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NEOPLASIA
From INSERM U517, Faculties of Medicine and Pharmacy,
Dijon, France and Research Centre of University of Montreal Hospital
Centre, Notre Dame Hospital, Montreal, QC, Canada.
Engagement of the plasma membrane receptor Fas can induce apoptosis
of leukemic cells. Signaling through Fas requires the formation of a
death-inducing signaling complex (DISC) that involves the cytoplasmic
domain of Fas, the adaptor molecule FADD/MORT-1, and procaspase-8. The
present study investigated whether another caspase, known as
procaspase-2L, played a role in Fas-mediated cell death. A series of
human leukemic variant cells was derived by stable transfection with a
CASP2L antisense construct (CASP2L/AS). Specific down-regulation of procaspase-2L decreased the sensitivity of
these cells to apoptosis induced by an agonistic anti-Fas antibody (Ab,
clone CH11), as determined by studying DNA fragmentation, chromatin
condensation, and externalization of phosphatidylserine on the plasma
membrane. In leukemic cells transfected with an empty vector, anti-Fas
Ab treatment activated caspase-8, decreased the expression of the BH3
domain-only protein Bid, triggered the release of cytochrome c from the
mitochondria to the cytosol, and activated caspase-3. All these events
could not be observed when CASP2L/AS cells were
similarly treated with anti-Fas Abs. CASP2L/AS
transfection did not inhibit the formation of the DISC and no direct
interaction between procaspase-2L and either Fas or FADD or
procaspase-8 was identified. Down-regulation of procaspase-2L inhibited
anti-Fas Ab-mediated cleavage of c-FLIP (FLICE-inhibitory protein), a
protein that interferes with the formation of a functional DISC. These
results suggest that the long isoform of caspase-2 plays a role in the
Fas-mediated pathway to cell death by contributing to caspase-8
activation at the DISC level.
(Blood. 2001;97:1835-1844) Apoptosis can be induced in many cell types
by engagement of the Fas (CD95/APO-1) receptor by its natural
ligand (FasL) or agonistic antibodies (anti-Fas Ab).1,2
Fas-mediated cell death was proposed to play a role in
T-cell3 and erythroid cell4 homeostasis. Fas
receptor was also suggested to be involved in the early phase of
cytotoxic drug-mediated leukemic cell apoptosis.5,6 Signaling through Fas requires its aggregation and the formation of an
intracellular protein complex referred to as the death-inducing signaling complex (DISC).7,8 This complex
consists of the cytoplasmic domain of Fas, the adaptor molecule
FADD/MORT-1, and the cysteine aspartic acid-specific protease
Mch5/Flice/Mach/procaspase-8.9-13 This latter
protein belongs to a family of structurally related cysteine proteases
that currently includes 14 enzymes in mammals.14 All are
synthesized as inactive proenzymes that have to be cleaved at key
aspartate residues to be activated.15 Caspases can be divided into 2 groups according to the length of their prodomain, those
with a short prodomain (procaspase-3, -6, and -7) and those with a long
prodomain (the remainder). These enzymes were shown to function as a
proteolytic cascade in which caspases with a long prodomain act
upstream of those with a short prodomain.16 In the
Fas-mediated cell death pathway, caspase-3 was suggested to play an
important role in the cascade, downstream of
caspase-8.17
The binding of procaspase-8 to FADD/MORT-1 is mediated through
tandem death effector domains (DED). The FLICE-inhibitory
protein, c-FLIP (Casper/I-Flice/Flame-1/Cash/Clarp/Mrit/Usurpin) that
contains similar DED but an inactive caspase domain, interferes with
the formation of a functional DISC.18-25 On recruitment to
the DISC, procaspase-8 is processed into its active subunits, which are released from the complex to cleave intracellular substrates and to
signal apoptosis.26 The amount of active caspase-8
generated at the DISC and the level of expression of procaspase-3 could determine whether downstream pathways involve the mitochondria or
not.27 Type I cells in which caspase-8 directly activates downstream caspases to induce the cell demise were distinguished from
type II cells in which caspases have to cleave the proapoptotic BH3
domain-only protein of the Bcl-2 family known as Bid to trigger cell
death.28,29 In this latter pathway, cleaved/activated Bid
migrates from the cytosol to the mitochondria and induces the opening
of the permeability transition pores, the loss of inner mitochondrial
transmembrane potential, and the release of apoptogenic factors such as
cytochrome c and apoptosis-inducing factor (AIF).30-33 In
the cytosol, cytochrome c triggers oligomerization of the adaptor
molecule APAF-1 in the presence of adenosine triphosphate (ATP).
