Cell Autonomous Apoptosis Defects in Acid Sphingomyelinase Knockout Fibroblasts*

José LozanoDagger , Silvia MenendezDagger , Albert MoralesDagger , Desiree Ehleiter§, Wen-Chieh Liao§, Rachel Wagman§, Adriana Haimovitz-Friedman§, Zvi Fuks§, and Richard Kolesnick*Dagger

From the Dagger  Laboratory of Signal Transduction and § Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, July 18, 2000, and in revised form, September 12, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A body of evidence suggests that stress-induced sphingomyelin hydrolysis to the second messenger ceramide initiates apoptosis in some cells. Although studies using lymphoblasts from Niemann-Pick disease patients or acid sphingomyelinase (ASMase)-deficient mice have provided genetic support for this hypothesis, these models have not been universally accepted as definitive. Here, we show that mouse embryonic fibroblasts (MEFs) prepared from asmase mice manifest cell autonomous defects in apoptosis in response to several stresses. In particular, asmase-/- MEFs failed to generate ceramide and were totally resistant to radiation-induced apoptosis but remained sensitive to staurosporine, which did not induce ceramide. asmase-/- MEFs were also partially resistant to tumor necrosis factor alpha / actinomycin D and serum withdrawal. Thus, resistance to apoptosis in asmase-/- MEFs was not global but rather stress type specific. Most importantly, the sensitivity to stress could be restored in the asmase-/- MEFs by administration of natural ceramide. Overcoming apoptosis resistance by natural ceramide is evidence that it is the lack of ceramide, not ASMase, that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by the product of the enzymatic deficiency provides proof that ceramide is obligate for apoptosis induction in response to some stresses.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The sphingomyelin (SM)1 pathway is an ubiquitous signaling system conserved from yeast to humans (1-3). Ceramide is the central molecule in this pathway, generated from SM by the action of a neutral sphingomyelinase or acid sphingomyelinase (ASMase) or by de novo synthesis coordinated through the enzyme ceramide synthase. Ceramide has been implicated as second messenger for cytokines signaling through the tumor necrosis factor (TNF) receptor superfamily and for diverse stresses (e.g. ionizing radiation, oxidative stress, ultraviolet C, and heat). Although in some cells ceramide may provide proliferative or differentiating signals, often the outcome of ceramide signaling is the induction of apoptosis. Evidence that ceramide is involved directly in the induction of apoptosis is summarized as follows: (i) agonist-induced ceramide elevations precede biochemical and morphological manifestations of apoptosis (4-7); (ii) elevation of cellular ceramide levels using natural ceramide (8-10), ceramide analogs, or exogenous SMases mimic the effects of stress on apoptosis (11-16); (iii) pharmacological agents which interfere with enzymes of ceramide metabolism and elevate cellular ceramide, such as the glucosylceramide synthase inhibitor D-threo-1-phenyl-decanoylamino-3-morpholino-1-propanol or the ceramidase inhibitor N-oleoylethanolamine, uniformly enhance apoptosis (17-19), whereas agents that reduce ceramide generation, such as the ceramide synthase inhibitor fumonisin B1, prevent apoptosis (10, 20, 21); and (iv) other lipids, including other sphingolipid metabolites (except perhaps sphingosine), fail to consistently signal apoptosis (12, 13, 16).

The most compelling evidence supporting ceramide as mediator of apoptosis is derived, however, from data using genetic models. Lymphoblasts from Niemann-Pick disease (NPD) patients, which have an inherited deficiency of ASMase activity, failed to respond to ionizing radiation with ceramide generation or apoptosis (22-24). These abnormalities were, however, reversed by transduction of the human asmase cDNA. Furthermore, asmase knockout mice also manifested defects in radiation-induced ceramide generation and apoptosis in endothelium of the lung and throughout the central nervous system (22, 25). asmase knockout mice were, however, normally sensitive to radiation-induced apoptosis in thymic cells. In contrast, p53 knockout mice were largely insensitive in the thymus but were not resistant to radiation-induced apoptosis in endothelium. Since these original findings, other reports have confirmed that the Niemann-Pick cells are resistant to ionizing radiation (23, 24). Whether the ASMase-deficient systems are resistant to Fas-/APO-1-/CD95-induced death has been a matter of debate. Testi and coworkers (26) claimed the Niemann-Pick cells manifested resistance to Fas-induced death, whereas Borst and coworkers (27) found no differences. Recently, Green and coworkers (28) reported partial resistance to Jo2 anti-Fas-induced liver failure and death in asmase knockout mice in vivo.

