated with TNF/CHX/zVAD (Fig. 4B ).

To account for potential nonspecific effects of the above inhibitors, we selectively targeted RIP1 expression in NIH3T3 cells by RNAi. As shown in Fig. 4C , down-regulation of RIP1 protein levels by independent siRNAs specific for RIP1 clearly diminished caspase-independent PCD in response to TNF/zVAD, further corroborating the idea that RIP1 is essential for induction of ceramide-mediated caspase-independent PCD. In summary, the above data suggest that ceramide and RIP1 mediate caspase-independent PCD through signaling pathways that are conserved across different cell types.

Participation of mitochondria in ceramide-mediated caspase-independent PCD
Mitochondria have been implicated as an additional factor in caspase-independent PCD and have been described as targets of ceramide in the induction of cell death (1) . As shown in Fig. 5 A, Jurkat cells overexpressing the antiapoptotic mitochondrial protein Bcl-2 (33) clearly displayed a higher resistance to TNF/CHX/zVAD-induced caspase-independent PCD than their parental counterparts. Likewise, overexpression of Bcl-2 can delay the onset of TNF-induced caspase-independent PCD in L929 cells (34) , suggesting that mitochondria do indeed represent another component of the signaling cascade by which ceramide elicits caspase-independent PCD.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Contribution of mitochondria to ceramide-induced caspase-independent PCD. A) Wild-type and Bcl-2-overexpressing Jurkat cells were stimulated with 100 ng/mL hTNF in combination with 50 µM zVAD-fmk and 2 µg/mL CHX for 20 h before cell death was determined by flow cytometric counting of PI-positive cells. Prior to stimulation, cells were preincubated for 30 min with 50 µM zVAD-fmk. The fraction of surviving cells is displayed relative to cells that were treated as above, except that no hTNF was added. B) L929 cells were stimulated with 100 ng/mL hTNF and 50 µM zVAD-fmk for 6 h in the presence or absence of 150 µM BHA or BHT before viability was determined by staining with crystal violet. Jurkat cells were analyzed identically, except that 5 µg/mL CHX were added and viability was determined by staining with MTT after 20 h. Values represent the means from 5 independent determinations, error bars indicate the respective SDs. C) Accumulation of ROS in L929 and Jurkat wild-type cells was induced by treatment of cells with 1 mM tert-butyl hydroperoxide (BuOOH) for 1 h in the absence or presence of 150 µM BHA or BHT. Cells were coincubated with 1 µM dihydrorhodamine 123 before the oxidation product rhodamine 123 was quantified by flow cytometry.

With regard to the molecular mechanisms by which mitochondria might elicit caspase-independent PCD, reactive oxygen species (ROS) represent a likely candidate. We therefore investigated the importance of ROS for ceramide-mediated caspase-independent PCD. In line with previous results (22) , pretreatment with the radical scavenger BHA protected L929 cells from TNF/zVAD-induced caspase-independent PCD, potentially arguing in favor of a causative function of ROS in the ceramide death pathway. However, the structurally closely related radical scavenger BHT did not protect L929 cells (Fig. 5B ). The same results have been observed in NIH3T3 cells (27) . Moreover, neither BHA nor BHT could protect Jurkat wild-type cells from TNF/CHX/zVAD-induced caspase-independent PCD (Fig. 5B ), although both radical scavengers were equally effective in scavenging ROS in both L929 and Jurkat wild-type cells (Fig. 5C ). Clearly, additional studies are required to fully unravel the mechanism by which ROS participate in the ceramide death pathway.