Oligomerized APAF-1 recruits and activates caspase-9 that, in
turn, activates caspase-3 and other downstream caspases in a
proteolytic cascade.34-36
The specific contribution of other caspases to Fas-mediated cell death
remains poorly understood. One of these is caspase-2, initially
described as Nedd2/Ich1, whose gene was highly expressed in mouse
embryonic brain and down-regulated in the adult.37,38 Mice
deficient in caspase-2 have shown that this enzyme acts both as a
positive and a negative regulator of cell death.39
Alternative splicing of the CASP2 gene37
generates at least 2 messengers that encode a long isoform, caspase-2L,
whose overexpression induces cell death, and a short isoform,
caspase-2S, that antagonizes cell death. Caspase-2L is the dominant
isoform that is expressed in most tissues. Its contribution to
apoptosis is attested to by several lines of evidence. Procaspase-2L
can be activated by cleavage early in the process of apoptosis, its
overexpression induces mammalian cell death, antisense-mediated
decrease of procaspase-2L expression delays cell death induced by
trophic factor deprivation in various cell lines, and oocytes from
caspase-2-deficient mice are resistant to chemotherapeutic
drug-induced apoptosis.39-42 The prodomain of procaspase-2
interacts with the TNFR1 receptor via the adaptor molecules TRADD, RIP,
and RAIDD/CRADD, suggesting its upstream involvement in the proteolytic
cascade that mediates cell death induced by tumor necrosis factor- To analyze the role of caspase-2L in Fas-mediated apoptosis, we have
derived a series of variant human leukemic cell populations by
transfection with a CASP2L antisense construct
(CASP2L/AS). Our results indicate that the presence
of caspase-2L contributes to caspase-8 activation and downstream
events, including Bid cleavage, cytochrome c release, caspase-3
activation, and DNA fragmentation in leukemic cells exposed to anti-Fas
Abs. Furthermore, c-FLIP cleavage was prevented by down-regulation of
caspase-2L, suggesting a potential mechanism for caspase-2-mediated
sensitization to Fas agonists.
Chemicals and reagents
Cell line and culture conditions
Complementary DNA construct and cell transfection CASP2L complementary DNA (cDNA) was generated by reverse transcriptase- polymerase chain reaction (RT-PCR) performed on RNA extracted from U937 cells,46 and subcloned in an antisense orientation (CASP2L/AS) into pTARGET vector (Promega, Charbonniëres, France). About 1.0 × 107 cells were electroporated at 270 kV with 10 µg pTARGET-CASP2L/AS or empty vector. The cells were cultured for 3 weeks in medium containing 1.0 mg/mL G418 to generate mixed cell populations. In addition, 48 hours after electroporation, some transfected cells were plated in 96-well plates at different cell densities and cultured during 4 weeks in medium containing 1.0 mg/mL G418. Clones were selected for subsequent analyses and maintained in 1.0 mg/mL G418 containing medium.Antibodies Monoclonal Abs used in this study include antihuman procaspase-3 (clone 19), procaspase-7 (clone 51), and FADD (clone 1) purchased from Transduction Laboratories (Lexington, KY); antihuman procaspase-2 (clone G310-1248), procaspase-8 (clone B9-2), and cytochrome c (clone 7H8.2C12) obtained from Pharmingen (San Diego, CA); and