These genetic models have not uniformly been accepted as definitive. The NPD lymphoblasts described above were transformed with Epstein-Barr virus, and the transformation process itself, as well as known Epstein-Barr virus strain-specific phenotypic differences (29-31), might have played an unknown role in the radiation resistance. Furthermore, the potential for a direct contribution of the mutated ASMase protein to the radiation resistance phenotype has not been assessed. Additionally, it remains possible that the resistance of endothelium to radiation-induced apoptotic death in vivo is not a cell autonomous defect but rather reflects a systemic process. To address these issues directly, we have generated murine embryonic fibroblasts (MEFs) from the asmase knockout mice. We observe complete resistance to radiation-induced apoptosis in these cells, partial resistance to serum withdrawal or TNFalpha / actinomycin D (ActD) treatment, yet complete sensitivity to staurosporine-induced apoptosis. Furthermore, sensitivity to radiation-, serum withdrawal-, and TNFalpha /ActD-induced death could be restored by pretreatment with minute amounts of natural ceramide. Rescuing the apoptotic phenotype without reversing the genotype by the product of the enzymatic deficiency indicates that ceramide is obligate, in particular for radiation, for apoptotic death.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Reagents-- Actinomycin D, Hoechst-33258, and staurosporine were from Sigma. Recombinant mouse TNFalpha was from Pharmingen. The fluorogenic substrate Ac-DEVD-AFC was purchased from Kamiya Biomedical Co. Anti-phospho-mitogen-activated protein kinase and anti-phospho-p38 antibodies were from New England Biolabs, whereas the anti-phospho c-Jun N-terminal kinase (JNK) antibody was from Promega. The anti-TNF receptor (TNFR) antibody was purchased from Santa Cruz Biochemicals. LK6D Silica Gel 60A TLC plates were from Whatman. C16-ceramide and C16-dihydroceramide were from Biomol.

MEF Preparation and Culture-- MEFs derived from asmase+/+ and asmase-/- day 12-13 embryos were prepared as described (32). Cells were subcultured every 3 days for a maximum of six passages. Only MEFs from passages 2-5 were used for experiments. 0.05-0.1 × 106 cells were seeded per 60-mm plate and grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. Unless otherwise indicated, cells were made quiescent by 12-h incubation in 0.5% FBS and then either irradiated using a 137Cs Shepherd Mark-I irradiator or treated with different inducers of apoptosis, as stated in the figure legends. For ceramide rescue, cells were pretreated for 30 min with C16-ceramide or C16-dihydroceramide in dodecane:ethanol (2:98, v/v; 0.05% final concentration).

Lipid Mass Analysis-- Total cellular lipids were extracted as described previously (13). To measure SM mass, equal aliquots of each sample were spotted on TLC plates, and SM was resolved using chloroform:methanol:acetic acid:water (50:30:8:3, v/v/v/v) as solvent. After chromatography, plates were sprayed with 35% (v/v) sulfuric acid, and lipids were charred by heating in an oven at 180 °C for 30 min and quantified by comparison with a concomitantly run standard curve composed on known quantities of SM. Ceramide was quantified by the diacylglycerol kinase assay as described previously (33). ASMase and neutral sphingomyelinase activities were measured as described by Lin et al. (34).

Bisbenzimide Staining-- At different times after treatment, cells were processed for nuclear staining as described (35). Briefly, the medium containing the floating dead cells was aspirated and pooled together with a PBS wash of the monolayer in a 15-ml Falcon conical tube. The remaining adherent cells were trypsinized, collected in the same tube, and centrifuged at 400 × g for 5 min. The pellets were gently vortexed and fixed in 400 µl of 10% buffered formalin phosphate for 10 min at room temperature. Nuclear apoptosis was assessed by staining of formalin-fixed cells with the DNA binding fluorochrome Hoechst-33258 as described (13). A minimum of 200 cells were scored at each point.