We examined whether there was evidence for an early decrease in mitochondrial membrane potential ({Delta}{Psi}m) or for an early leakage of mitochondrial cytochrome c in ceramide-mediated caspase-independent PCD. Concurring with reports on TNF- or FasL-induced caspase-independent PCD in L929sAhFas and Jurkat wild-type cells (28 , 35) , a decrease of {Delta}{Psi}m in TNF/zVAD- or TNF/CHX/zVAD-treated L929, NIH3T3, and Jurkat wild-type cells became apparent only in the late to end, but not the early, stages of cell death (Fig. 6 A), coinciding with loss of plasma membrane integrity (Figs. 1C , 2B, E ).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 6. Ceramide-mediated caspase-independent PCD does not depend on early loss of {Delta}{Psi}m or early release of cytochrome c. Cells were either left untreated or incubated with 100 ng/mL hTNF and 20 µM zVAD-fmk (L929, NIH3T3) or 100 ng/mL hTNF, 50 µM zVAD-fmk, and 2 µg/mL CHX (Jurkat wild-type) for the indicated times to elicit caspase-independent PCD. A) Reduction of {Delta}{Psi}m is shown as percentage of cells with decreased CMTMRos staining. B) Cytosolic extracts were prepared and cytochrome c protein was detected by immunoblotting. M, mitochondrial extracts of untreated cells were loaded for comparison. Equal loading was verified by immunodetection of ß-actin (not shown).

Similarly, release of cytochrome c did not occur early in either L929, NIH3T3, or Jurkat wild-type cells (Fig. 6B ). Identical results have been obtained by us and others for caspase-independent PCD of L929/L929sAhFas cells induced by TNF alone, where cytochrome c release has been observed in the late phases of cell death but never in the early stages (ref 35 ; not shown). This observation is also in line with the absence of detectable caspase-8 and -3 activity in TNF/zVAD-treated L929 cells (Supplementary Fig. 1). In summary, mitochondrial changes characteristic for caspase-dependent apoptosis such as early release of cytochrome c and early dissipation of {Delta}{Psi}m were not detected, suggesting that mitochondria contribute to ceramide-mediated caspase-independent PCD by mechanisms other than these.

Rapid externalization of phosphatidylserine is not essential for caspase-independent PCD induced by ceramide
To investigate more closely and precisely the role of ceramide in caspase-independent PCD, we monitored the kinetics of phosphatidylserine externalization vs. loss of plasma membrane integrity in L929, NIH3T3, and Jurkat wild-type cells. As observed above for loss of {Delta}{Psi}m, when undergoing ceramide-mediated caspase-independent PCD all three cell lines concurrently stained positive with both annexin V and 7-amino-actinomycin D (7-AAD) in the late phase of cell death (Fig. 7 ). Therefore, the rapid externalization of phosphatidylserine that has been described in caspase-dependent apoptosis of these cells (28 , 35) apparently is not crucial for caspase-independent PCD induced by ceramide.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. Flow cytometric analysis of L929, NIH3T3, and Jurkat wild-type cells stained with 7-AAD and annexin V after induction of ceramide-mediated caspase-independent PCD. Cells were treated with 100 ng/mL hTNF and 20 µM zVAD-fmk (L929, NIH3T3) or 100 ng/mL hTNF, 50 µM zVAD-fmk, and 2 µg/mL CHX (Jurkat wild-type). The percentage of cells staining positively with annexin V or 7-AAD is shown.