antihuman procaspase-8 from Biosource International (Camarillo, CA). Affinity purified polyclonal Abs used included antihuman caspase-2 and antihuman HSC-70 from Santa Cruz Biotechnology (Santa Cruz, CA), antihuman Bid from R&D Systems (Oxon, United Kingdom), antihuman PARP from Boerhinger Mannheim (Meyla, France), antihuman caspase-10 and antihuman c-FLIP from Pharmingen, and another antihuman c-FLIP from Upstate Biotechnology (Souffelweyersheim, France). The rabbit polyclonal anticaspase-3 serum, which recognizes the proenzyme (p32) and its p19 and p17 subunits, was kindly provided by Dr D. Nicholson (Merck, Toronto, ONT, Canada). Goat horseradish peroxidase-conjugated antimouse or antirabbit Abs were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Enhanced chemiluminescence (ECL) reagents were from Amersham.Western blot, coimmunoprecipitation, and fractionation analysis For Western blot analysis, cells were washed twice in ice-cold phosphate-buffered saline (PBS), lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% Na-sodium dodecyl sulfate [SDS], 0.5% Na-desoxycholate) in the presence of protease inhibitors (0.1 mM PMSF, 2.5 µg/mL pepstatin, 10 µg/mL aprotinin, 2.5 µg/mL trypsin inhibitor, 5 µg/mL leupeptin) for 30 minutes on ice, then centrifuged at 15 000g for 20 minutes at 4°C. Supernatants were collected and 50 µg proteins were incubated in loading buffer (125 mM Tris-HCl, pH 6.8, 10% -mercaptoethanol, 4.6% SDS, 20% glycerol,
and 0.003% bromophenol blue) prior to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) on a 12% to 15% SDS polyacrylamide gel.
Following electrophoresis, proteins were electroblotted to polyvinylidene difluoride (PVDF). After blocking nonspecific
binding sites overnight with 5% nonfat milk in TPBS (PBS, 0.1% Tween
20), membranes were incubated for 2 to 3 hours at room temperature with
the primary Abs. After 2 washes in TPBS, membranes were incubated with
the corresponding peroxidase-conjugated secondary Abs for 30 minutes at
room temperature. Immunoblots were revealed by ECL and autoradiography.
For coimmunoprecipitation studies, cells were collected by
centrifugation, washed twice with ice-cold PBS, and then homogenized in
a lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM
NaVO4; 1% bovine serum albumin [BSA]; 1% NP-40; 2 mM
phenylmethylsulfonyl fluoride [PMSF]; 1.0 mg/mL aprotinin) at
4°C for 30 minutes. The extracts were then centrifuged and supernatants were collected. The antihuman caspase-8
(Pharmingen), caspase-2 (Santa Cruz), and Fas (clone APO1-3,
Alexis Biochemicals, San Diego, CA) were added overnight, which was
followed by incubation with protein A/G agarose for 1 hour and
centrifugation. Immunoprecipitates were washed in lysis buffer without
NP-40 and analyzed by Western blotting. For subcellular fractionation,
cells were resuspended in ice-cold buffer A (250 mM sucrose, 20 mM
HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
EGTA, 1 mM DDT, 17 µg/mL PMSF, 8 µg/mL aprotinin, 2 µg/mL
leupeptine) and dounced briefly on ice. Unlysed cells and
nuclei (750g for 10 minutes) and mitochondria (10 000g for 25 minutes) were pelleted by sequential
differential centrifugation. Supernatants were further clarified by
centrifugation at 100 000g for 60 minutes (S100 fraction).