Caspase 3 Assay-- The fluorogenic substrate Ac-DEVD-AFC was used to measure caspase 3 activity according to the manufacturer's instructions. Briefly, cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate plus 10 µg/ml leupeptin and 10 µg/ml soybean trypsin inhibitor), and the protein concentration was measured. 20 µg of cell lysate were mixed with 8 µM Ac-DEVD-AFC in 200 µl of caspase 3 buffer (50 mM HEPES, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and 10 mM dithiothreitol) in a 96-well plate. Fluorescence generated by the release of the fluorogenic group AFC on cleavage by caspase 3 was measured in a PerkinElmer LS50B luminescence spectrometer by excitation at 400 nm and emission at 505 nm.

Immunoblot Assays-- Mitogen-activated protein kinase, JNK, and p38 activation were detected by Western blot with anti-phospho-specific antibodies. After 12 h in 0.5% FBS, cells were stimulated with 100 ng/ml epidermal growth factor, 100 ng/ml platelet-derived growth factor, or 100 ng/ml TNFalpha for 10 min, washed with PBS, and lysed in 0.2 ml of Nonidet P-40 lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 2 mM EDTA, 10% glycerol, and 1% Nonidet P-40 plus protease and phosphatase inhibitors). Western blots were performed as described (36).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of asmase-/- MEFs-- To investigate whether ASMase-deficient cells displayed cell autonomous defects in apoptosis, we generated MEFs from wild-type and ASMase-deficient mice. No measurable ASMase activity was detected in the asmase-/- MEFs (Table I), whereas neutral sphingomyelinase activity was unchanged. As a consequence of the ASMase deficiency, asmase-/- MEFs manifested a modest 20% increase in the SM content and a 12% decrease in ceramide levels (Table I). Both wild-type and knockout MEFs grew at similar rates (Table I) and manifested identical morphology (data not shown). The content and distribution of caveolin-1, the primary protein within the sphingolipid-rich microdomains termed caveolae, were similar in asmase+/+ and asmase-/- MEFs (37). Furthermore, the proportion of detergent-insoluble caveolar membranes isolated and the signaling components found within these membranes appeared similar in asmase+/+ and asmase-/- MEFs (37). Activation of p38, c-Jun N-terminal, and extracellular signal-regulated kinases in response to platelet-derived growth factor, TNFalpha , and epidermal growth factor were also identical in asmase+/+ and asmase-/- MEFs (see Table I and Fig. 3B). Thus, by numerous criteria, asmase+/+ and asmase-/- MEFs appeared similar.


                              
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Table I
Comparison of asmase+/+ and asmase-/- MEFs

asmase-/- MEFs Are Resistant to Radiation-induced Apoptosis-- Our previous studies demonstrated that NPD lymphoblasts failed to respond to ionizing radiation with ceramide generation or apoptosis (22-24). To evaluate whether asmase-/- MEFs display similar defects, cells were made quiescent by incubation in 0.5% FBS for 12 h, irradiated, and assessed for apoptosis at various times after irradiation. A 12-h quiescence period was selected to minimize the basal level of apoptosis induced by serum withdrawal, which under these conditions was typically in the range of 4-5%. Fig. 1A, left panel, shows that exposure of asmase+/+ cells to a dose of 20 Gy resulted in time-dependent apoptosis, as measured by bisbenzimide staining. Apoptosis was apparent by 3 h and maximal 12 h after irradiation, when 25-30% of the population manifested chromatin condensation or nuclear segmentation, or both, morphologic characteristics of apoptotic cells. In contrast, asmase-/- cells were resistant to radiation-induced apoptosis (Fig. 1A) for as long as 72 h (data not shown). To determine whether a higher radiation dose might induce apoptosis in the asmase-/- cells, we treated the wild-type and knockout cells with increasing radiation doses and measured apoptosis after 24 h (Fig. 1A, right panel). As little as 1 Gy increased apoptosis to 13% of the asmase+/+ population, and a maximal effect was obtained with a 20-Gy dose. Quantitatively similar results were reported by Santana et al. (22) after irradiation of Epstein-Barr virus-transformed lymphoblasts from Niemann-Pick patients. In contrast, asmase-/- MEFs were resistant to radiation up to 30 Gy.