Inhibition of caspases, but not cathepsins, enhances the ceramide death pathway
We investigated how zVAD-fmk enhances ceramide accumulation in TNF-induced caspase-independent PCD—specifically, whether this was a caspase-mediated effect or whether lysosomal cathepsins were implicated (zVAD-fmk inhibits both types of enzymes at the concentration of 20 µM employed in the previous experiments; ref 36 ). L929 cells were treated with TNF in combination with zVAD-fmk, the cathepsin B/L inhibitor zFA-fmk, the cathepsin B inhibitor CA-074 Me, or the broad spectrum cysteine protease inhibitor E-64d (20 µM each). Of all inhibitors, only zVAD-fmk strongly amplified the cytotoxic response of TNF (Fig. 8 A). When added at 1 µM, a concentration that only affects caspases but not cathepsins (36) , zVAD-fmk still substantially enhanced the cytotoxic effects of TNF (not shown), suggesting that the inhibition of caspases rather than cathepsins by zVAD-fmk is responsible for the observed enhancement of ceramide accumulation and for caspase-independent PCD.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. Ceramide-mediated caspase-independent PCD is enhanced by inhibition of caspases, but not cathepsins. A) L929 cells were incubated with 100 ng/mL hTNF for 5 h in the absence or presence of 20 µM zVAD-fmk, zFA-fmk, CA-074 Me, or E-64d before cell death was measured by flow cytometric counting of PI-positive cells. Incubation with the inhibitors without TNF was not toxic (not shown). B) L929 cells were left untreated or stimulated with 100 ng/mL hTNF for 5 or 20 h with optional addition of 20 µM zDEVD-fmk, zIETD-fmk or zVAD-fmk. C) L929 or NIH3T3 cells were left untreated or subjected to treatment with 100 ng/mL hTNF in combination with the indicated concentrations of zDEVD-fmk, zIETD-fmk, or zVAD-fmk for 3 h before intracellular ceramide levels were quantified by the charring method. Jurkat wild-type cells were analyzed identically after 16 h, except that 2 µg/mL CHX were added to the stimulations. D) L929, NIH3T3, and Jurkat wild-type cells were left untreated or apoptosis was induced by adding either 100 ng/mL hTNF and 2 µg/mL CHX (L929, NIH3T3, Jurkat wild-type), a stimulus that leads to activation of caspases in all 3 cell lines (Supplementary Fig. 1; ref 27 ) or 25 ng/mL FLAG-tagged hFasL and 1 µg/mL anti-FLAG antibody as an enhancer (NIH3T3, Jurkat wild-type). For NIH3T3 cells, 2 µg/mL CHX were added to hFasL to sensitize the cells. After 5 (L929) or 16 h (NIH3T3, Jurkat wild-type), intracellular ceramide levels were quantified by the diacylglycerol kinase assay.

To further specify the caspase responsible for this effect, we analyzed the impact of the inhibitors zIETD-fmk (specific for the initiator caspase-8) and zDEVD-fmk (inhibiting the effector caspase-3). Whereas zVAD-fmk still was the most potent enhancer of TNF cytotoxicity, zIETD-fmk and, to a lesser extent zDEVD-fmk, likewise accelerated TNF-dependent cell death (Fig. 8B ). Parallel experiments demonstrated accumulation of ceramide in L929, NIH3T3, and Jurkat cells after treatment with TNF in combination with low amounts of zVAD-fmk as well as with zDEVD-fmk or with zIETD-fmk (Fig. 8C ). Garcia-Calvo and co-workers have demonstrated that the DEVD tetrapeptide is a potent inhibitor not only for caspase-3, but also for caspase-8 (37) . Therefore, the effect of zDEVD-fmk on caspase-independent PCD and ceramide accumulation may result from inhibition of the initiator caspase-8 rather than the effector caspase-3, implicating caspase-8 as a potential candidate responsible for suppression of ceramide-mediated caspase-independent PCD under physiological conditions.

To address the question of whether in consequence, activators of caspases also suppress ceramide formation, we examined ceramide levels in apoptotic L929, NIH3T3, and Jurkat wild-type cells undergoing caspase-dependent cell death. As shown in Fig. 8D , induction of apoptosis in all three cell lines did not suppress but rather induced ceramide formation, in line with a large body of evidence implicating the accumulation of intracellular ceramide in apoptotic cell death (38 , 39) . Thus, while activation of caspases such as caspase-8 may suppress ceramide-mediated caspase-independent PCD (and therefore ceramide production through the caspase-independent death pathway), it simultaneously induces ceramide production as part of the apoptotic death pathway. Although an apparent paradox at first glance, the induction of ceramide by either death pathway may assist a cell in committing suicide regardless of whether this is accomplished by caspase-dependent or -independent mechanisms.

