Acid Sphingomyelinase Activation Requires Caspase-8 but Not P53 nor Reactive Oxygen Species during Fas-Induced Apoptosis in Human Glioma Cells

Experimental Cell Research 273, 157–168 (2002) doi:10.1006/excr.2001.5437, available online at http://www.idealibrary.com on

Acid Sphingomyelinase Activation Requires Caspase-8 but Not P53 nor Reactive Oxygen Species during Fas-Induced Apoptosis in Human Glioma Cells Motoshi Sawada,* ,1 Shigeru Nakashima,† Tohru Kiyono,‡ Jun Yamada,* Shigeru Hara,* Masanori Nakagawa,* Jun Shinoda,* and Noboru Sakai* *Department of Neurosurgery and †Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan; and ‡Laboratory of Viral Oncology, Research Institute, Aichi Cancer Center, 1-1 Kanokoden, Chikusaku, Nagoya 464-8681, Japan

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val 138 induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS. © 2002 Elsevier Science (USA) Key Words: acid sphingomyelinase; caspases; glioma; p53; reactive oxygen species.

Ceramide has emerged as an important component of signal transduction pathways involved in a variety of cellular processes [1] and is mainly produced by the hydrolysis of sphingomyelin (SM). 2 Fas (CD95/APO-1) is a potent inducer of apoptosis [2]. Engagement of Fas 1 To whom reprint requests should be addressed at Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan. Fax: ⫹81-58-265-9025. E-mail: nohgeka@cc. gifu-u.ac.jp. 2 Abbreviations used: SM, sphingomyelin; SMase, sphingomyelinase; A-SMase, acid sphingomyelinase; N-SMase, neutral and magnesiumdependent sphingomyelinase; ROS, reactive oxygen species; CHX, cycloheximide; H 2DCF-DA, 2⬘,7⬘-dichlorofluorescein diacetate; PDTC, pyrrolidinedithiocarbamate; L-NAME, L-nitroarginine methyl ester; SIN-1, 3-morpholinosydnomine; z-IETD-fmk, benzyloxycarbonylIle-Glu-Thr-Asp fluoromethyl ketone; Ac-IETD-AMC, acetyl-Ile-Glu-

results in the activation of multiple death signal transduction pathways [3–21]. Although the role of the FADD/caspase-8 system during Fas-induced apoptosis is well documented [3–9], data regarding the kinetics of the ceramide response and the sphingomyelinases (SMases) involved are conflicting. Some investigators have found a rapid and transient ceramide response, within several minutes, which has been attributed to acid SMase (A-SMase) [10 –12]. Other investigators, however, have revealed only a late, more sustained response, occurring over a period of hours after receptor stimulation [13, 14]. This ceramide generation was attributed to a neutral and magnesium-dependent SMase (N-SMase) activity [14]. Therefore, these results suggest the cell-type specific involvement of ceramide and SMase(s) during Fas-mediated cell death. However, the role of ceramide in Fas-induced apoptosis of human gliomas remains poorly understood. Recently, several components involving apoptotic cell death have been extensively studied, including caspases, reactive oxygen species (ROS), and p53 [6 – 8, 20 –26]. The previous reports suggest that increased production of ROS serves as a pivotal step in Fasmediated apoptosis in neutrophils and T lymphocytes [15–17]. A number of data, however, indicate that ROS may be important but not common and obligatory mediators of all forms of apoptosis [27–29]. p53 has been proposed as the “guardian of the genome.” A recent study has reported that p53 may regulate ceramide accumulation only in apoptotic pathways induced by Thr-Asp-7-amino-4-methylcoumarin; SR33557, ((2-isopropyl-1-(4[3-N-methyl-N-(3,4-dimethoxy-␤-phenethyl)amino]propyloxy)benzenesulfonyl))indolizine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GSH, reduced glutathione; HPTLC, highperformance thin-layer chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPV, human papillomavirus; NOS, nitric oxide synthase; NO, nitric oxide; NAC, N-acetylcysteine; MTp53ts, temperature-sensitive human p53 val138 mutant; AcDEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; z-DEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone; TNF, tumor necrosis factor.

