Published online before print April 1, 2004
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* General Pathology and Immunology Section,
Pharmacology Section, and
Hematology Section, Department of Clinical and Experimental Medicine, and
Otolaryngology Section, Department of Medical and Surgical Specialities, University of Perugia, Italy
1Correspondence: Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, University of Perugia, Via Brunamonti, General Hospital-Monteluce 06100 Perugia, Italy. E-mail: marimmun{at}unipg.it
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Key Words: thiols • survival • caspases
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Thiols have been proposed as important molecules for apoptosis control in many cell types, but no information is available about their potential role in regulating B cell apoptosis. This topic deserves investigation, as in addition to the well-known survival stimuli, signals also triggered by thiol molecules could, at least in part, contribute to B cell survival. Recent findings have demonstrated that thiols can directly modulate the activity of several proteins with a regulatory role for apoptosis, including signal-transduction molecules [10 11 12 13 ], transcription factors [14 15 16 ], and cysteine proteases [17 18 19 ]. This can occur by thiol-disulfide exchange reactions at reactive cysteine-residue levels in the protein catalytic sites and is one of the most important mechanisms in the regulation of protein function [13 , 16 , 20 ]. Therefore, thiols, in addition to scavenging the reactive oxygen species (ROS), could act as signaling molecules to directly activate molecular events essential for cell survival.
Recently, it has been demonstrated that the simple exogenous thiol, N-acetyl-L-cysteine (NAC), promotes survival of different cell types by activating the retrovirus-associated sequence/extracellular-regulated kinase signaling pathway [10
, 11
] and inducing specific gene expression [21
]. Consistent with this last observation, thiols can also up-regulate the DNA-binding activity of some transcription factors including activated protein-1 [22
], nuclear factor (NF)-
B [23
], and NF of activated T cells [24
], which are known to control the gene expression of some antiapoptotic proteins belonging to the Bcl-2 family [25
26
27
] and inhibitors of apoptosis family [28
, 29
].
Based on these observations about the ability of thiols to interact with intracellular signaling pathways, we investigated the effect of NAC on in vitro spontaneous apoptosis of tonsillar B lymphocytes (TBL) and focused on some molecular events, which occur during the apoptotic process. Results provide the first evidence that NAC inhibits TBL apoptosis and maintains their survival in vitro by inhibiting caspase-3 and -7 processing. NAC also induces changes in several regulatory components of the apoptotic process.
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Cell isolation and culture
Mononuclear cells from surgically removed tonsils were isolated by gentle mincing followed by centrifugation on Ficoll-Hypaque density gradients (Amersham Pharmacia, Uppsala, Sweden). Cell suspension was T cell-depleted by two cycles of rosetting with neuraminidase-treated sheep erythrocytes and monocyte-depleted by plastic adherence. This B cell-enriched population was further fractionated on a discontinuous gradient of 60%, 50%, and 30% Percoll (Amersham Pharmacia). Cells recovered between the 60% and 50% layers, considered high-density cells, were used in this study. These B cells were shown to be >98% pure on flow cytometry by CD19 staining with <1% CD14+ and <2% CD3+. Cells were cultured at 37°C in a humid 5% CO2 atmosphere at a concentration of 2 x 106 cells/ml in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. NAC or the caspase inhibitors were added at the beginning of culture, and CHX was added to TBL 1 h before NAC. In some experiments, NAC was added after different times from the start of culture or added at time 0 and then removed at different times when cells were washed and reincubated in fresh medium for further times.
Assessment of viability and apoptosis
The number of viable cells was counted by the trypan blue dye exclusion assay. Apoptosis was assessed by flow cytometric quantitation of nuclei with hypodiploid DNA content and by DNA fragmentation assay. Hypodiploid DNA content was evaluated as described by Nicoletti et al. [30
]. Briefly, 3 x 105 cells were harvested, permeabilized in PI staining solution (PI, 50 µg/ml; sodium citrate, 0.1%; Triton X-100, 0.1%), and stored at 4°C in the dark overnight. The PI fluorescence of individual nuclei was measured by flow cytometry with standard FACScan equipment (Becton Dickinson, San Jose, CA). Preparation of DNA for agarose gel electrophoresis was performed as follows: TBL (5x106) were dissolved in hypotonic lysing buffer [100 mM NaCl, 10 mM Tris, 1 mM EDTA, 1% sodium dodecyl sulfate (SDS), 200 mg/ml proteinase K, pH 7.5]. The lysates were deproteinized by extraction, twice with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1), then precipitated overnight at –20°C in ethanol in the presence of 0.3 M sodium acetate, and recovered by centrifugation. Pellets were air-dried and resuspended in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (Tris-EDTA buffer), at 4°C. DNA was then analyzed in a 2% agarose gel stained with ethidium bromide.
Western blot analysis
Cells were washed with cold phosphate-buffered saline and resuspended in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM phenylmethanesulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 50 mM sodium fluoride, and 1 mM sodium orthovanadate. After 30 min incubation at 4°C, the lysates were centrifuged at 12,000 g for 20 min at 4°C, and the protein content in supernatants was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). For each experiment, a constant amount of protein was separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Keen, NH). Equal protein loading was controlled by Ponceau red staining of membranes. After blocking, the membranes were incubated with the following anti-human primary antibodies (Ab): caspase-8 (mouse monoclonal, clone 1C12, New England Biolabs, Cambridge, MA), caspase-9, -3, and -7, poly(adenosine 5'-diphosphate-ribose)polymerase (PARP), BID (all rabbit polyclonal, New England Biolabs), DNA-fragmentation factor (DFF)45 (rabbit polyclonal, c-terminal, PharMingen, San Diego, CA), Bcl-2 (mouse monoclonal, clone 124, Dako, Hamburg, Germany), Bcl-xS/L (rabbit polyclonal, clone S-18, Santa Cruz Biotechnology, Santa Cruz, CA), and ß-tubulin (mouse monoclonal, clone Tub 2.1, Sigma Chemical Co.). Bands were visualized using appropriate horseradish peroxidase-conjugated secondary Ab: goat anti-rabbit (New England Biolabs), sheep anti-mouse (Amersham Pharmacia), and the enhanced chemiluminescence system (Amersham Pharmacia). For some analyses, the blots were reprobed after exposure to stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.8) for 30 min at 55°C. Lysates, from Jurkat T cells pretreated for 1 h with Z-IETD-fmk and then stimulated for 20 h with CD95 CH11-triggering Ab (Upstate Biotechnology, Lake Placid, NY), were used as positive control for the Z-IETD-induced inhibition of BID cleavage and mitochondrial cytochrome c release.
Densitometric analysis
Blots were scanned at a resolution of 400 pixels per inch using an Agfa Arcus II scanner with Agfa FotoLook software (v3.0) and imported into Adobe Photoshop (v5.5). The scanned images were saved as grayscale PICT files and imported into Fuji Photo Film Co. image gauge software (v3.0). An area of 84 square pixels was then placed around each band, and the software determined the pixel density. Values were plotted as pixel density per unit area and are displayed as arbitrary units.
Subcellular fractionation for detection of mitochondrial cytochrome c release
Cytosolic and mitochondrial fractions were prepared by resuspending cell pellets in 300 µl hypotonic extraction buffer [250 mM sucrose, 20 mM HEPES, pH 7.2, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, and a protease inhibitor cocktail (Sigma Chemical Co.)] added at 1:100 dilution. After 30 min incubation on ice, cells were homogenized by 50–60 passages through a 25-gauge needle. Unbroken cells and nuclei were pelleted by centrifugation at 760 g for 10 min at 4°C. Supernatants were then centrifuged at 13,000 g for 30 min at 4°C to obtain the cytosolic fraction (supernatant) and the fraction enriched in mitochondria (pellet). Western blot analysis to detect mitochondrial cytochrome c release into the cytosol was performed, as described above, on 40 µg extracts using mouse monoclonal anti-human cytochrome c (clone 7H8.2C12, PharMingen). To control the purity of the cytosolic fraction and the specificity of cytochrome c release, anticytochrome c oxidase subunit IV (COX IV; Molecular Probes, Eugene, OR) was used.