Immunocytofluorometry and confocal microscopy analysis Relative level of expression of Fas in control and transfected variant lines was determined by cytofluorometry and confocal microscopy analysis. Nonpermeable cells were incubated in the absence and presence of antihuman Fas (clone DX2 from Pharmingen) as previously described,5 and the fluorescence intensity distribution was studied by using a FACScan flow cytometer (Becton Dickinson, Le Pont de Claix, France). Cells were also fixed for confocal microscopy analysis as previously described.47Identification of apoptosis Staining of nuclear chromatin with Hoechst 33342 for 30 minutes at 37°C was used for identification of morphologic changes by fluorescence microscopy (each percentage was established by counting 400 cells). DNA fragmentation was measured using a previously described DNA filter elution assay48 and expressed as percentage of fragmented relative to total DNA. Exposure of phosphatidylserine on the outer plasma membrane leaflet was identified by annexin V-fluorescein isothiocyanate (FITC) staining in the presence of propidium iodide (Boehringer Ingelheim, Heidelberg, Germany). Briefly, cells were resuspended in 10 mM HEPES, pH7.4, 140 mM NaCl, 2.5 mM CaCl2, in the presence of annexin V-FITC (1.0 mg/mL) and propidium iodide (1.0 mg/mL) for 10 minutes in the dark and analyzed using a FACScan flow cytometer (Becton Dickinson).Caspase activity Cytosolic extracts were prepared by resuspending the cells at indicated times in a lysis buffer containing 100 mM HEPES (pH 7.5), 5 mM EDTA, 5 mM DTT, 20% glycerol, and 0.3% NP-40. Samples were centrifuged at 10 000g for 15 minutes at 4°C and supernatants were collected as cytosolic extracts. Caspase activities were measured as described17 by monitoring fluorescence continuously in a dual luminescence fluorimeter (LS 50B PerkinElmer, Courtaboeuf, France) using specific excitation and emission wavelengths for Ac-DEVD-AMC and z-IETD-AFC peptide derivative substrates, respectively. Reactions were carried out at 37°C using a water-jacketed sample compartment. The assay mixture contained 100 mM HEPES (pH7.5), 20% glycerol, 5 mM DTT, 5 mM EDTA, 200 µM fluorogenic peptides, and 200 µg proteins per assay. Enzyme activities were determined as initial velocities (intensity/min/mg) and related to results obtained in untreated cells.
Stable transfection of CASP2L/AS in U937 human leukemic cells Exposure of U937 cells to 50 ng/mL CH11 anti-Fas Abs in the presence of 0.8 µg/mL CHX induces simultaneous activation of caspases 2L and 8, as demonstrated by the decreased expression of procaspase-2L, the appearance of caspase-8 active fragments (Figure 1, insert), and the increased ability of cell extracts to cleave VDVAD-AFC and IETD-AFC substrates (Figure 1). To evaluate the contribution of caspase-2L to Fas-mediated cell death, the full-length CASP2L cDNA was subcloned into an antisense orientation in pTARGET plasmid vector that contains a neomycin-resistance gene. U937 cells were stably transfected by electroporation with the NEO or CASP2L/AS vectors and stable clones and mixed populations were derived under G418 selection. Western blot analysis of transfected cells using an antihuman caspase-2 Ab revealed the presence of a 48-kd protein in cells transfected with the empty vector (Figure 2A; U937/neo). The expression of the 48-kd long isoform of procaspase-2 was decreased in the 2 mixed cell populations (Figure 2A; U937/AS1 and U937/AS2) as well as in a selected clone (Figure 2B; U937/AS3). The antisense constructs did not significantly modify the level of expression of Fas, FADD, and various procaspases including procaspase-3, -7, and -8 (Figure 2B). The lack of influence of CASP2L/AS construct on the plasma membrane expression of Fas was demonstrated by flow cytometry (Figure 2C). Confocal microscopy analysis showed that the clustering of Fas on stimulation with recombinant soluble Fas-L was similar in U937/neo and U937/AS3 cells (Figure 2D).
Decreased expression of procaspase-2L delays anti-Fas Ab-induced apoptosis in U937 cells To determine the role of procaspase-2L in cell response to Fas agonists, we have exposed U937/neo and U937/AS3 clones to 50 ng/mL anti-Fas (clone CH11) Ab in the presence of 0.8 µg/mL CHX. In the tested conditions, neither anti-Fas Ab nor CHX was toxic to the cells.49 The occurrence of DNA fragmentation, monitored by a DNA filter elution assay, was delayed in U937/AS3 compared to U937/neo, with approximately 25% and 50% of fragmented DNA 15 hours after treatment, respectively (Figure 3A). Trypan blue exclusion assays indicated that in most cells, plasma membranes were able to exclude the dye, even 24 hours after treatment, suggesting that the observed DNA fragmentation was related to apoptosis rather than necrosis (not shown). Apoptotic cell death was further demonstrated by Hoechst 33342 staining of nuclear chromatin in anti-Fas-Ab-treated cells (Figure 3B). The morphologic study confirmed that anti-Fas Ab-induced cell death was delayed in U937/AS3 compared to U937/neo cells. Externalization of phosphatidylserine on the outer leaflet of the plasma membrane is an early event in cells that are induced to undergo apoptosis.50 Using FITC-labeled annexin V and flow cytometry analysis, we observed that the appearance of annexin V+ cells was also delayed in U937/AS3 compared to U937/neo cells after anti-Fas Ab treatment (Figure 3C). In parallel, U937/neo and U937/AS3 cells remained unlabeled by propidium iodide, further indicating that the plasma membrane was not disrupted (Figure 3C). Similar modulation in kinetics of apoptosis induction was observed in U937/AS1 and U937/AS2 cells (data not shown). Altogether, these results indicated that decreased expression of procaspase-2L in U937 cells delayed anti-Fas Ab-induced apoptosis.