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genotoxic stress [30]. Although p53 is shown to play a critical role in Fas-mediated apoptosis in lymphoid cells [18, 19], little is known about the relationship between p53 and ceramide in Fas-induced apoptosis in other types of cells. Initiator caspase-8 activation by FADD is an essential step in the Fas-mediated death signaling pathway [3–9]. However, the relationship between caspase activation and ceramide production in Fas-mediated apoptosis signaling is not well defined. In the current study, we have examined whether ceramide generation plays a role in Fas-induced apoptosis of human glioma cells and which type(s) of SMases is activated. Furthermore, the involvements of p53, ROS, and caspases, if any, in ceramide formation are investigated. The data obtained indicate that ceramide, which is produced through caspase-8-dependent activation of A-SMase, may be involved in Fasmediated apoptosis signaling of human glioma cells. MATERIALS AND METHODS Materials. Cycloheximide (CHX), 2⬘,7⬘-dichlorofluorescein diacetate (H 2DCF-DA), pyrrolidinedithiocarbamate PDTC), and L-nitroarginine methyl ester (L-NAME) were obtained from Sigma (St. Louis, MO). Anti-Fas monoclonal antibody (CH-11 clone) was from Medical & Biological Lab. (Nagoya, Japan). 3-Morpholinosydnomine (SIN-1) was from Tocris Cookson (Langford, UK). Anti-rat p21 monoclonal antibody was from Santa Cruz Biotech. (Santa Cruz, CA). Anti-mouse p53 (Ab-6) monoclonal antibody, anti-caspase-8 mouse monoclonal antibody (Ab-3), and selective caspase-8 inhibitor, benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone (zIETD-fmk), were from Calbiochem-Novabiochem (Cambridge, MA). The tetrapeptide substrates for caspase-8, acetyl-Ile-Glu-Thr-Asp-7-amino4-methylcoumarin (Ac-IETD-AMC), were from Peptide Institute (Osaka, Japan). ((2-Isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-␤phenethyl)amino]propyloxy)benzenesulfonyl))indolizine (SR33557) was kindly supplied from Dr. J.-P. Jaffrezou (Claudius Regaud Center, France). Other reagents were obtained from previously noted sources [31–33]. Cells and transfections. The human U-87 MG, U-373 MG, and T-98 G glioblastoma cell lines were obtained from American Type Culture Collection (Rockville, MD). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 ␮g/ml streptomycin (FBS/DMEM) in a humidified atmosphere containing 5% CO 2 at 37°C. The retrovirus vector pLXSN-16E6SD was constructed by inserting a 16E6SD BamHI fragment from pLRHL-16E6SD [34] into the BamHI site of pLXSN. pLXSN-16E6SD-8S9A10T was constructed by inserting an EcoRI-SspI fragment from pLXSN-16E6-8S9A10T [35] and an SspI-BamHI fragment between EcoRI and BamHI sites of pLXSN. Preparation of E6- and LXSN-retroviruses and infection protocols have been described [36] except that an amphotrophic packaging cell line FLYA13 [37] was used instead of PG13. U87LXSN, U87-Wild E6, and U87-Mut E6 cell lines were obtained by infecting U-87 MG cells with LXSN, LXSN-16E6SD, and LXSN16E6SD-8S9A10T retroviruses, respectively, followed by clonal selection with 800 ␮g/ml G418 for 2 weeks. The mutant E6 (16E6SD8S9A10T) is inactive for degrading p53 but active for other known E6 functions [38]. The p53ts val 138 encodes a mutant protein with a substitution from wild-type alanine to valine at position 138. The wild-type human p53 cDNA cloned into pCMV was kindly provided by Dr. T. Takahashi

(Aichi Cancer Center, Japan). The p53ts val 138 was constructed from the wild-type p53 by site-directed mutagenesis. U-373 MG and T-98 G cells were stably transfected with p53ts val 138 in pCMV using Trans-It. After selection with 800 ␮g/ml G418 for 2 weeks, resistant cells were cloned by limiting dilution. Fluorescent microscopy. Apoptotic cells stained with Hoechst 33258 were quantified by fluorescent microscopic analysis [32, 39]. Briefly, cells were fixed in 1% glutaraldehyde for 30 min. The cells were then stained with 10 mM Hoechst 33258 for 10 min. Nuclear morphology was observed under a fluorescent microscope (Olympus BX60, Tokyo, Japan). Measurement of intracellular GSH level. Intracellular reduced gluthatione (GSH) content was determined according to the previously described method [41]. In brief, the harvested cells were suspended in 150 ␮l of water and 5-sulfosalicyclic acid was added to a final concentration of 2%. The precipitated proteins were pelleted by centrifugation at 2000g for 10 min at 4°C. Aliquots of the soluble supernatant were mixed with 125 mM sodium phosphate buffer (pH 7.5) containing 6.3 mM EDTA, 0.21 mM NADPH, and 0.6 mM 5, 5-dithiobis(2-nitrobenzoic acid) in a total volume of 1 ml. On addition of glutathione reductase, the increase in absorption at 412 nm was monitored to determine the amount of GSH in the sample. A reference curve was generated with the known amounts of GSH standards. Detection of intracellular ROS. Intracellular production of ROS was measured by using the H 2DCF-DA [41]. H 2DCF-DA dissolved in ethanol was added to the washed cells at a final concentration of 5 mM in Hank’s balanced salt solution. After a 5-min incubation, cells were collected in a microcentrifuge. DCF fluorescence was measured using a spectrofluorometer (Hitachi F-3000, Japan) with excitation and emission wavelengths set at 488 and 520 nm, respectively. In some cases, ROS-induced intracellular DCF fluorescence was visualized with a Leica laser confocal scanning microscope (Heidelberg, Germany). SMase assay. Membrane fraction was prepared as described previously [32, 33]. The activities of both neutral and acid SMases were determined using a mixed micelle assay system [32, 42]. For measuring neutral SMase activity, the membrane fractions (20 ␮g protein) were mixed with [methyl- 14C]SM (40,000 cpm in 1 nmol of bovine brain SM in 0.25% Triton X-100 solubilized by sonication) in 0.1 M Tris/HCl buffer (pH 7.4) containing 6 mM MgCl 2 and the reaction mixture was incubated for 30 min at 37°C. A-SMase activity in membrane was measured as above except that the Tris/HCl buffer was replaced with 0.1 M sodium acetate buffer (pH 5.5) containing 5 mM EDTA. Measurement of ceramide level by Escherichia coli diacylglycerol kinase. Lipids extracted from cells were first treated in 0.1 M KOH in chloroform:methanol (1:2, v/v) at 37°C for 1 h [33]. Ceramide was converted to ceramide 1-[ 32P]phosphate by E. coli diacylglycerol kinase in the presence of [␥- 32P]ATP, and then lipids were separated on high-performance thin-layer chromatography (HPTLC) plates [33]. Following autoradiography, spots corresponding to ceramide 1-phosphate were scraped into vials and the radioactivity was counted in a scintillation counter (Beckman LS-6500). Quantitation of ceramide was based on a standard curve of known amounts of ceramide. The changes in ceramide content were normalized based on total protein. Measurement of [ 14C]ceramide and [ 14C]SM in [ 14C]serine-labeled cells. To label sphingolipids, C6 cells (5 ⫻ 10 5 cells/120 ml) were cultured for 72 h in the medium containing 25 ␮Ci of [ 14C]serine [33]. The extracted lipids were treated with 0.1 M KOH and then separated on HPTLC plates using the solvent system of chloroform: methanol:water (70:30:50, v/v) for SM or chloroform:methanol (95:5, v/v) for ceramide. The radioactivity of individual lipids was determined as described above. The changes in [ 14C]ceramide and [ 14C]SM contents were normalized based on total protein.

MECHANISMS OF ACID SPHINGOMYELINASE ACTIVATION

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Activity of caspase proteases. Cells were harvested after exposure to anti-Fas antibody plus CHX for indicated periods of time and washed three times with PBS, and then suspended in buffer containing 50 mM Tris/HCl (pH 7.4), 1 mM EDTA, and 10 mM EGTA. After addition of 10 ␮M digitonin, cells were incubated at 37°C for 10 min. Lysates were centrifuged at 900g for 3 min, and the resulting supernatant (40 ␮g protein) was incubated with 50 ␮M of a peptide substrate at 37°C for 1 h. Levels of released AMC were measured using spectrofluorometers (Hitachi F-3000 and F-2000, Japan) with excitation at 380 nm and emission at 460 nm [32]. Excitation and emission slit widths were adjusted to 10 and 20 mm, respectively. One unit was defined as the amount of enzyme required to release 0.22 nmol AMC/min at 37°C. Western blot analysis. Cells were solubilized with ice-cold lysis buffer containing 1% Triton X-100, 50 mM NaCl, 25 mM Hepes (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 ␮g/ml E-64. Extracted proteins (60 ␮g/well) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10, 13, or 15% polyacrylamide gels, and were electrophoretically transferred onto Immobilon-P membrane. Blocking was performed in Tris-buffered saline containing 5% skimmed-milk powder and 0.1% Tween 20. The membranes were probed with antibodies against p53, p21, CPP32 (procaspase-3), FLICE (caspase8), or actin proteins. Detection was performed with an ECL system. The protein content was determined with BCA protein assay using bovine serum albumin as a standard. Statistical analysis. Data are expressed as means ⫾ SD. Significance was assessed by two-way ANOVA, followed by Scheffe’s post hoc test. P values less than 0.01 were considered as significant.

RESULTS

Anti-Fas antibody induces apoptosis via a P53-independent manner in human glioma cells. We first examined whether p53 is implicated in the Fas-induced apoptosis in human glioma cells. Inactivation of p53 can be accomplished by the expression of the human papillomavirus (HPV) E6 protein, which binds p53 and accelerates its proteolytic degradation through the ubiquitin pathway [43]. Using the retroviral gene transfer technique, wild-type or mutant E6 gene and the empty vector (LXSN) were introduced into U-87 MG cells (designated as U87-Wild E6, U87-Mut E6, and U87-LXSN, respectively). When U87-LXSN cells were treated with the increasing concentrations of anti-Fas monoclonal antibody (CH-11) in combination with 10 ␮g/ml CHX for 12 h, the number of apoptotic cells increased in proportion to the concentrations of anti-Fas antibody (data not shown). In contrast, cells treated with anti-Fas antibody alone or CHX alone displayed no sign of cytotoxity even after incubation for 24 h (data not shown). Therefore, we decided to use 100 ng/ml CH-11 with 10 ␮g/ml CHX in the following experiments. In U87-LXSN and U87-Mut E6 cells, p53 expression was induced by etoposide (VP-16), a DNA topoisomerase II inhibitor. In contrast, in U87-Wild E6 cells, p53 expression by etoposide was completely abrogated by functional E6 protein (Fig. 1B). However, anti-Fas antibody failed to induce p53 expression in U87-LXSN (Fig. 1A). In addition to cell lines constructed with the retrovi-

FIG. 1. Fas-induced apoptosis of human glioma cells. (A) Immunoblot analysis of p53 protein in U87-LXSN, U87-Mut E6, and U87Wild E6 cells treated with 100 ng/ml anti-Fas antibody (CH-11) plus 10 ␮g/ml CHX or 40 ␮g/ml etoposide (VP-16) for 24 h. Data are representative of three separate experiments with compatible outcomes. (B) U87-LXSN (E), U87-Mut E6 (Œ), U87-Wild E6 (F), U-373 MG (䊐), and T-98 G (■) cells were treated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX for the indicated periods. For assessment of apoptotic cell death, the cells with fragmented and condensed nuclei were counted in over 1000 cells under a fluorescent microscope. Data are means ⫾ SD from three independent experiments, each performed in duplicate.

ral gene transfer technique, U-373 MG and T-98 G human gliomas which express mutant p53 were also used to examine their susceptibility to Fas-mediated apoptosis. When these human glioma cells expressing different p53 status, due either to mutation or to the expression of HPV E6 protein, were exposed to antiFas antibody with CHX, a nearly equivalent time-dependent pattern of increase in apoptotic cells was observed (Fig. 1A), although VP-16 induced apoptotic cell death in a p53-dependent manner (Fig. 1B). These findings suggest that wild-type p53 is not required for apoptosis triggered by engagement of Fas in human glioma cells. Fas-mediated ceramide generation is independent of p53. The changes of intracellular ceramide level during Fas-mediated apoptosis in human glioma cells

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tion was observed in human glioma cells. In addition, significant changes were not observed in N-SMase activity during the time course examined. Fas-induced activation of A-SMase, but not N-SMase, was also observed in other cell lines shown in Fig. 2 (data not shown). To further examine the role of ceramide during

FIG. 2. Fas-induced ceramide accumulation and sphingomyelin hydrolysis. U87-LXSN (E), U87-Mut E6 (Œ), U87-Wild E6 (F), and U-373 MG (䊐) cells were treated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX for the indicated periods. (A) Ceramide content was measured by the E. coli diacylglycerol kinase assay. (B) Changes of [ 14C]SM were measured in [ 14C]serine-labeled cells. The radioactivity of [ 14C]SM in untreated control cells (Time 0) was designed as 100%. Data are means ⫾ SD from three independent experiments, each performed in duplicate.

were measured by the enzymatic analysis with E. coli diacylglycerol kinase as well as by the metabolic labeling of cells with [ 14C]serine. In all the cell lines tested, U87-LXSN, U87-Mut E6, U87-Wild E6, and U-373 MG cells, a marked increase in ceramide content was observed as early as 2 h after exposure to anti-Fas antibody (Fig. 2A). The maximal level was observed at 6 h (4.5-fold increase over the control level). A similar profile of [ 14C]ceramide generation was observed in cells labeled with [ 14C]serine (data not shown). Concurrently, an initial decrease in [ 14C]SM was observed at 2 h after engagement of Fas and then its level declined to approximately 60% of the control level at 12 h (Fig. 2B). On careful examination, rapid ceramide responses within 30 min could not be detected in human glioma cells (Fig. 2), although a biphasic increase in ceramide levels at the early phase after Fas activation has been reported in Jurkat T cells [44]. SR33557 inhibits Fas-mediated A-SMase activation and cell death. In U87-LXSN and U-373 MG cells, anti-Fas antibody gave rise to a time-dependent increase in A-SMase activity and its activity surpassed by approximately 4-fold that of control culture at 6 h after treatment (Fig. 3A). Although we carefully examined A-SMase activity during the time course over the first few minutes for 6 h after Fas activation, neither a rapid increase at the early phase nor a biphasic eleva-

FIG. 3. Effects of SR33557 on Fas-induced activation of acid sphingomyelinase and apoptosis in U-87 MG cells. (A) U87-LXSN (■), U87-Mut E6 (u), U87-Wild E6 (3), and U-373 MG (䊐) cells were treated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX for the indicated periods. The activity of A-SMase was determined using a mixed micelle assay system with [methyl-14C]SM at pH 5.5. Data are means ⫾ SD from three independent experiments, each performed in duplicate. (B) U-87 MG cells were incubated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX in the presence or absence of 10 ␮M SR33557 for 6 h. The activities of A-SMase and N-SMase were determined using a mixed micelle assay system with [methyl- 14C]SM at pH 5.5 and 7.5, respectively. (C) U-87 MG cells were treated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX in the presence or absence of the indicated concentrations of SR33557 for 6 h. The cells with fragmented and condensed nuclei were counted in over 1000 cells under a fluorescent microscope. Data are means ⫾ SD from three independent experiments, each performed in duplicate. *P ⬍ 0.05 and **P ⬍ 0.01 versus anti-Fas antibody alone: two-way ANOVA followed by Scheffe’s post hoc test.

MECHANISMS OF ACID SPHINGOMYELINASE ACTIVATION

Fas-mediated apoptosis in human glioma cells, a potent inhibitor of A-SMase, SR33557 [45, 46] was employed. U-87 MG cells were pretreated for 30 min with or without SR33557 and then incubated with anti-Fas antibody plus CHX. SR33557 (10 ␮M) prevented Fasinduced activation of A-SMase below the control level, but it had no effect on N-SMase activity (Fig. 3B). SR33557 inhibited CD95-induced apoptosis in a concentration-dependent manner (Fig. 3C). Furthermore, 10 ␮M SR33557 blocked the characteristic pattern (laddering) of DNA fragmentation (Fig. 4A) and abrogated typical apoptotic features such as condensation and fragmentation of nuclei (Fig. 4B) upon Fas stimulation. Fas-mediated apoptosis and ceramide generation through A-SMase activation proceed without ROS formation. The requirement of ROS for Fas signaling pathways has not yet been clearly defined. Therefore, changes of intracellular GSH and ROS levels were first measured during Fas-induced apoptosis in U87-LXSN cells expressing functional p53 and cells lacking functional p53 such as U87-Wild E6 and U-373 MG. In all cell lines tested, significant changes in GSH levels were not observed during the time course examined (Fig. 5A). Accordingly, a sign of ROS formation assessed by a ROS-sensitive fluorophore DCF-DA was not obtained. In contrast, exposure of cells to VP-16 resulted in a decrease of GSH contents with a reciprocal increase in DCF fluorescence (Fig. 5A). In addition, an inhibitor of nitric oxide synthase (NOS), L-NAME, or a nitric oxide (NO) donator, SIN-1, failed to modulate Fas-induced apoptosis of U-87 MG cells. Whereas, L-NAME and SIN-1 had effects on ROS-mediated apoptosis induced by VP-16 as a suppressor and an enhancer, respectively (Fig. 5B). Moreover, two chemically distinct antioxidants, 25 mM N-acetylcysteine (NAC) and 200 ␮M PDTC had no effects on A-SMase activation and ceramide accumulation upon Fas ligation (Table 1), although these antioxidants significantly inhibited ceramide accumulation by N-SMase activation through ROS formation in response to 40 ␮M VP-16 for 12 h, as previously described [32]. A-SMase activation is not involved in ROS-mediated apoptosis by induction of functional P53 using Mtp53ts. To further confirm the possibility that ceramide generation through A-SMase activation requires neither p53 nor ROS, U-373 MG and T-98 G cells expressing mutant p53 were transfected with an expression plasmid encoding the temperature-sensitive p53 val 138 (MTp53ts) that assumes mutant conformation at 37.5°C and wild-type conformation at 32.5°C [47]. Consistent with the previous report [48], U-373 MG cells expressing MTp53ts caused DNA fragmentation and classical apoptotic morphological features

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FIG. 4. Effects of SR33557 on DNA laddering and nuclear morphological changes induced by anti-Fas antibody in U-87 MG cells. (A) Agarose gel electrophoresis of oligonucleosomal DNA fragments (DNA laddering). U-87 MG cells were incubated in the presence or absence of anti-Fas antibody (100 ng/ml), CHX (10 ␮g/ml), SR33557 (10 ␮M), or the indicated combinations for 12 h. DNA extracted from the cells was subjected to conventional agarose gel electrophoresis. MK: Molecular weight marker. (B) U-87 MG cells were incubated in the presence or absence of 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX with and without 10 ␮M SR33557 for 12 h. Cells were stained with Hoechst 33258 and photographed under a fluorescent microscope. Photographs are representatives from at least 10 different cultures.

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FIG. 5. Fas-induced apoptosis occurs independent of reactive oxygen species in human glioma cells. (A) Time-dependent changes in intracellular GSH level and ROS formation. U87-LXSN (‚, Œ), U87-Wild E6 (■, 䊐), and U-373 MG ({, }) cells were exposed to 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX for the indicated periods. U87-LXSN cells (E, F) were also exposed to 40 mg/ml VP-16 for the indicated periods. The content of GSH (E, ‚, 䊐, {) was calculated as mg GSH per mg protein. DCF fluorescence (F, Œ, ■, }) of untreated control cells (Time 0) and that of U-87 MG cells treated with 1 mM H 2O 2 for 12 h were designed as 0 and 100%, respectively. (B) U-87 MG cells were treated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX (F, 䊐, ‚) or 40 ␮g/ml VP-16 (E, ■, Œ) in the presence or absence of 1 mM L-NAME or 100 ␮M SIN-1 for the indicated periods. The cells with fragmented and condensed nuclei were counted in over 1000 cells under a fluorescent microscope. Data are means ⫹ SD from three independent experiments, each performed in duplicate.

at 48 h after incubation at 32.5°C (data not shown). As shown in Fig. 6A, the function of the induced p53 protein was confirmed by the expression of p21 WAF1/ CIP1 , a product of p53-responsive genes [49]. During p53-induced apoptosis in U-373 MG cells, an increase in DCF fluorescence and ceramide accumula-

FIG. 6. Temperature-sensitive p53-induced ROS formation, sphingomyelinase activation, ceramide accumulation, and apoptotic cell death in U-373 MG cells. (A) Immunoblot analyses of p53 and p21 expression up to 48 h after incubation at 32.5°C in U-373 MG cells transduced with MTp53ts. Actin was used as loading control. Relative DCF fluorescence (DCF), ceramide content (Cer), and the rate of apoptosis (Apo) up to 48 h postinduction. DCF fluorescence of unincubated U-373 MG cells (control) was designated as 0% and that of cells incubated at 32.5°C for 48 h was designated as 100%. Data are means from two independent experiments, each performed in duplicate. (B) Time-dependent changes of N-SMase and A-SMase activities in U-373 MG cells transduced with MTp53ts after induction at 32.5°C. Data are means ⫾ SD from three independent experiments, each performed in duplicate.

tion were observed in proportion to the levels of p53 expression in MTp53 transfectants (Fig. 6A). In these cells, activity of N-SMase time dependently increased (Fig. 6), resulting in the accumulation of ceramide (data not shown). However, significant changes were not observed in A-SMase activity during the induction process (Fig. 6B). Strong DCF fluorescence at 48 h postinduction in MTp53ts trans-

TABLE 1 Effects of Antioxidants on SMase Activation and Ceramide Formation Induced by Anti-Fas Ab or VP-16 Treatments

A-SMase (% of control)

N-SMase (% of control)

Ceramide (% of control)

Anti-Fas Ab Anti-Fas Ab ⫹ 25 mM NAC Anti-Fas Ab ⫹ 200 ␮M PDTC VP-16 VP-16 ⫹ 25 mM NAC VP-16 ⫹ 200 ␮M PDTC

401 406 395 102 104 98

98 80 96 308 125 108

428 438 430 348 132 118

Note. U-87 MG cells were treated with 40 ␮g/ml etoposide for 12 h or 100 ng/ml anti-Fas Ab plus 10 ␮g/ml CHX for 6 h in the presence or absence of antioxidants.

MECHANISMS OF ACID SPHINGOMYELINASE ACTIVATION

fectants was effectively abrogated by pretreatment with 25 mM NAC, whereas no significant changes in DCF fluorescence was observed by pretreatment with 10 ␮M SR33557 (Fig. 7A). In addition, 25 mM NAC significantly inhibited p53-induced apoptosis by MTp53ts transfection, but 10 ␮M SR33557 had no effects on apoptosis (Fig. 7B) and ceramide formation (data not shown). Similar findings were also observed in T-98 G cells transfected with MTp53ts (data not shown). These results suggest that the activation of A-SMase is not involved in ROS-mediated apoptosis induced by functional p53. Caspase-8, but not caspase-3, regulates A-SMase activation during Fas-induced apoptosis. The Fas/FasL system recruits caspase-8/FLICE/Mch5 [6] to the Fas death-inducing signaling complex, where it is activated [7]. Subsequently, the cytosolic caspase-3/ CPP32/Yama becomes activated [8]. Since several studies suggest a crucial function of caspases on A-SMase activation during Fas-mediated apoptosis [11–13, 50], we tested the possibility that caspases are involved in the regulation of A-SMase activation. Exposure of U-87 MG cells to anti-Fas antibody resulted in time-dependent processing of caspases-8 and -3, as assessed by immunoblot analyses (Fig. 8A). Activation of caspases-8 and -3(-like) proteases was further confirmed by the hydrolysis of their selective substrates, Ac-IETD-AMC (Fig. 8B) and acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) (Fig. 8C), respectively. The processing of caspase-8 preceded that of caspase-3. The former was detectable within 2 h but the latter became evident at 4 h. Pretreatment of cells with SR33557 had no effects on the proteolytic processing of procaspase-8 and IETD-AMC cleavage (Figs. 8A and 8B), but largely prevented processing of caspase-3 (Figs. 8A and 8C), suggesting that ASMase activation occurs between steps of sequential activation of caspases-8 and -3. z-IETD-fmk, a selective inhibitor of caspase-8, inhibited A-SMase activity as well as caspase-3 activity in a concentration-dependent manner (Figs. 8B and 9) and the inhibitory effect of A-SMase activity by z-IETD-fmk well correlated with that of apoptotic cell death (Fig. 9). Benzyloxycarbonyl-Asp-Glu-ValAsp fluoromethyl ketone (z-DEVD-fmk), a selective inhibitor of caspase-3, also prevented cells from Fasinduced apoptosis. Therefore, caspase activation is an essential step in Fas-mediated apoptosis of human glioma cells, as previously reported with various nonglial cells [20, 21, 51]. However, 200 ␮M z-DEVD-fmk hardly affected Fas-induced proteolytic processing of caspase-8 (Fig. 8), and A-SMase activation (Fig. 9).

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DISCUSSION

Accumulating evidence suggests that ceramide plays a role in apoptosis signaling. There are at least two classes of SMases (N- and A-SMases), which are responsible for hydrolysis of SM to ceramide. Although the role of N-SMase during apoptosis induced by genotoxic stress is well documented [1, 32, 52], the role of A-SMase in Fas- and tumor necrosis factor (TNF)-mediated apoptosis is controversial. A-SMase plays an important role in lipid metabolism, as evidenced from the disease phenotype of A-SMase-deficient NiemannPick disease (NPD) type A and B patients and A-SMase⫺/⫺ mice [53, 54]. Several reports question the role of A-SMase in the Fas-mediated signaling pathway. For example, Epstein-Barr virus-transformed B cells [12] and Jurkat T cells [14] exhibited a late ceramide response attributed to N-SMase activated by Fas. Whereas, a number of recent studies have shown that the early ceramide elevations after Fas activation are abrogated in A-SMase knockout systems [55–57]. In addition, Fas-induced activation of caspases and apoptosis were not equivalent in the wildtype and A-SMase knockout cells [12], suggesting that A-SMase contributes directly to the Fas-induced apoptotic signaling pathway in lymphoid cells. Interestingly, a recent study indicated a role for A-SMase in some but not all forms of Fas-mediated cell death [58]. The present study indicates that the cross-linking of Fas caused a relatively late and sustained elevation of ceramide with activation of A-SMase, but not N-SMase in human glioma cells. The data obtained from using SR33557, a selective inhibitor of A-SMase, point to an involvement of the A-SMase in Fas-triggered cell death of human glioma cells. In contrast, Wagenknecht et al. [59] reported that ceramide generation was not involved in Fas-induced apoptosis of LN-18 and LN-229 human glioma cells. However, they measured ceramide formation only in [ 14C]serine-labeled cells at 6 h and showed no kinetics of ceramide formation or apoptosis. Thus, their results do not necessarily exclude a crucial role for ceramide in Fas-induced apoptosis of glioma cells. Our present data using z-IETD-fmk show that caspase-8 is an upstream regulator of the A-SMase, linking the Fas receptor to ceramide formation, as observed in other types of cells [51, 60]. FADD may be involved in this process, since FADD-dependent activation of A-SMase has been demonstrated [3, 61]. However, z-DEVD-fmk had no effect on Fas-induced ASMase activation. In contrast, SR33557 effectively suppressed the activation of caspase-3. These results indicate that Fas-induced ceramide formation occurs upstream, but is not the consequence of caspase-3 activation. We have previously reported that exogenous C 2-ceramide induced the proteolytic cleavage of the

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FIG. 8. Fas-induced processing of caspases-8 and -3 in U-87 MG cells. (A) U-87 MG cells were exposed to 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX for the indicated periods in the absence or presence of SR33557 (10 ␮M), z-IETD-fmk (200 ␮M), or z-DEVD-fmk (200 ␮M). Cellular proteins were subjected to SDS-PAGE and immunoblotted with antibodies against caspases-8 and -3. Data shown are representative of three separate experiments with compatible outcomes. (B, C) U-87 MG cells were harvested after exposure to 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX in the presence or absence of the indicated concentrations of z-IETD-fmk, 200 ␮M zDEVD-fmk, or 10 ␮M SR33557 for the indicated periods. The activities of caspase-8(-like) (B) and caspase-3(-like) (C) were measured spectrofluorometrically using 50 ␮M Ac-IETD-MCA and 50 ␮M AcDEVD-MCA, respectively. Data are means ⫾ SD from three independent experiments, each performed in duplicate.

FIG. 7. Different effects of SR33557 and PDTC on ROS formation and apoptosis induced by temperature-sensitive p53. Cells transfected with empty vector or with MTp53 were incubated in the absence or presence of 10 ␮M SR33557 or 200 ␮M PDTC at 32.5°C for 48 h. (A) DCF fluorescence of the cells was photographed under a confocal scanning microscope. Photographs are representatives from at least 10 different cultures. (B) The cells with fragmented and condensed nuclei were counted for over 1000 cells under a fluorescent microscope. Data are means ⫾ SD from three independent experiments, each performed in duplicate.

executor caspase-3 through the release of mitochondrial cytochrome c and caspase-9 activation in C6 glioma cells [32]. Taken together, these results suggest that endogenous ceramide induced by Fas and exogenous short-chain ceramide give rise to activation of caspase-3, a key enzyme at the commitment step to apoptosis in glioma cells. Although p53-dependent induction of Fas was documented [62– 64], the role of p53 in apoptosis signaling downstream of Fas ligation is not clearly understood. In the present study, Fas-induced ceramide formation and apoptosis proceeded regardless of their p53 status

MECHANISMS OF ACID SPHINGOMYELINASE ACTIVATION

FIG. 9. Effects of caspase inhibitors on Fas-induced acid sphingomyelinase activation in U-87 MG cells. U-87 MG cells were treated with 100 ng/ml anti-Fas antibody plus 10 ␮g/ml CHX in the presence or absence of the indicated concentrations of z-IETD-fmk or 200 ␮M z-DEVD-fmk for 12 h. The activity of A-SMase was determined using a mixed micelle assay system with [methyl- 14C]SM at pH 5.5. For assessment of the rate of apoptosis (Apo), the cells with fragmented and condensed nuclei were counted in over 1000 cells under a fluorescent microscope. Data are means ⫾ SD from three independent experiments, each performed in duplicate.

in human glioma cell lines examined. We further showed that p53-induced apoptosis by MTp53ts gave rise to ceramide accumulation through the activation of N-SMase but not A-SMase. These results indicate that p53 may not be involved in Fas-mediated apoptosis signaling including A-SMase activation. Similarly, the role of p53-independent A-SMase activation was also demonstrated in apoptosis of lung endothelial cells induced by ionizing irradiation [65]. ROS plays a pivotal role in the regulation of cell death [22, 25]. The possible involvement of ROS in Fas-mediated apoptosis is documented in neutrophils and T lymphocytes [15, 17]. In murine fibrosarcoma cells, human histiocytic cells, and human fibroblasts, however, Fas-mediated apoptosis is not affected by antioxidants and can proceed under nearly anaerobic conditions where no ROS are generated [27–29]. In the current study, no significant changes in both intracellular GSH content and ROS levels were observed during Fas-induced apoptosis of human glioma cells. Neither NOS inhibitor nor NO donator affected Fasinduced apoptosis. Moreover, the antioxidants, which inhibited N-SMase activation by genotoxic stress [32, 66], had no effect on Fas-induced ceramide generation and A-SMase activation. These results suggest that ROS formation may not be required for Fas-induced apoptosis and A-SMase activation in human glioma cells. Conversely, during ROS-mediated apoptosis induced by Mtp53ts, the activity of A-SMase was not changed. These findings are consistent with those in a previous report that GSH depletion functions as an important factor in the activation of N-SMase, but does not affect the activation of A-SMase [66]. Finally, in the present study 10 ␮M SR33557 com-

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pletely inhibited Fas-induced ceramide formation and A-SMase activation. However, this A-SMase inhibitor even at 50 ␮M blocked but failed to abolish apoptotic cell death. In addition, SR33557 incompletely blocked caspases-3 processing. These results suggest the presence of alternative apoptotic signaling pathways independent of ceramide. For example, active caspase-8 directly engages the caspase cascade by activating caspase-3 [67] or indirectly leads, through cleavage of Bid, to the release of cytochrome c and activation of caspases-9 and -3 [68]. In summary, we would like to propose a hypothetical signaling sequence during Fas-induced glial apoptosis (Fig. 10). Triggering of Fas (CD95/APO-1) results in recruitment of a adaptor molecule, FADD. The FADD in turn binds to caspase-8 leading to oligomerization and activation of caspase-8. A-SMase is mainly activated through activated caspase-8. Ceramide released by the A-SMase activation may stimulate the executor caspases, in particular caspase-3. Alternatively, caspase-8 may directly or indirectly, by cleaving cytosolic factors such as Bid, activate the effector caspase-3. The mechanisms regulating A-SMase are important issues, but still undefined at present. Its precise delineation and evaluation of its role in apoptosis induction and ceramide generation by Fas will require further extensive investigation using pharmacological or genetical approaches.

FIG. 10. A hypothetical scheme for regulation of Fas-induced acid sphingomyelinase activation by caspases in human glioma cells. Activation of Fas (CD95/APO-1) results in recruitment of a adapter molecule, FADD. The FADD in turn binds to caspase-8, leading to oligomerization and activation of caspase-8. Caspase-8 may directly or indirectly stimulate the acid sphingomyelinase (A-SMase). Ceramide released by A-SMase activation may stimulate the effector caspase-3. Alternatively, caspase-8 may directly activate the effector caspase-3 or can act indirectly by cleaving cytosolic factors such as Bid, leading to subsequent activation of the effector caspase-3.

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We are grateful to Dr. T. Takahashi (Aichi Cancer Center, Japan) for wild-type human p53 plasmid, to Dr. Y. Takeuchi (Chester Beatty Laboratories, ICR, UK) for FLYA13 cells, and to Dr. J.-P. Jaffrezou (Claudius Regaud Center, France) for SR33557. This work was supported in part by Grants-in-Aid for Scientific Research (B) (11557104) and (C) (11671364, 12671347) from the Ministry of Education, Science, Sports, and Culture of Japan, Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan, the Naito Foundation, and the ONO Medical Research Foundation.

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