Ceramide-mediated caspase-independent PCD reduces the clonogenic survival of tumor cells
We wanted to determine whether induction of ceramide-mediated caspase-independent PCD represents a viable strategy for the elimination of tumor cells that have become refractory to drugs eliciting apoptotic cell death. We therefore analyzed the clonogenic survival of untreated L929, NIH3T3, and Jurkat wild-type cells in comparison to cells that were treated with TNF/zVAD (Jurkat wild-type cells: TNF/CHX/zVAD). This treatment simulated apoptosis resistance of cells by inhibiting caspases through zVAD-fmk while simultaneously inducing ceramide-dependent caspase-independent PCD. As shown in Fig. 9 , clonogenicity was clearly decreased in all three cell lines, confirming that induction of caspase-independent PCD by manipulation of intracellular ceramide levels may represent a promising additional option for the development of novel tumor therapies.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 9. Impact of ceramide-mediated caspase-independent PCD on clonogenic survival of cells. A) L929, NIH3T3, and Jurkat wild-type cells were left untreated (0) or incubated with 100 ng/mL hTNF and 20 µM zVAD-fmk (100 ng/mL hTNF, 50 µM zVAD-fmk and 2 µg/mL CHX for Jurkat wild-type cells) for 5 h (L929), 16 h (NIH3T3), or 20 h (Jurkat wild-type). Subsequently, their ability to form colonies was analyzed by soft agarose cloning. Values represent the means from 3 independently counted wells per condition. Error bars indicate the respective SDs. B) Visual representation of L929 and 3T3 cells. L929 cells were cloned into soft agarose and stained with MTT (left). Since NIH3T3 cells form only small colonies in soft agarose, cells were treated as above, plated in medium without agarose and stained with crystal violet after 7 days. The nonadherent Jurkat wild-type cells also formed only small colonies and therefore were omitted from the visual representation. Images were obtained using a Konica Digital Revio KD-500Z digital camera.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
While there is ample evidence implicating ceramide in apoptotic cell death (38 , 39) , the issue whether ceramide represents an essential inducer or is generated merely in consequence of apoptosis has been, and still is, subject to debate (5 ,6 ). There is no doubt that ceramide is an essential inducer of apoptosis in certain tissues, e.g., in the lung (39) or in oocytes (40) , but apparently this is not the case in others, e.g., in the thymus (39) . Although the role of ceramide in apoptosis has been studied extensively by many groups for many years, it remains a complex issue that probably will not be definitely clarified in the near future.

Here, we have focused on investigating the relevance of ceramide in caspase-independent PCD. In line with caspase-independent PCD being the more ancient form of cell death clearly predating the appearance of caspases in evolution (1 , 41) , ceramide signaling likewise predates apoptosis (40) . Therefore, it is tempting to speculate that in the ancient pathways to cell death, ceramide might have represented one of the central mediators. Subsequently, with the evolution of caspase-dependent apoptosis, the original system, including ceramide, may have partially or completely transferred its functions to caspase-dependent mechanisms, resulting in the differential relevance of ceramide for apoptotic cell death seen in current organisms. However, under conditions where the classical apoptosis machinery fails (e.g., in tumor cells that have become resistant to death signals inducing apoptosis), intracellular generation of ceramide may regain importance by acting as part of an ancient backup system that enables a cell to still commit suicide by caspase-independent PCD.

A pivotal role of ceramide in caspase-independent PCD is supported by our observation that ceramide consistently accumulates well before the onset of caspase-independent death in multiple cell systems. Interference with ceramide generation by multiple approaches such as overexpression of AC, pharmacological intervention, RNAi, or genetic ablation of A-SMase or RIP1 uniformly conferred protection regardless of the cell type studied. Moreover, A-SMase-deficient cells could be resensitized for caspase-independent PCD by reconstitution of A-SMase, providing compelling evidence for ceramide as a central mediator of caspase-independent PCD.

Unlike a previous study reporting that fumonisin B1 can protect L929 cells against TNF cytotoxicity (42) , we did not observe protection against TNF/zVAD in this or against TNF in a previous (14) study. This may be explained by differences of the L929 cells used. However, our results using fumonisin B1 are consistent within L929, NIH3T3, and Jurkat wild-type cells and in line with the data using AC-overexpressing or A-SMase-deficient cells, implicating A-SMase as the main enzyme responsible for the observed accumulation of ceramide.