Decreased expression of procaspase-2L prevents procaspase-3 activation and cytochrome c release in anti-Fas Ab-treated U937 cells We have shown previously that Fas-induced cell death was associated with procaspase-3 cleavage/activation in U937 cells.51 Using a rabbit polyclonal Ab raised against the long subunit of caspase-3, we observed that procaspase-3 was rapidly cleaved into a p19 and a p17 fragment in Fas-treated U937/neo cells. This cleavage, which suggested caspase-3 activation, was not observed in U937/AS3 cells (Figure 4A). The cleavage of poly(ADP-ribose) polymerase, a well-known target for active caspase-3, was also delayed in U937/AS3 compared to U937/neo cells (not shown). To confirm that decreased expression of procaspase-2L prevented caspase-3 activation, we measured the ability of lysates from untreated and anti-Fas Ab-treated U937/neo and U937/AS3 cells to cleave the DEVD-AMC substrate that mimics the specific target site for some effector caspases, mainly caspase-3. This activity was strongly increased in U937/neo cells exposed to anti-Fas Abs, whereas it remained low in U937/AS3 clone (Figure 4B).
To delineate more precisely the pathway of caspase-3 activation during anti-Fas Ab treatment, cytochrome c release from the mitochondria to the cytosol was monitored. After a 6-hour exposure to anti-Fas Abs, the cytochrome c release observed in U937/neo cells was not identified in U937/AS3 cells treated in similar conditions (Figure 4C). In addition, the time-dependent decrease of Bid expression observed in U937/neo cells was delayed in U937/AS3 cells (Figure 4D). Altogether, these results suggested that caspase-2L might interfere with Fas-mediated apoptosis pathway upstream of the mitochondrial release of proapoptotic molecules and Bid cleavage. Decreased expression of procaspase-2L inhibits anti-Fas Ab-mediated caspase-8 activation Because caspase-8 activation has been shown to play a central role in Fas-mediated cell death,9-13,52 we monitored procaspase-8 expression and caspase-8 activity in U937/neo and U937/AS3 cells exposed to anti-Fas Abs. The decrease of procaspase-8 expression, suggesting its cleavage into active fragments, was observed to be almost inhibited in U937/AS3 compared to U397/neo cells (Figure 5A). As a control, procaspase-10 expression remained unchanged in anti-Fas Ab-treated U937/neo and U937/AS3 cells (Figure 5A). The hypothesis that down-regulation of caspase-2L expression could prevent caspase-8 activation in response to Fas agonists was confirmed by measuring IETDase activity in U937/neo and U937/AS cells following exposure to anti-Fas Abs. IETDase activity demonstrated a biphasic kinetics in U397/neo cells that was not observed in U937/AS3 cells (Figure 5B).