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Fig. 1.   asmase-/- MEFs are resistant to radiation-induced apoptosis. A, asmase+/+ and asmase-/- MEFs were grown in 0.5% FBS for 12 h, irradiated, and apoptosis was measured by bisbenzimide staining at various times after a 20 Gy irradiation (left) and at 24 h after irradiation with various doses (right). Data (mean ± S.E.) represent three studies performed in duplicate. At least 200 cells were evaluated for each point. B, time course of caspase 3-like activity after 20 Gy measured as cleavage of the fluorogenic substrate Ac-DEVD-AFC. Cells were made quiescent, irradiated as in A, and then cell lysates were prepared in radioimmunoprecipitation assay buffer, and the DEVDase activity was measured. Data (mean ± S.E.) represent three studies performed in duplicate. C, ceramide levels in asmase+/+ and asmase-/- MEFs at 15 min after 20 Gy of irradiation were determined by the diacylglycerol kinase assay. Data (mean ± S.E.) represent three independent studies performed in duplicate. *, p < 0.05 versus unirradiated controls or irradiated asmase-/- MEFs.

To confirm the results of Fig. 1A using a biochemical assay, caspase 3 activity was measured in both asmase+/+ and asmase-/- MEFs at various times after irradiation. As shown in Fig. 1B, we detected a 2.8 ± 0.1-fold increase in caspase activity at 6 h after 20 Gy irradiation (p < 0.001 versus zero time control) in asmase+/+ MEFs and a maximal 3.4 ± 0.1-fold increase at 10 h. In agreement with the results shown in Fig. 1A, we could not detect any increase in caspase 3 activity in the asmase-/- MEFs at any time point. These results show that lack of ASMase renders cells resistant to radiation-induced apoptosis.

To confirm that ASMase deficiency resulted in resistance to ceramide generation, ceramide levels were measured after irradiation of asmase+/+ and asmase-/- MEFs. In asmase+/+ cells, the ceramide content increased 1.5-fold by 15 min after 20 Gy irradiation (Fig. 1C; p < 0.05) and returned to baseline within 1 h. In contrast, no ceramide elevation was observed in the asmase-/- MEFs under the same conditions (Fig. 1C) at any time point up to 4 h. These data indicate that, as in NPD cells, lack of ASMase renders MEFs resistant to radiation-induced ceramide generation and apoptosis. Although these studies are consistent with a role for ceramide accumulation in the apoptotic response to ionizing radiation, they do not constitute proof that the apoptosis resistance resulted from lack of ceramide production.

asmase-/- MEFs Are Sensitive to Staurosporine-induced Apoptosis-- To evaluate whether the mutation leading to the asmase-/- phenotype resulted in a universal inactivation of the apoptotic apparatus, MEFs were exposed to another type of proapoptotic stress. Staurosporine is a potent protein kinase inhibitor (38) that induces apoptosis in many cell types (39-41). In the wild-type MEFs, a dose as low as 0.01 µM staurosporine induced apoptosis in 50% of the population after 24 h, whereas 95% apoptosis was observed with a dose of 0.1 µM staurosporine (Fig. 2A, left panel). In contrast to the irradiation experiments, the asmase-/- MEFs showed no resistance to staurosporine-induced cell death, and the level of apoptosis was essentially identical to that of the asmase+/+ MEFs. In fact, if there was any difference, the asmase-/- MEFs died slightly faster (Fig. 2A, right panel); at 6 h after addition of staurosporine 37.6 ± 1.7% of the asmase-/- MEFs were undergoing apoptosis, whereas only 24.6 ± 0.1% of the asmase+/+ cells displayed apoptosis. To confirm these results, caspase 3 activation was also measured in the staurosporine-treated MEFs. By 8 h after treatment, we detected an ~6.5-fold increase in caspase 3 activity in the asmase+/+ MEFs, in good agreement with staurosporine as a more potent inducer of apoptosis than ionizing radiation in MEFs (compare Fig. 1A with Fig. 2A). Caspase 3 activation in asmase-/- MEFs was very similar to that in the asmase+/+ MEFs (Fig. 2B). In contrast to radiation, staurosporine-induced apoptosis did not appear associated with ceramide generation. Staurosporine failed to generate ceramide at any time within the first 30 min of treatment in either asmase+/+ or asmase-/- MEFs (data not shown). These studies indicate that asmase-/- MEFs are fully competent in generating an apoptotic response and suggest that the lack of apoptosis to radiation may be specifically associated with ASMase deficiency.