With regard to the associated signal transduction pathways, RIP1 appears to be a central initiator since blockade of RIP1 function by genetic ablation, RNAi, or pharmacological intervention protected from TNF-induced caspase-independent PCD. Since inhibition of ceramide accumulation clearly diminished caspase-independent PCD but not as completely as inhibition of RIP1, ceramide obviously represents a central, but most likely not the only, factor transmitting the death signals generated by RIP1 in response to TNF.

Our own results and data from others using different Bcl-2 overexpressing cell lines implicate mitochondria in the ceramide death pathway (34 , 35) . In line, mitochondria have been described as targets of ceramide in vitro (43) and in vivo (1) . The generation of ROS represents one of the mechanisms that have been suggested for the induction of caspase-independent PCD by mitochondria (3) . Cauwels and co-workers recently demonstrated that zVAD-fmk does not alleviate, but rather exacerbates, TNF toxicity by a caspase-independent, ROS-mediated pathway in mice, providing strong evidence for a role of ROS in caspase-independent PCD in vivo (2) . In accordance, our own data demonstrate protection of L929 cells by the radical scavenger BHA. However, the structurally very similar scavenger BHT did not protect L929 cells, and Jurkat wild-type cells were not protected by either scavenger. The different response of these two cell lines indicates that the involvement of mitochondria in ceramide-mediated caspase-independent PCD may not be universal. Therefore, the precise contribution of ROS to caspase-independent PCD in different cell types will have to be addressed in further studies. With regard to cytochrome c, our data clearly do not support a role for this molecule in caspase-independent PCD. The slow dissipation of {Delta}{Psi}m (and likewise, the late positive annexin V-staining) observed in TNF(/CHX)/zVAD-treated L929, NIH3T3, and Jurkat wild-type cells coinciding with loss of plasma membrane integrity has also been described in caspase-independent PCD induced by other stimuli (TNF or FasL; refs 28 , 35 ). This suggests that loss of {Delta}{Psi}m (as well as positive staining for phosphatidylserine) is a consequence rather than the cause of ceramide-mediated caspase-independent PCD. Therefore, aside from ROS as potential mediators, mitochondria may participate in the ceramide death pathway by mechanisms and factors other than early release of cytochrome c or early dissipation of {Delta}{Psi}m. The function of apoptosis-inducing factor as such a mitochondrial candidate molecule is subject of current investigation. As for the mechanism by which Bcl-2 protects from ceramide-mediated caspase-independent PCD, Denecker and co-workers have implicated complexation of the proapoptotic Bcl-2 family member BNIP3 by Bcl-2, and alternatively, the ability of Bcl-2 to prolong the integrity of mitochondrial oxidative phosphorylation (35) .

Our data indicate that inhibition of caspases (rather than cathepsins) appears to be responsible for the amplification of TNF-mediated caspase-independent PCD by zVAD-fmk in L929 cells, in line with previous data. In addition to zVAD-fmk, both zIETD-fmk and zDEVD-fmk induced accumulation of ceramide and accelerated TNF-dependent cell death in L929 cells. Since zDEVD inhibits caspase-8 in addition to caspase-3 (37) , our data argue for the initiator caspase-8 as a likely candidate molecule whose inhibition (e.g., by infecting viruses attempting to block apoptotic suicide) might amplify the execution of the caspase-independent death program, thereby accelerating rather than blocking cell suicide. A similar amplification of caspase-independent PCD has been described after transfection of a dominant negative caspase-8 construct (27) . Alternatively, inhibition of caspase-8 may inhibit a signaling pathway that protects from caspase-independent PCD. For instance, caspase-8 is able to proteolyze RIP1. Therefore, inhibition of caspase-8 might prevent cleavage of RIP1 and thus propagate caspase-independent PCD (44 , 45) .

Finally, our results demonstrate that induction of ceramide-mediated caspase-independent PCD leads to a pronounced reduction of clonogenic survival, providing additional evidence for the concept that modulation of ceramide metabolism may represent an attractive strategy to augment conventional tumor therapies. This may be especially valuable for the elimination of tumor cells that have become resistant to drugs inducing apoptotic death signals (46 , 47) .