Stable transfection of CASP2L/AS also interferes with Fas-mediated cell death in Jurkat and CEM leukemic cell lines The previous experiments were repeated in mixed cell populations obtained by stably transfecting 2 other human leukemic cell lines with either the empty vector (Jurkat/neo and CEM/neo) or the CASP2L/AS-containing vector (Jurkat/AS1, Jurkat/AS2, and CEM/AS). Western blot analysis demonstrated that procaspase-2L expression was decreased in Jurkat and CEM cell populations transfected with the antisense construct (Figure 6A). When exposed to CH11 anti-Fas Abs (in these cells, CHX is not required to potentiate CH11 activity), Jurkat/neo and CEM/neo cells underwent apoptosis, as demonstrated by Hoechst 33342 staining of nuclear chromatin (Figure 6B,C). Contrary to U937 cells, CHX was not required for CH11 Abs to induce apoptosis in Jurkat and CEM cells. Dose-response and time-course experiments confirmed the results obtained in CASP2L/AS- transfected U937 cells by showing that decreased expression of procaspase-2L in Jurkat/AS and CEM/AS cells significantly delayed Fas-mediated apoptosis (Figure 6B,C). In addition, by using DEVD-AMC and IETD-AFC as substrates to measure caspase activities, we observed that activation of these enzymes was delayed in cells transfected with the antisense construct (Figure 7A). This was in accordance with Western blot analyses showing that the decreased expression of procaspase-3, procaspase-8 and Bid as well as the cleavage of the 116-kd PARP into a 85-kd fragment, observed in Jurkat/neo cells exposed to anti-Fas Abs, was delayed in 2 Jurkat/AS mixed populations treated under similar conditions (Figure 7B). The same observations were made by comparing CEM/neo and CEM/AS cells (data not shown).
The caspase-2 inhibitor z-VDVAD prevents Fas-induced apoptosis in U937 and Jurkat cells When added to cell culture in combination with CH11 anti-Fas Abs, the permeant peptide inhibitor Z-VDVAD-fmk, designed to inhibit caspase-2,53,54 prevented apoptosis in both U937 and Jurkat parental cell lines (Figure 8A,B). This peptide also prevented activation of the proteases that cleave DEVD-AMC and IETD-AFC substrates (Figure 8C). These results further argued for a role of caspase-2 in Fas-mediated apoptosis.
Procaspase-2L does not interact with the DISC components Results from the previous experiments had suggested that caspase-2L could facilitate procaspase-8 activation during Fas-induced apoptosis in U937 cells. To determine whether this effect was a consequence of a direct interaction of procaspase-2L with procaspase-8 or the adaptor protein FADD, we performed immunoprecipitation experiments in control and anti-Fas Ab-treated U937/neo cells. Even by using high concentrations of Abs, no interaction was identified between procaspase-2L and either procaspase-8 or FADD (Figure 9A,B). As described previously,55 under these conditions of Ab excess, an interaction between procaspase-8 and FADD was detected in both untreated and anti-Fas-treated U937/neo cells (Figure 9B). To determine whether caspase-2L was required for the DISC formation, we realized immunoprecipitation experiments using an anti-Fas Ab in control and FasL-treated cells. This experiment, performed in Jurkat/neo and Jurkat/AS1 cell populations, demonstrated that transfection with CASP2L/AS did not prevent interaction of Fas with FADD and procaspase-8 in response to FasL. Recruitment of procaspase-8 was decreased, whereas no interaction with procaspase-2L could be detected (Figure 10). Similar results were obtained by comparing U937/neo and U937/AS clones exposed to FasL in the presence of CHX (data not shown). In these conditions of immunoprecipitation, no interaction between Fas and FADD or procaspase-8 was detected in untreated cells.
Decreased expression of procaspase-2L prevents the cleavage of c-FLIP We also evaluated the effect of caspase-2L on c-FLIP (FLICE-inhibitory protein), a potent inhibitor of procaspase-8 activation at the DISC. Western blot experiments performed with a first polyclonal anti-c-FLIP Ab (Upstate Biotechnology) indicated that the decreased expression of c-FLIP observed in U937/neo cells treated with anti-Fas Abs was delayed in U937/AS3 cells (Figure 11A). By using another polyclonal anti-c-FLIP Ab (Pharmingen), we identified a 12-kd cleavage fragment in U937/neo cells exposed to anti-Fas Abs. This cleavage fragment was not observed in U937/AS3 cells treated under the same conditions (Figure 11B). Similar results were obtained in U937/AS1 and AS2 cells (not shown).
CASP2L/AS transfection also delays TRAIL-mediated apoptosis To determine whether caspase-2L could play a role in the apoptotic pathway triggered by engagement of another death receptor, we tested the influence of CASP2L/AS transfection on TRAIL-mediated apoptosis in Jurkat (TRAIL: 100 ng/mL) and CEM (TRAIL: 200 ng/mL) cells. In both cell lines, decreased expression of procaspase-2L was associated with a delay in TRAIL-induced apoptosis, as determined by Hoechst 33342 staining of nuclear chromatin (Figure 12).
The present study suggests that the long isoform of caspase-2
plays a role in the Fas-mediated pathway to cell death by contributing to caspase-8 activation at the DISC level. The role of caspase-2 in
cell death pathways appears to depend on both the apoptotic stimulus
and the cell type. Analysis of caspase-2 The pathway leading to Fas-mediated apoptosis depends on the clustering of Fas receptor that triggers the death signal. Decreased expression of procaspase-2L did not influence Fas receptor expression on the plasma membrane of leukemic cells. When analyzed by confocal microscopy, the clustering of Fas receptor triggered by Fas-L on the plasma membrane of leukemic cells47 was not influenced by procaspase-2L down-regulation. In these cells, Fas-mediated cell death involves the decreased expression of the native form of the BH3 domain-only protein Bid,28,29 the release of cytochrome c from the mitochondria to the cytosol, and the activation of caspase-3. Caspase-mediated cleavage of Bid28,57 was proposed to mediate, either directly or through Bax dimerization, the mitochondrial damage observed in some cells exposed to Fas agonists.29,58 Because caspase-2L down-regulation was observed to delay all these events, the enzyme was supposed to interfere with Fas-mediated cell death pathway somewhere between Fas clustering at the plasma membrane and caspase-mediated cleavage of Bid.28,29 Death signaling through Fas receptor involves the recruitment of FADD and procaspase-8 to form the DISC.8 Recruitment of procaspase-8 to the DISC leads to its proteolytic activation.12,13 Gene ablation studies in mice have shown that the presence of caspase-8 was an essential requirement for Fas-mediated cell death.52 We observed that down-regulation of procaspase-2L expression prevented Fas-mediated caspase-8 activation. One possible explanation for that observation was that procaspase-2L could be recruited to the DISC to facilitate caspase-8 activation on Fas stimulation. Procaspase-2L had been shown to associate via its amino-terminal CARD domain with RAIDD, a death adaptor molecule that is involved in death signaling through the TNF-R1 complex via association with the death domain proteins RIP and TRADD.44 Immunoprecipitation experiments failed to identify any direct interaction between procaspase-2L and either Fas or FADD or procaspase-8. These experiments also showed that decreased procaspase-2L expression did not prevent the formation of the DISC but could interfere with procaspase-8 recruitment. These observations suggest that caspase-2L could indirectly modulate the kinetics of caspase-8 recruitment and activation in the DISC, for example, through the cleavage products of a target protein. The mechanism of this modulation remains to be clarified. Another molecule that interferes with the Fas-mediated cell death pathway is c-FLIP, a protein structurally related to procaspase-8 but lacking a catalytic active site and the residues that form the substrate binding pocket.18-25 The protein is predominantly found as a 55-kd isoform in most tissues and cell lines,19 although a minor species of 27/28 kd was also detected in some cell types.18,59 In hematopoietic cells, mainly lymphoid cells, overexpression of c-FLIP could involve a cell surface-mediated signaling pathway through a member of the immunoglobulin gene superfamily named Toso.60 The role of c-FLIP in controlling Fas susceptibility of peripheral T cells and other cell types remains controversial.59,61 Overexpression of c-FLIP was proposed to specifically prevent death receptor-induced apoptosis through inhibition of procaspase-8 processing without affecting other pathways of cell death.18-21,59,62,63 However, some reports have suggested that c-FLIP could behave as a proapoptotic molecule whose effect can be inhibited by a dominant-negative caspase-8 mutant, various caspase inhibitors, and the antiapoptotic proteins of the Bcl-2 family.22-25 Another controversial issue concerns the consequences of the caspase-mediated cleavage of c-FLIP. Caspase-2 is one of the caspases that have been shown to cleave c-FLIP at Asp-341 to generate a p43 N-terminal and a p12 C-terminal fragments in vitro.21 Some authors have proposed that the cleavage of c-FLIP was required for its antiapoptotic activity,59 whereas others have reported that up-regulation of the native molecule was essential for its activity.64,65 In the present study, we show that the decreased expression of c-FLIP induced by anti-Fas Ab treatment of leukemic cells is prevented by procaspase-2L down-regulation. A faint cleavage fragment of c-FLIP can be identified in anti-Fas Ab-treated control cells, whereas this fragment is not detected in anti-Fas Ab-treated CASP2L/AS-transfected cells. Although inhibition of c-FLIP cleavage could account for the decreased sensitivity of CASP2L/AS-transfected cells to Fas-mediated cell death, we cannot rule out the possibility that this inhibition is a nonspecific consequence of the decreased activation of caspase-8 and downstream caspases.21 To summarize, decreased expression of procaspase-2L in human leukemic cell lines prevented the activation of procaspase-8 and downstream events induced by agonistic anti-Fas Abs, including decreased expression of Bid, cytochrome c release from the mitochondria, procaspase-3 activation, apoptotic DNA fragmentation, and externalization of phosphotidylserine on the plasma membrane. These results strongly suggest that caspase-2L plays a role in caspase-8 activation at the DISC level. It remains to be determined how this caspase is activated in response to Fas agonists and whether the correlation observed between Fas-induced caspase-8 activation and c-FLIP expression is directly related to caspase-2L activity.
The authors wish to thank Francois Martin and Ali Bettaieb for helpful advice and discussion. We are grateful to Dr D. Nicholson (Merck, Toronto, Canada) and A. Fontana (Lausanne, Switzerland) for providing us the polyclonal anti-caspase-3 Ab and Fas-L Neuro2A transfected cells, respectively.
Submitted March 20, 2000; accepted November 9, 2000.
Supported by grants from the committees (Cote d'Or, Niëvre, Saone et Loire) of the Ligue Nationale Contre le Cancer, the Association pour la Recherche contre le Cancer (no. 9567) and the Conseil Régional de Bourgogne to E.S. and from the Medical Research Council of Canada (MT-15019) to R.B. N.D. obtained a studentship from the Ministëre de l'Education Nationale, de la Recherche et de la Technologie (France) and from the Sociètè Francáise d'Hématologie. R.B. is a scholar from the Medical Research Council of Canada and the Cancer Research Society (Canada).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Eric Solary, INSERM U517, Faculties of Medicine and Pharmacy, BP 87900, 21079 Dijon Cedex, France; e-mail: esolary{at}u-bourgogne.fr.
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© 2001 by The American Society of Hematology.
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  Y. Murakami, E. Aizu-Yokota, Y. Sonoda, S. Ohta, and T. Kasahara Suppression of Endoplasmic Reticulum Stress-induced Caspase Activation and Cell Death by the Overexpression of Bcl-xL or Bcl-2 J. Biochem., March 1, 2007; 141(3): 401 - 410. [Abstract] [Full Text] [PDF]
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A. Holleman, M. L. d. Boer, K. M. Kazemier, H. B. Beverloo, A. R. M. von Bergh, G. E. Janka-Schaub, and R. Pieters Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia Blood, September 1, 2005; 106(5): 1817 - 1823. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(TNF-
)
and IETD-AFC (
) substrates was measured and the expressions of
procaspase-2L, procaspase-8, and caspase-8 active fragments were
studied by Western blot. An antihuman HSC70 Ab was used for
loading control.
) and U937/AS3 (
) cells. Data points, means of 2 independent
experiments in triplicate; bars indicate SD. (B) Morphologic aspect of
control and anti-Fas Ab-treated (CH11 50 ng/mL, CHX 0.8 µg/mL; 24 hours) U937/neo and U937/AS3 cells stained by Hoeschst 33342 (magnification × 40). (C) Flow cytometry profile of FITC-conjugated
annexin V and propidium iodide (PI)-labeled U937/neo and U937/AS3 at
various times after anti-Fas Ab treatment. Percentages of annexin
V+ cells are indicated. The percentage of labeled cells
with PI did not exceed 10%, 15 hours after treatment.
), and U937/AS2 (
) cells. Enzyme activities were
measured as initial velocities and expressed as relative to control
untreated cells.
), CEM/neo (
/