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Fig. 2.   Staurosporine induces apoptosis in asmase-/- MEFs. A, staurosporine dose response at 24 h (left) and time course using 0.04 µM staurosporine (right) of apoptosis measured by bisbenzimide staining as in Fig. 1A. Data (mean ± S.D.) are from two experiments performed in duplicate. B, increase in caspase activity after 0.04 µM staurosporine treatment as measured in Fig. 1B. Data (mean ± S.D.) are from one representative of three independent studies performed in duplicate.

asmase-/- MEFs Are Partially Resistant to Apoptosis after TNFalpha /ActD Treatment-- Whereas ASMase activation and ceramide accumulation have been reported in response to TNFalpha in numerous cell types (12, 35, 42), studies were carried out to explore whether asmase-/- MEFs showed a defect in TNFalpha responsiveness. Many cell types, including MEFs, require previous sensitization for TNFalpha to become toxic (43-45). As in previous studies, a maximal dose of TNFalpha (1 ng/ml) was unable to signal apoptosis in wild-type MEFs. However, the combination of TNFalpha and ActD effectively induced time- and dose-dependent apoptosis. As little as 0.2 µg/ml ActD in combination with 1 ng/ml TNFalpha induced apoptosis in 50% of the asmase+/+ MEFs (Fig. 3A) after 24 h, while 75% apoptosis was achieved with 0.3% µg/ml ActD and TNFalpha . Apoptosis of asmase+/+ MEFs in response to TNFalpha /ActD was detectable by 8 h and maximal at 24 h of treatment (data not shown). At all times from 8 to 24 h the apoptotic response of asmase-/- MEFs to TNFalpha /ActD was reduced by 25-50% compared with asmase+/+ MEFs (shown in Fig. 3A at 24 h), indicating partial resistance to this stress. asmase-/- MEFs were also resistant to ceramide generation. In contrast to the transient ceramide elevation in asmase+/+ MEFs after ionizing radiation (Fig. 1C), TNFalpha (1 ng/ml)/ActD (0.3 µg/ml) induced a persistent 40-50% ceramide increase beginning 15 min after irradiation and lasting for at least 1 h. asmase-/- MEFs, however, displayed no ceramide elevation up to 6 h after irradiation. These data suggest that only part of the apoptotic response to TNFalpha /ActD is mediated via ASMase.



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Fig. 3.   TNFalpha -dependent apoptosis in asmase-/- MEFs. A, MEFs were treated with TNFalpha (1 ng/ml) and increasing concentrations of ActD, and apoptosis was measured after 24 h as in Fig. 1A. Data (mean ± SD) are from one representative of three independent studies performed in duplicate. B, 30 µg of total cell lysates prepared from asmase+/+ and asmase-/- MEFs were resolved by 7.5% SDS-polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane, TNFR expression was analyzed by Western blot with an anti-TNFR antibody (left panel). To measure JNK, p38, and mitogen-activated protein kinase activation, cells were grown in low serum (0.5% FBS) for 24 h and then either left untreated or stimulated with 100 ng/ml TNFalpha , epidermal growth factor, or platelet-derived growth factor for various times. Cell lysates were prepared, and 50 µg of protein from each sample were analyzed by Western blot with anti-phospho-specific antibodies (right panel). Note that the anti-phospho-JNK antibody recognizes both the JNK1 and JNK2 activated isoforms. In the phospho-extracellular signal-regulated kinase studies, cells were stimulated for 10 min. Similar results were obtained in two independent experiments.

To exclude the possibility that the difference in the response of asmase+/+ and asmase-/- MEFs to TNFalpha /ActD was attributable to a deficiency in TNF receptors, we examined the level of TNFRs by Western blot with an anti-TNFR antibody. Fig. 3B, left panel, shows that asmase+/+ and asmase-/- MEFs express similar levels of TNFRs. Despite similar receptor levels, it was possible that TNFRs might not be functional in asmase-/- MEFs. To rule out this possibility, we treated asmase+/+ and asmase-/- MEFs with TNFalpha and examined the status of three major signaling pathways, the JNK, p38, and extracellular signal-regulated kinase 1 and 2 cascades, with antibodies specific for the activated kinases. Fig. 3B, right panel, shows that TNFalpha induced a robust activation of C-terminal Jun and p38 protein kinases in both types of MEFs after 10 min of treatment. We did not detect extracellular signal-regulated kinase 1 and 2 activation by TNFalpha , although it was rapidly activated by growth factors such as epidermal growth factor and platelet-derived growth factor in both asmase+/+ and asmase-/- MEFs (Fig. 3B). Nuclear factor kappa B nuclear translocation in response to TNFalpha treatment was also normal in ASMase-deficient MEFs (Table I). These results suggest that the differences in apoptosis observed between asmase+/+ and asmase-/- MEFs are not attributable to an alteration in the level or the functionality of membrane receptors but rather to a selective deficiency in ASMase activity.

asmase-/- MEFs Are Partially Resistant to Serum Withdrawal-induced Apoptosis-- To investigate the response of ASMase-deficient MEFs to induction of apoptosis by serum withdrawal, MEFs from wild-type and knockout animals were plated in 15% FBS for 24 h and then switched to low serum. After 48 h, wild-type MEFs displayed more apoptosis than asmase-/- at all concentrations from 0.1 to 4% FBS (Fig. 4). Differences were more pronounced in cells challenged with very low doses of serum (<1%). (Note that in all previous experiments, cells were serum deprived with 0.5% FBS for only 12 h before any treatment.)



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Fig. 4.   Low serum-induced apoptosis in asmase-/- MEFs. Cells were grown for 24 h in 15% FBS and then switched to low serum (0.1-4% FBS). After 48 h, cells were analyzed for apoptosis as in Fig. 1A. Data (mean ± S.D.) are from two independent studies performed in duplicate.

Natural Ceramide Can Revert the Apoptotic Response to Radiation in the asmase-/- MEFs-- To provide definitive evidence that the lack of ceramide is the cause of the defects in apoptosis, we pretreated asmase-/- cells with natural C16-ceramide before apoptosis induction. Fig. 5 shows that low doses of C16-ceramide (0.05-1.0 µM) had no direct effect on apoptosis at 24 h of treatment. In fact, doses up to 1 µM C16-ceramide were without effect for as long as 48 h (data not shown). However, these doses completely restored apoptosis in response to 20 Gy of ionizing radiation (XRT + 0.5 or 1.0 µM C16-ceramide, p < 0.001 versus 0.5 or 1.0 µM C16-ceramide alone). In contrast, up to 1.0 µM C16-dihydroceramide, the precursor of C16-ceramide, was without effect (data not shown), indicating a specificity for ceramide in this response. Similarly, C16-ceramide rescued the apoptosis defects of asmase-/- MEFs after treatment with TNFalpha /ActD and serum withdrawal (data not shown).



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Fig. 5.   Natural C16-ceramide restores radiation sensitivity to asmase-/- MEFs. For these studies, cells were pretreated for 30 min with C16-ceramide or diluent (0.05% final concentration) before irradiation with 20 Gy (XRT). Apoptosis was evaluated in 300 cells per point as in Fig. 1A after 24 h. 95% confidence limits for each point are shown in one representative of three independent experiments.



    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present investigations provide definitive evidence for cell autonomous defects in apoptosis in ASMase-deficient cells. Similar to the defects observed in endothelial apoptosis in the lung (22) and throughout the central nervous system (25), MEFs from asmase-/- animals failed to generate ceramide within the first 30 min of ionizing radiation treatment and displayed almost complete resistance to induction of apoptosis. In contrast to the marked resistance to ionizing radiation, the sensitivity to staurosporine, which appears to be ceramide independent, was unchanged in asmase-/- MEFs. Thus, resistance to apoptosis in asmase-/- MEFs was not global but rather stress type specific. Consistent with this notion, asmase-/- MEFs displayed partial resistance to TNFalpha /ActD and serum withdrawal. The fact that apoptosis resistance of asmase-/-MEFs to radiation, and the partial resistance to TNFalpha / ActD, could be overcome by natural ceramide provides compelling evidence that it is the lack of ceramide and not the lack of ASMase that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by providing the product of the enzymatic deficiency is proof that ceramide is obligate for apoptosis induction in cells that use the SM pathway.