   ACKNOWLEDGMENTS
 
We thank J. Blenis, B. Seed, and S. Korsmeyer for the FADD- and RIP1-deficient as well as for the Bcl-2-overexpressing Jurkat cells, T. Ogita for scyphostatin, and M. Hein and A. Hethke for technical assistance. We are especially indebted to E. Gulbins for kindly and generously providing A-SMase-deficient mice, and we explicitly wish to thank D. Kabelitz for his continuous support and encouragement. Supported by a grant of the Deutsche Forschungsgemeinschaft to D.A. (SFB 415, A4).

Received for publication January 26, 2005. Accepted for publication August 10, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Leist, M., Jäättelä, M. (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol. 2,589-598[CrossRef][Medline]
  2. Cauwels, A., Janssen, B., Waeytens, A., Cuvelier, C., Brouckaert, P. (2003) Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2. Nat. Immunol. 4,387-393[CrossRef][Medline]
  3. Jäättelä, M., Tschopp, J. (2003) Caspase-independent cell death in T lymphocytes. Nat. Immunol. 4,416-423[CrossRef][Medline]
  4. Johnstone, R. W., Ruefli, A. A., Lowe, S. W. (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108,153-164[CrossRef][Medline]
  5. Hofmann, K., Dixit, V. M. (1998) Ceramide in apoptosis—does it really matter?. Trends Biochem. Sci. 23,374-377[CrossRef][Medline]
  6. Kolesnick, R., Hannun, Y. A. (1999) Ceramide and apoptosis. Trends Biochem. Sci. 24,224-225[CrossRef][Medline]
  7. Ramos, B., Lahti, J. M., Claro, E., Jackowski, S. (2003) Prevalence of necrosis in C2-ceramide-induced cytotoxicity in NB16 neuroblastoma cells. Mol. Pharmacol. 64,502-511[Abstract/Free Full Text]
  8. Zhao, S., Yang, Y. N., Song, J. G. (2004) Ceramide induces caspase-dependent and -independent apoptosis in A-431 cells. J. Cell. Physiol. 199,47-56[CrossRef][Medline]
  9. Pirianov, G., Colston, K. W. (2001) Interactions of vitamin D analogue CB1093, TNFalpha and ceramide on breast cancer cell apoptosis. Mol. Cell. Endocrinol. 172,69-78[CrossRef][Medline]
  10. Maurer, B. J., Melton, L., Billups, C., Cabot, M. C., Reynolds, C. P. (2000) Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism. J. Natl. Cancer Inst. 92,1897-1909[Abstract/Free Full Text]
  11. Metkar, S. S., Anand, M., Manna, P. P., Naresh, K. N., Nadkarni, J. J. (2000) Ceramide-induced apoptosis in Fas-resistant Hodgkin’s disease cell lines is caspase independent. Exp. Cell Res. 255,18-29[CrossRef][Medline]
  12. Jones, B. E., Lo, C. R., Srinivasan, A., Valentino, K. L., Czaja, M. J. (1999) Ceramide induces caspase-independent apoptosis in rat hepatocytes sensitized by inhibition of RNA synthesis. Hepatology 30,215-222[CrossRef][Medline]
  13. Mengubas, K., Fahey, A. A., Lewin, J., Mehta, A. B., Hoffbrand, A. V., Wickremasinghe, R. G. (1999) Killing of T lymphocytes by synthetic ceramide is by a nonapoptotic mechanism and is abrogated following mitogenic activation. Exp. Cell Res. 249,116-122[CrossRef][Medline]
  14. Strelow, A., Bernardo, K., Adam-Klages, S., Linke, T., Sandhoff, K., Krönke, M., Adam, D. (2000) Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death. J. Exp. Med. 192,601-611[Abstract/Free Full Text]
  15. Santana, P., Peña, 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[CrossRef][Medline]
  16. Grassmé, H., Kirschnek, S., Riethmueller, J., Riehle, A., von Kurthy, G., Lang, F., Weller, M., Gulbins, E. (2000) CD95/CD95 ligand interactions on epithelial cells in host defense to pseudomonas aeruginosa. Science 290,527-530[Abstract/Free Full Text]
  17. Schuermann, M. (1990) An expression vector system for stable expression of oncogenes. Nucleic Acids Res. 18,4945-4946[Free Full Text]