Statistical analysis
Experiments using the trypan blue exclusion method and flow cytometric DNA analysis in the presence of NAC or inhibitors were repeated in eight different TBL isolates, except those (12) to evaluate the effect of Z-IETD-fmk on spontaneous TBL apoptosis. Results are shown as the means ± SD of eight or 12 experiments. Statistical differences were evaluated using paired Student’s t-test. Experiments of agarose gel electrophoresis and Western blot were performed in five different TBL isolates. Data reported are those of a typical experiment.
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40% remained viable after 8 days (Fig. 1A) . TBL cell death occurred by apoptosis, as confirmed by the hypodiploid DNA content of PI-stained TBL (Fig. 1B)
and the characteristic internucleosomal DNA fragmentation (Fig. 1C) . In TBL cultured with NAC, at 24 and 48 h, there was a strong reduction of the hypodiploid DNA content with respect to that of controls (Fig. 1B)
. DNA fragmentation was also inhibited in TBL cultured in the presence of NAC with respect to controls (Fig. 1C)
.
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Figure 1. NAC delays cell death and inhibits nuclear hypodiploidy and DNA fragmentation in TBL. Freshly isolated TBL were cultured with or without the indicated concentrations of NAC. At different time-points, TBL were harvested, and the percentage of viable cells (A), hypodiploid nuclei (B), and DNA fragmentation (C) was determined as described in Materials and Methods. (A) The results are shown as means ± SD of eight experiments. *, P < 0.01, and , P < 0.05 (NAC-treated TBL vs. controls at each time-point), according to Student’s t-test. The effect of NAC, 1 mM, is not significant. (B and C) Representative data from one of five experiments with similar results.
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A series of nonthiol antioxidants including catalase, ascorbic acid [31 ], and the cell-permeable Mn(III)tetrakis(4-benzoic acid)porphyrin, which has superoxide dismutase (SOD) and catalase mimetic activity [32 ], failed to inhibit TBL apoptosis (data not shown).
Kinetics of NAC-induced inhibition of TBL apoptosis
Kinetic studies were performed to evaluate the effect of delayed NAC addition or NAC removal on TBL apoptosis. In the first experiments, NAC was added to TBL after 6, 12, 24, and 48 h from the start of culture. After a total of 24, 48, and 72 h from the start of culture, apoptosis was evaluated and compared with that of TBL cultured with NAC added at time 0 and controls. As shown in Figure 2A
, a 6-h delay in adding NAC to TBL significantly (P<0.01) reduced, with respect to addition at time 0, its antiapoptotic activity at each time examined. A progressive reduction (P<0.01) in NAC antiapoptotic activity was observed at each time examined if the addition was delayed 12 or 24 h (Fig. 2A)
. However, even when added at these time-points, NAC was still able to inhibit TBL apoptosis (P<0.01 when added at 12 h and P<0.05 when added at 24 h; Fig. 2A
). Delayed addition of NAC for 48 h completely abrogated the ability of NAC to inhibit TBL apoptosis (Fig. 2A)
.
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Figure 2. Effect of delayed NAC addition and NAC removal on TBL apoptosis. (A) Freshly isolated TBL were cultured for 24, 48, and 72 h in medium or with NAC, 20 mM, added at time 0. In some cultures, NAC was added after different times from the start of culture. (B–E) Freshly isolated TBL were cultured in medium or with NAC, 20 mM, for 6 (B), 12 (C), 24 (D), and 48 (E) h. At these times, in some cultures after NAC was removed, cells were washed and reincubated with fresh medium for a further 6, 12, and 24 h. (A–E) At the indicated times, apoptosis was evaluated, measuring the percentage of hypodiploid nuclei by flow cytometry. The results are shown as means ± SD of eight experiments. Statistics were performed by Student’s t-test. °, P < 0.01 (delayed NAC addition vs. NAC added at time 0); *, P < 0.01, and , P < 0.05 (NAC-treated TBL vs. controls).
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To evaluate whether the antiapoptotic activity of NAC requires new protein synthesis, experiments with the protein synthesis inhibitor CHX were performed. We initially determined that 10 µg/ml was the concentration of CHX required to inhibit by >95% the incorporation of [35S]-methionine into trichloracetic acid–precipitable proteins in TBL (data not shown). CHX was added to TBL 1 h before NAC, and the cultures were evaluated for apoptosis 24 and 48 h later. CHX did not affect the antiapoptotic activity of NAC (data not shown). Also, when tested in the absence of NAC, CHX did not alter TBL apoptosis (data not shown).
NAC inhibits caspase-3 and -7 but not -8 and -9 proteolytic processing
To assess the involvement of caspases in TBL apoptosis, we tested the effect of the broad-spectrum caspase inhibitor, Z-VAD-fmk and the more selective (caspase-2, -3, and -7) inhibitor Z-DEVD-fmk. Cytofluorimetric analysis showed that Z-VAD-fmk and Z-DEVD-fmk (P<0.01) significantly inhibited TBL apoptosis at 24 and 48 h, with respect to controls (Fig. 3A
), suggesting that caspases and in particular, caspase-3 and/or a closely related caspase are involved in TBL apoptosis.
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Figure 3. NAC inhibits caspase-3 and -7 but not -8 and -9 proteolytic processing. (A) The involvement of caspases in TBL apoptosis was tested in cells cultured with Z-VAD-fmk (50 µM), Z-DEVD-fmk (50 µM), or 0.05% DMSO as control. At the indicated time-points, apoptosis was determined measuring the percentage of hypodiploid nuclei by flow cytometry. The results are shown as means ± SD of eight experiments. *, P < 0.01 (TBL treated with Z-VAD or Z-DEVD vs. controls at each time-point), according to Student’s t-test. (B–E) The processing of the indicated caspases was analyzed by Western blot in 25 µg total protein obtained from TBL cultured for the indicated times with NAC, 20 mM, at pH 7.4 (B, C, E), with the indicated caspase inhibitors, each at 50 µM (D), with NAC, 20 mM, at pH 8.3 (E), NAC, 20 mM, diluted from a stock solution stored at 1 M, pH 6 (E), and with medium at pH 7 (E). Arrows indicate the procaspases and cleaved products (sizes expressed in kDa). Separate panels are shown when long X-ray film exposure was necessary to detect the cleaved products. One representative experiment of five with similar results is shown. (B and C) Densitometry was performed, and the density of each active band, expressed as arbitrary units, is given as the mean ± SD of the five blots. Values were normalized using ß-tubulin as an internal standard. Lane designations are identical for blots and histograms. *, P < 0.01 (NAC-treated TBL vs. controls at each time-point), according Student’s t-test.
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Processing of procaspase-3 into its p17 active subunit was detected in TBL at 12 h after in vitro culture and increased progressively at 24 and 48 h (Fig. 3C) . Caspase-7 cleavage into its p20 subunit also occurred in TBL with kinetics similar to that of caspase-3 but to a greater extent (Fig. 3C) . To examine whether caspase-8 and -9 were involved in caspase-3 and -7 processing, the effect of caspase-8 inhibitor Z-IETD-fmk and caspase-9 inhibitor Z-LEHD-fmk was tested. The results in Figure 3D show that neither inhibitor affected the cleavage of caspase-3 and -7.
When we examined TBL cultured with NAC, the following results were obtained: proteolytic processing of caspase-8 was unchanged with respect to that of controls during the first 24 h but was increased at 48 h (Fig. 3B) , cleavage of caspase-9 remained unchanged at all times tested (Fig. 3B) , and cleavage of caspase-3 and -7 was strongly inhibited (Fig. 3C) . Inhibition of caspase-3 and -7 processing was also associated with a decrease in the total amount of Ac-DEVD-pNA-cleaving activity recovered in the cells (data not shown).
When we tested the effect of NAC at pH 8.3 and that of medium at pH 7 on caspase-3 and -7 cleavage, the cleavage of both caspases was unchanged with respect to controls (Fig. 3E) . On the contrary, NAC added from a stock solution stored at 1 M, pH 6, strongly inhibited caspase processing similar to NAC at pH 7.4 (Fig. 3E) .
NAC inhibits DFF45 cleavage
DFF45 is a subunit of a heterodimeric DNase complex DFF40/DFF45 critical for induction of DNA fragmentation in several apoptosis models [33
, 34
]. In the following experiments, we examined whether DFF45 was cleaved in TBL apoptosis and if so, whether it depended on caspase-3 and -7. Western blot analysis showed that DFF45 was processed in TBL apoptosis as revealed by the appearance of the 11-kDa fragment at 24 and 48 h (Fig. 4A
). When TBL were incubated with the caspase-3-like inhibitor Z-DEVD-fmk, DFF45 cleavage (Fig. 4A)
and also DNA fragmentation (Fig. 4B)
were inhibited. Results in Figure 4A
show that cleavage of DFF45 was inhibited in TBL cultured with NAC.
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Figure 4. In TBL, DFF45 cleavage is dependent on caspase-3-like activities and is inhibited by NAC. (A) DFF45 cleavage was analyzed by Western blot in 30 µg total protein obtained from TBL cultured for the indicated times in medium in the presence of Z-DEVD-fmk (50 µM) or NAC (20 mM). ß-Tubulin was used as a loading control. (B) Effect of Z-DEVD-fmk on DNA fragmentation, evaluated as described in Materials and Methods. (A and B) One representative experiment of five with similar results is shown.
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Figure 5. In TBL, PARP cleavage is dependent on caspase-8 and is increased by NAC. PARP cleavage was analyzed by Western blot in 25 µg total protein obtained from TBL cultured for the indicated times with or without NAC, 20 mM (A), or with the indicated caspase inhibitors, each at 50 µM (B). (A and B) ß-Tubulin was used as a loading control, and one representative experiment of five with similar results is shown. (A) Densitometry was performed, and the density of p85 band, expressed as arbitrary units, is given as the mean ± SD of the five blots. Values were normalized using ß-tubulin as an internal standard. Lane designations are identical for blots and histograms. Statistics were performed by Student’s t-test. , P < 0.01 (controls at 48 h vs. controls at 2 h); *, P < 0.01 (NAC-treated TBL vs. controls at each time-point); °, P < 0.01 (NAC-treated TBL at 48 h vs. NAC treated at 2 h). (C) Effect of Z-IETD-fmk on TBL apoptosis, determined measuring the percentage of hypodiploid nuclei by flow cytometry. The results are shown as means ± SD of 12 experiments. Z-IETD-induced inhibition of TBL apoptosis is not significant.
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Figure 6. NAC inhibits BID cleavage and mitochondrial cytochrome c release. (A and B) Freshly isolated TBL were cultured for different times in medium, in the presence of Z-IETD-fmk (50 µM) or NAC 20 mM. (C and D) Jurkat T cells, pretreated for 1 h with Z-IETD (50 µM), were then cultured for 20 h with CD95 CH11-triggering Ab (100 ng/ml). (A and C) BID cleavage was analyzed by Western blot in 25 µg total protein, and ß-tubulin was used as a loading control. (B and D) The presence of cytochrome c (Cyt. C) in the cytosol and mitochondrial fraction (MF) was analyzed by Western blot in 40 µg extracts. The blots were reprobed with Ab anti-COX IV to control the purity of cytosolic fraction and the specificity of cytochrome c release and with anti-ß-tubulin as a loading control in cytosolic extracts. (A–D) One representative experiment of five with similar results is shown.
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Figure 7. NAC increases Bcl-2 and BclXL protein expression. Western blot was performed on 25 µg total protein obtained from TBL cultured for the indicated times with or without NAC 20 mM. ß-Tubulin was used as a loading control. One representative experiment of five with similar results is shown.
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Time-course studies of NAC addition to the culture indicate that the antiapoptotic activity of NAC is progressively reduced with delayed addition. However, NAC significantly inhibits TBL apoptosis, even if added up to 12 and 24 h after the start of culture but not when added after 48 h. Kinetics experiments to study the effect of NAC removal on TBL apoptosis show that at each time-point when NAC is removed, apoptosis is slowed down for the next 12 h, but at 24 h after each removal time, it reverts to the original rate, suggesting that the effect of NAC is reversible.
The protein synthesis inhibitor CHX does not inhibit antiapoptotic activity of NAC, suggesting that de novo protein synthesis is not required for NAC antiapoptotic activity (data not shown).
In spontaneous TBL apoptosis, caspase-3 and -7 are cleaved and seem to play a central role. In fact, their inhibitor Z-DEVD-fmk strongly reduces DFF45 cleavage and DNA fragmentation, suggesting their involvement in these molecular events. As observed in other studies, caspase-3 and -7 could be connected to DNA fragmentation by cleaving DFF45, allowing the nuclease DFF40 to enter the nucleus and degrade DNA [33 , 34 ]. We demonstrate that NAC inhibits proteolytic processing of caspase-3 and -7, DFF45 cleavage, and DNA fragmentation. Given the relevance of these molecular events in TBL apoptosis, their inhibition could be the major mechanism by which NAC rescues TBL from apoptosis. However, we can only speculate about how NAC exerts inhibitory effects on these molecular events. The most likely explanation for the inhibition of caspase-3 and -7 cleavage by NAC is that the upstream activity responsible for this cleavage is inhibited. Some of our results suggest that this putative activity is not caspase-8 and -9. In fact, even if caspase-8 and -9 are processed during TBL apoptosis, they are not involved in caspase-3 and -7 cleavage. Furthermore, cleavage of caspase-8 is increased by NAC, and caspase-9 is not affected. However, even if other undefined caspases are involved in caspase-3 and -7 cleavage, studies of other authors suggest that a direct, inhibitory effect of NAC on this putative activity would be unlikely. In fact, it has been demonstrated that reducing agents favor the activity of caspases in vitro [17 18 19 ]. This occurs, as to function, these redox-sensitive enzymes require reduction of a catalytic cysteine-residue site as well as other cysteine residues around the catalytic site [44 ]. On the contrary, their activity is lost in a thiol-oxidized state [45 , 46 ].
As in TBL apoptosis caspase-3 and -7 seem to be responsible for DFF45 cleavage and DNA fragmentation, the inhibition of these molecular events by NAC could be the consequence of impaired cleavage of these caspases. Conversely, a direct, inhibitory effect of NAC on DFF complex is rather improbable. In fact, it has been shown in a cell-free system that DFF45-dependent production of a functional DFF40 is enhanced in the presence of reducing agents [47 ]. Furthermore, even if human DFF40 has a high cysteine-residue content [34 ], DNase activity is barely affected by reducing or oxidizing agents [47 ].
A characteristic event in apoptosis induced by a variety of stimuli is the proteolytic cleavage of PARP by caspases, especially caspase-3-like activities [48 ]. However, recent studies have demonstrated that PARP can be cleaved even in the absence of caspase-3 and -7 [49 50 51 ]. Kinetics analysis of PARP and caspase cleavage during TBL apoptosis indicate that PARP is already cleaved at 2 h when caspase-3 and -7 are not yet processed, suggesting that at least at this time, caspase-3 and -7 are dispensable for PARP cleavage. Experiments performed with caspase inhibitors suggest that caspase-8 is mainly responsible for the cleavage of PARP occurring during TBL apoptosis and that at 24 h, also caspase-3 and -7 are involved but in a minimal part. Therefore, the inhibition of caspase-3 and -7 cleavage by NAC should not reduce PARP cleavage significantly. Indeed, in TBL cultured with NAC, PARP cleavage is similar to controls up to 24 h, and at 48 h, it is increased. As at this time caspase-8 cleavage is also increased by NAC, and caspase-3 and -7 cleavage is inhibited, these data seem to reinforce the possible correlation between caspase-8 and PARP, but further studies are needed to understand the role of caspase-8-mediated cleavage of PARP in TBL apoptosis and the significance of these two NAC effects in TBL survival. However, as NAC strongly protects TBL from apoptosis, these two effects cannot be considered proapoptotic. In addition, the cause of caspase-8 activation in TBL apoptosis is unclear. Indeed, although caspase-8 activation has been shown to be a critical event in death receptor-mediated apoptosis [36 , 37 ], in TBL, it is not a result of a Fas signal, as there is no detectable cell-surface expression of Fas and FasL in these cells (data not shown). Furthermore, TBL apoptosis is not affected in the presence of the CD95 ZB4-blocking Ab (data not shown), which inhibits Fas/FasL interaction.
It is well known that caspase-8 induces the cleavage of the proapoptotic protein BID, which generates an active fragment able to translocate to mitochondria, where it induces the opening of the permeability transition pore, a decrease in mitochondrial membrane potential, and the release of cytochrome c into the cytoplasm [36 , 37 ]. In TBL apoptosis, BID is cleaved, and cytochrome c is released from mitochondria. NAC is able to inhibit both these molecular events. These results conflict with the fact that caspase-8 cleavage is not inhibited by NAC. The explanation we suggest is that in TBL, BID cleavage does not seem to depend on caspase-8, as instead occurs in Fas-treated Jurkat T cells. In addition, kinetics analysis of caspase-8 and BID cleavage shows that at 2 h, in the presence of a cleaved caspase-8, BID is in its intact form. However, further studies are necessary to identify the possible activity responsible for BID cleavage and cytochrome c release in TBL and to clarify how NAC interferes with these molecular events.
It has been suggested that the antiapoptotic proteins Bcl-2 and BclXL can antagonize BID cleavage and proapoptotic effects of BID, including cytochrome c release [37 , 52 ]. In TBL, the constitutive levels of Bcl-2 and BclXL seem unable to antagonize these proapoptotic events. NAC increases Bcl-2 and BclXL protein expression. However, this effect is induced only after 24 h, and NAC exerts its antiapoptotic activity already during the first 12 h. These data seem to suggest that the increased expression of Bcl-2 and BclXL does not play a significant role in the antiapoptotic activity of NAC during the first 12 h. However, further studies are necessary to discern their possible involvement in mediating this process.
Collectively, our results show for the first time that NAC inhibits TBL apoptosis by inhibiting caspase-3 and -7 processing and also induces changes in several regulatory components of the apoptotic process.
The evidence that B lymphocytes survive in response to thiol molecules poses the question of whether microenvironment thiols may in part contribute to in vivo B cell survival.
Received April 10, 2003; revised March 3, 2004; accepted March 4, 2004.
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promotes survival of human primary B-lymphocytes via phosphatidylinositol 3-kinase Biochem. Biophys. Res. Commun. 284,583-586[CrossRef][Medline]B Free Radic. Biol. Med. 28,1317-1327[CrossRef][Medline]B signal transduction pathway Proc. Natl. Acad. Sci. USA 91,11527-11531B activation in primary hippocampal neurons J. Biol. Chem. 274,8531-8538B family directly activates expression of the apoptosis inhibitor Bcl-XL Mol. Cell. Biol. 20,2687-2695B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation Science 281,1680-1683-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis J. Biol. Chem. 273,15540-15545This article has been cited by other articles:
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