The present investigations extend our knowledge of the mechanisms of radiation-induced apoptosis. Our studies originally proposed that in addition to double-stranded breaks, damage to the plasma membrane and the generation of the proapoptotic lipid ceramide signaled apoptosis in some cell types (13). This was supported by the demonstration that ionizing radiation was capable of SMase activation in isolated membranes devoid of DNA altogether. Since these initial studies, numerous reports confirm the early generation of ceramide in response to irradiation (14, 23, 46-55) and by a variety of biochemical, pharmacological, and genetic approaches have provided support to the critical role of this event in the ensuing apoptotic response (14, 22, 23, 35, 46, 48, 54, 55). Here, we show that ionizing radiation-induced apoptosis of MEFs requires at least two components, one of which is ceramide, as doses of natural ceramide that alone are without effect restore apoptosis to irradiated cells. Recent studies by Zundel and Giaccia (23) and Zundel et al. (24) may shed light on the mechanism of this ceramide-dependent step. These investigators reported that ionizing radiation induced ceramide-dependent complexation of the antiapoptotic protein phosphoinositide 3-kinase to caveolin-1, resulting in phosphoinositide 3-kinase inactivation. Whether the ceramide-dependent step in MEFs requires phosphoinositide 3-kinase inactivation and the nature of the ceramide-independent step require further investigation.

Nix and Stoffel (56) recently reported marked biochemical alterations and membrane dysfunction in cells derived from their asmase knockout mice, which were generated separately from our colony. Although both knockouts are maintained in a C57BL/6 × 129 background, the substrains of the 129 line are different (22, 57, 58). Furthermore, these ASMase-deficient mouse lines were developed using nonisogenic stem cell clones (22, 57, 58). MEFs derived from their mice displayed dramatic reduction in caveolin content and resistance to isolation of caveolae (56). Further, hepatocytes displayed a 2-fold increase in plasma membrane SM content accompanied by reduced signaling through tyrosine kinases in T lymphocytes, lymphopenia, the absence of proliferation in response to anti-CD3, reduced expression of the antiapoptotic adaptor FLIP, and a paradoxic increase in apoptosis of anti-CD3 pretreated splenocytes upon activation of CD95 (56). The conclusion of these studies was that disruption of membrane microdomains in response to altered sphingolipid metabolism has significant impact on signaling. We have repeated all of these studies and do not find any of these abnormalities in cells derived from our asmase knockout colony (Ref. 37 and Table I). Phenotypic differences also exist between the two asmase knockout mouse colonies. While their mice die from NPD by 4 months of age (57), our animals do not manifest even the earliest sign of NPD, a resting tremor, until 12-16 weeks of age and survive to 9.6 ± 0.4 (mean ± S.D.) months of age (22, 25, 37). Extragenic strain-specific enhancers and suppressors, such as polymorphisms or background mutations, and epigenic environmental factors are well known to modify the expression of genetic phenotypes in mice. Whatever the reason for the differences, it is clear that the asmase knockout mice of Stoffel and coworkers (56, 57) develop NPD more rapidly than our knockout mice. For these reasons, we urge that investigators interested in using these models to study apoptosis select reagents carefully.

The present investigations demonstrate cell autonomous defects in apoptosis in ASMase-deficient cells. These defects are stress type specific; although MEFs were completely resistant to ionizing radiation, they were only partially resistant to TNFalpha /Act D or serum withdrawal, and not at all resistant to staurosporine. Furthermore, the ability of natural ceramide to rescue the apoptosis phenotype without reversing the genotype provides evidence that ceramide is obligate for induction of apoptosis in some stress response systems.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA85714 (to R. K.) and CA52462 (to Z. F.), a Spanish Ministry of Education Fellowship (to J. L.), and a Spanish Ministry of Education-Fulbright Fellowship (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7558; Fax: 212-639-2767.

Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006353200


    ABBREVIATIONS

The abbreviations used are: SM, sphingomyelin; ASMase, acid sphingomyelinase; TNF, tumor necrosis factor; TNFR, TNF receptor; NPD, Niemann-Pick disease; MEF, murine embryonic fibroblast; ActD, actinomycin D; JNK, c-Jun N-terminal kinase; FBS, fetal bovine serum.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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