  18. Dressler, K. A., Kolesnick, R. N. (1990) Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin. J. Biol. Chem. 265,14917-14921[Abstract/Free Full Text]
  19. Perry, D. K., Hannun, Y. A. (1999) The use of diglyceride kinase for quantifying ceramide. Trends Biochem. Sci. 24,226-227[CrossRef][Medline]
  20. Jensen, J. M., Schütze, S., Förl, M., Krönke, M., Proksch, E. (1999) Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier. J. Clin. Invest. 104,1761-1770[Medline]
  21. 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[CrossRef][Medline]
  22. Vercammen, D., Beyaert, R., Denecker, G., Goossens, V., van Loo, G., Declercq, W., Grooten, J., Fiers, W., Vandenabeele, P. (1998) Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J. Exp. Med. 187,1477-1485[Abstract/Free Full Text]
  23. 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[CrossRef][Medline]
  24. Hurwitz, R., Ferlinz, K., Sandhoff, K. (1994) The tricyclic antidepressant desipramine causes proteolytic degradation of lysosomal sphingomyelinase in human fibroblasts. Biol. Chem. Hoppe Seyler 375,447-450[Medline]
  25. Tanaka, M., Nara, F., Suzuki-Konagai, K., Hosoya, T., Ogita, T. (1997) Structural elucidation of scyphostatin, an inhibitor of membrane-bound neutral sphingomyelinase. J. Am. Med. Assoc. 119,7871-7872
  26. Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., Merrill, A. H., Jr (1991) Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J. Biol. Chem. 266,14486-14490[Abstract/Free Full Text]
  27. Lüschen, S., Ussat, S., Scherer, G., Kabelitz, D., Adam-Klages, S. (2000) Sensitization to death receptor cytotoxicity by inhibition of Fas-associated death domain protein (FADD)/caspase signaling—requirement of cell cycle progression. J. Biol. Chem. 275,24670-24678[Abstract/Free Full Text]
  28. Holler, N., Zaru, R., Micheau, O., Thomé, M., Attinger, A., Valitutti, S., Bodmer, J. L., Schneider, P., Seed, B., Tschopp, J. (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1,489-495[CrossRef][Medline]
  29. Juo, P., Woo, M. S., Kuo, C. J., Signorelli, P., Biemann, H. P., Hannun, Y. A., Blenis, J. (1999) FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ 10,797-804[Abstract/Free Full Text]
  30. 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]
  31. Ting, A. T., Pimentel-Muinos, F. X., Seed, B. (1996) RIP mediates tumor necrosis factor receptor 1 activation of NF-kappa B but not Fas/APO-1-initiated apoptosis. EMBO J. 15,6189-6196[Medline]
  32. Vanden Berghe, T., Kalai, M., van Loo, G., Declercq, W., Vandenabeele, P. (2003) Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis. J. Biol. Chem. 278,5622-5629[Abstract/Free Full Text]
  33. Armstrong, R. C., Aja, T., Xiang, J., Gaur, S., Krebs, J. F., Hoang, K., Bai, X., Korsmeyer, S. J., Karanewsky, D. S., Fritz, L. C., et al (1996) Fas-induced activation of the cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors. J. Biol. Chem. 271,16850-16855[Abstract/Free Full Text]
  34. Hennet, T., Bertoni, G., Richter, C., Peterhans, E. (1993) Expression of Bcl-2 protein enhances the survival of mouse fibrosarcoid cells in tumor necrosis factor-mediated cytotoxicity. Cancer Res. 53,1456-1460[Abstract/Free Full Text]
  35. Denecker, G., Vercammen, D., Steemans, M., Vanden Berghe, T., Brouckaert, G., van Loo, G., Zhivotovsky, B., Fiers, W., Grooten, J., Declercq, W., et al (2001) Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria. Cell Death Differ 8,829-840[CrossRef][Medline]
  36. Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M., Jäättelä, M. (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153,999-1009[Abstract/Free Full Text]
  37. Garcia-Calvo, M., Peterson, E. P., Leiting, B., Ruel, R., Nicholson, D. W., Thornberry, N. A. (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273,32608-32613[Abstract/Free Full Text]
  38. Pettus, B. J., Chalfant, C. E., Hannun, Y. A. (2002) Ceramide in apoptosis: an overview and current perspectives. Biochim. Biophys. Acta 1585,114-125[Medline]
  39. Kolesnick, R. N., Krönke, M. (1998) Regulation of ceramide production and apoptosis. Annu. Rev. Physiol. 60,643-665[CrossRef][Medline]
  40. Kolesnick, R. (2002) The therapeutic potential of modulating the ceramide/sphingomyelin pathway. J. Clin. Invest. 110,3-8[CrossRef][Medline]
  41. Koonin, E. V., Aravind, L. (2002) Origin and evolution of eukaryotic apoptosis: the bacterial connection. Cell Death Differ 9,394-404[CrossRef][Medline]
  42. Dbaibo, G. S., El-Assaad, W., Krikorian, A., Liu, B., Diab, K., Idriss, N. Z., El-Sabban, M., Driscoll, T. A., Perry, D. K., Hannun, Y. A. (2001) Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death. FEBS Lett. 503,7-12[CrossRef][Medline]
  43. Ghafourifar, P., Klein, S. D., Schucht, O., Schenk, U., Pruschy, M., Rocha, S., Richter, C. (1999) Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state. J. Biol. Chem. 274,6080-6084[Abstract/Free Full Text]
  44. Vanden Berghe, T., van Loo, G., Saelens, X., van Gurp, M., Brouckaert, G., Kalai, M., Declercq, W., Vandenabeele, P. (2004) Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD. J. Biol. Chem. 279,7925-7933[Abstract/Free Full Text]
  45. Yu, L., Alva, A., Su, H., Dutt, P., Freundt, E., Welsh, S., Baehrecke, E. H., Lenardo, M. J. (2004) Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304,1500-1502[Abstract/Free Full Text]
  46. Senchenkov, A., Litvak, D. A., Cabot, M. C. (2001) Targeting ceramide metabolism—a strategy for overcoming drug resistance. J. Natl. Cancer Inst. 93,347-357[Abstract/Free Full Text]
  47. Reynolds, C. P., Maurer, B. J., Kolesnick, R. N. (2004) Ceramide synthesis and metabolism as a target for cancer therapy. Cancer Lett. 206,169-180[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
S. Uhlig and E. Gulbins
Sphingolipids in the Lungs
Am. J. Respir. Crit. Care Med., December 1, 2008; 178(11): 1100 - 1114.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
E. L. Smith and E. H. Schuchman
The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases
FASEB J, October 1, 2008; 22(10): 3419 - 3431.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. J. May and L. A. Madge
Caspase Inhibition Sensitizes Inhibitor of NF-{kappa}B Kinase beta-deficient Fibroblasts to Caspase-independent Cell Death via the Generation of Reactive Oxygen Species
J. Biol. Chem., June 1, 2007; 282(22): 16105 - 16116.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. E. van Vlerken, Z. Duan, M. V. Seiden, and M. M. Amiji
Modulation of Intracellular Ceramide Using Polymeric Nanoparticles to Overcome Multidrug Resistance in Cancer
Cancer Res., May 15, 2007; 67(10): 4843 - 4850.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Ramu, Y. Xu, and Z. Lu
Inhibition of CFTR Cl- channel function caused by enzymatic hydrolysis of sphingomyelin
PNAS, April 10, 2007; 104(15): 6448 - 6453.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
Q. He, H. Suzuki, N. Sharma, and R. P. Sharma
Ceramide Synthase Inhibition by Fumonisin B1 Treatment Activates Sphingolipid-Metabolizing Systems in Mouse Liver
Toxicol. Sci., December 1, 2006; 94(2): 388 - 397.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Supplementary Figures
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thon, L.
Right arrow Articles by Adam, D.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Thon, L.
Right arrow Articles by Adam, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS