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Institut National de la Santé et de la Recherche Médicale, Unité 131 and Unité 542, and Claude Bernard Research Center, Clamart, France; and Hôpital Paul Brousse, Villejuif, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Caspase-8 activation has been extensively studied in apoptosis mediated
by members of the death domain receptor family such as Fas/CD95 and
TNFR (9). The activation of Fas by its natural ligand or
agonist Ab ligands results in Fas-associated death domain protein
(FADD)4
recruitment via its corresponding death domain (10, 11).
Interactions between Fas and FADD via their C-terminal death domains
expose the N-terminal death effector domain (DED) of FADD, which can
interact with DED domains in the caspase-8 proform, resulting in the
oligomerization of this protease and its subsequent autocleavage and
activation (12, 13). Thus, FADD appears to be the final
common link between the death domain-containing receptors and
caspase-8. However, recent studies have suggested that alternative
pathways may also be responsible for caspase-8 activation. Indeed, it
has been observed that TGF-
-mediated caspase-8 activation in Burkitt
cells does not involve death domain receptors (14), and
FADD-independent caspase-8 activation by anticancer drugs has been
reported by Wesselborg et al. (15). However, the nature
and regulation of these FADD-independent pathways of caspase-8
activation remain unknown. Caspase-8 cleaves the proapoptotic member of
the Bcl-2 protein family, Bid. The cleaved form of Bid is then
responsible for changes in the mitochondria, including the opening of
the permeability transition pore, a decrease in mitochondrial membrane
potential (
m) and the release of cytochrome c into the
cytoplasm (16, 17). Caspase-3 can be activated via
mitochondria-dependent or -independent pathways (18). One
of these pathways of caspase-3 activation is dependent on the release
by mitochondria of cytochrome c which, in the presence of
ATP, associates with the cytoplasmic Apaf1 protein and inactive
proforms of caspase-9, forming a complex called the apoptosome
(19, 20). Caspase-9 undergoes autocleavage and activation
in this complex. Caspase-9 then directly cleaves and activates
caspase-3 proforms. An alternative pathway, observed in type I Jurkat T
cells in response to Fas ligation, is independent of mitochondrial
activation and requires the direct cleavage of caspase-3 proforms by
activated caspase-8 (21, 22).
B cell receptor (BCR)-mediated apoptosis of human Burkitt’s lymphoma cells and of normal B cells is associated with and dependent on caspase-3 activation (1, 3, 4, 5, 6). Interestingly, soluble anti-µ Ab activates mitochondria and triggers the cytochrome c-dependent activation of caspases-9 and -3, but no caspase-8 activation has been observed in these conditions and mitochondrial activation appears to occur via a caspase-independent pathway that has not yet been well defined (5, 6). We have previously reported that BCR-mediated B cell apoptosis either occurs in the presence of soluble anti-µ Ab or requires further cross-linking with a secondary Ab, depending on cell type (23). To better understand the molecular basis of BCR-induced apoptosis, we therefore investigated whether the apoptotic pathways triggered by different levels of BCR cross-linking were similar and studied caspase involvement in mitochondrial activation.
We report here that the apoptosis of the Burkitt’s lymphoma BL41 cells
obtained in the presence of cross-linked anti-µ Ab is associated
with, and dependent on, caspase-8 activation. This in turn regulates
Bid cleavage, the loss of m, and the activation of caspase-9 and
caspase-3. We found that the caspase-8 activation mediated by BCR was
independent of the DED of FADD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine DA44 (anti-human IgM, IgG1) was obtained from hybridoma cell lines (American Type Culture Collection (ATCC), Manassas, VA) and was purified from ascitic fluids on protein A-Sepharose columns (Pharmacia, Uppsala, Sweden). The F(ab')2 of goat anti-mouse IgG (CL), anti-human Fas CH11, and the ZB4 mAbs were obtained from Immunotech (Marseille, France), and G28.5 mAb was a gift from Dr. E. Clark (University of Washington, Seattle, WA). Recombinant human TNF-related apoptosis-inducing ligand (TRAIL) was from R&D Systems (Wiesbaden, Germany). zAEVD-fmk, zIETD-fmk, zAEVD-p-nitroanaline (pNA), and zIETD-pNA were purchased from R&D Systems, and zVAD-fmk was obtained from Bachem Biochimie SARL (Voisin le Bretonneux, France). Stock solutions of zAEVD-fmk (100 mM), zIETD-fmk (20 mM), and zVAD-fmk (20 mM) were prepared in DMSO and stored at -20°C. The working dilutions were prepared immediately before use. 3,3'-Dihexylocarbocyanin iodide (DiOC6) was purchased from Molecular Probes (Leiden, The Netherlands).
Cell lines
The Burkitt’s lymphoma BL41 cell line, kindly provided by Drs. A. Calender and G. Lenoir (Institut National de la Santé et de la Recherche Médicale, Lyon, France) does not contain the EBV genome. Ramos and Jurkat cell lines were obtained from ATCC, and the Ramos-NS7 subclone was isolated in the laboratory by limiting dilution techniques and screened for its response to DA44 Ab (2.5 µg/ml) and CL Ab (20 µg/ml) (DA44 plus CL) stimulation. All cell lines were cultured in RPMI 1640 with Glutamax 1 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, sodium pyruvate, and nonessential amino acids (Life Technologies, Glasgow, U.K.).
Cell transfection
The PCDNA3.0/FADD-DN vector (kindly provided by Dr. V.
Dixit,Genentech, San Francisco, CA) carries a truncated FADD
cDNA (aa 80/208) lacking the DED region. It was used to transfect BL41
cells by electroporation (960 µF, 240 V in a Bio-Rad apparatus;
Bio-Rad Laboratories, Hercules, CA). Stable transfectants were selected
by incubating the cells with 1 mg/ml G418 (Life Technologies) for 
3
wk. Stable clonal transfectants were isolated from G418-resistant cells
using the limiting dilution technique and FADD and FADD-DN protein
levels in the various clones were analyzed by Western blotting using a
rabbit anti-FADD Ab (StressGen Biotechnologies, Victoria,
Canada).
Detection of apoptotic cells
Cells were washed in PBS, pelleted, and resuspended in PBS. Their dot-blot light scatter profiles were analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Shrunken cells with relatively high side scatter and low forward scatter properties were considered to be apoptotic and were counted. The number of apoptotic cells was then expressed as a percentage of the total population.
Analysis of m
m was evaluated by staining cells
(106) with DiOC6 at a final concentration of 40
nM (stock solution 1 µM in ethanol) for 15 min at 37°C in the dark.
The fluorescence emitted by cells was analyzed with a FACScan flow
cytometer (BD Biosciences) using the fluorescence signal 1 channel.
Assay of caspase-8 activity
Caspase-8 activity was determined using a colorimetric caspase assay (R&D Systems). Briefly, cells treated with DA44 Ab (2.5 µg/ml) and CL Ab (20 µg/ml) for various periods of time were collected and lysed according to the manufacturer’s instructions. Caspase-8 colorimetric substrates (IETD-pNA or AEVD-pNA) were added to the cell lysate, and assays were performed in a final volume of 100 µl in 96-well flat-bottom plates. The plates were incubated for 1 h at 37°C, and then OD was measured on a microplate reader at a wavelength of 405 nm. OD reading was standardized using free colorimetric substrate. The results are expressed as fold increase in caspase activity in stimulated cells, using unstimulated cells as the reference.
Western blot analysis
Cells were lysed by incubation in modified Laemmli buffer (60 mM
Tris (pH 6.8), 10% glycerol, and 2% SDS, without -mercaptoethanol
or bromphenol blue) and sonication for 15 s on ice. The samples
were centrifuged for 5 min at 4°C at 15,000 x g. The
supernatants were boiled for 3 min and frozen at -80°C or used
immediately. Aliquots of the supernatants were used for protein
determination (microBCA protein assay; Pierce Chemical, Rockford, IL).
-Mercaptoethanol and bromphenol blue were added, and cell lysate
proteins (30 µg/lane) were resolved by SDS-PAGE. Proteins were then
electroblotted onto nitrocellulose filters with 0.45-µm pores, and
the filters were blocked by incubation for 1 h with 5% nonfat
milk powder in TBST. The filters were then incubated for 1 h at
room temperature or overnight at 4°C with anti-caspase-8 mAb
(clone 5F7; Upstate Biotechnology, Lake Placid, NY), anti-FADD mAb
(StressGen Biotechnologies), anti-Bid Ab (R&D), anti-cleaved
caspase-9 Ab (New England Biolabs, Beverly, MA), anti-caspase-3 Ab
(polyclonal rabbit anti-Caspase-3 antiserum; BD PharMingen, San
Diego, CA) or anti-actin Ab (Sigma, St. Louis, MO). Blots were
washed three times for 10 min each in TBS, 0.1% Tween 20 and were then
incubated for 1 h with peroxidase-labeled anti-mouse or
anti-rabbit immunoglobulin. Blots were developed using the ECL
detection system (Pierce Chemical).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BCR-mediated apoptosis is observed in various types of human B
cells, including Burkitt’s lymphoma cells such as Ramos cells, which
are sensitive to apoptotic stimuli provided by soluble anti-µ Ab
(1, 6, 23, 24). We screened various Burkitt’s cell lines
and found that BL41 cells were insensitive to soluble anti-µ
Ab-induced apoptosis (Fig. 1
). Because we had previously shown that BCR-mediated apoptosis in
Ramos-EBV-positive cells (Ramos AW) depends on the degree of BCR
cross-linking (23), we investigated whether the
cross-linking of the murine monoclonal anti-µ Ab (DA44; 2.5
µg/ml) with F(ab')2 goat anti-murine Ig Ab (CL) (20 µg/ml)
promoted apoptosis in BL41 cells. Such cross-linking of DA44 Ab (Fig. 1A, DA44 plus CL) did indeed induce an apoptotic response in
BL41 cells which was quantified by assessing cell shrinkage (Fig. 1A), as well as annexin V staining, chromatin condensation,
and DNA fragmentation (data not shown). In parallel experiments, we
verified that the increase in the percentage of apoptotic cells in DA44
plus CL-stimulated BL41 cells was associated with a greater total
number of apoptotic cells in these cultures than in control cultures of
nonstimulated BL41 cells (data not shown). This apoptosis was
associated with a loss of m, as quantified by staining with DiOC6
(Fig. 1A). In contrast, as previously reported, apoptosis in
Ramos cells was maximal in the presence of soluble DA44 Ab, and
supplementary cross-linking of DA44 Ab did not significantly modify the
level of apoptosis and loss of m observed in these cells (Fig. 1B) (6, 23).
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m in the presence and absence of the broad spectrum
caspase inhibitor zVAD-fmk. Both the cell shrinkage and decrease in
m (DIOC6) induced by DA44 plus CL in BL41 cells were
significantly reduced by the presence of 100 µM zVAD-fmk (Fig. 2A). In contrast, Ramos cell shrinkage mediated by soluble
DA44 Ab was sensitive to zVAD-fmk, whereas loss of m was not
(Fig. 2B), consistent with previous demonstrations that
soluble anti-µ Ab-induced apoptosis in B cells is associated with
caspase-independent activation of the mitochondria (5, 6).
These data suggest that, depending on the degree to which it is
cross-linked, BCR can promote caspase-independent and caspase-dependent
mitochondrial activation in Ramos and BL41 cells, respectively.
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We next studied the nature of the caspases responsible for the
loss of m observed in BL41. Caspase-8 has been reported to
regulate mitochondrial activation in various models (10, 17). We investigated whether various specific inhibitors
(IETD-fmk and AEVD-fmk) of caspase-8 could modulate the
caspase-dependent loss of m induced by DA44 plus CL in BL41 cells
(Fig. 3A). Indeed, both IETD-fmk and AEVD-fmk (75 µM) were able to
inhibit the DA44 plus CL-mediated loss of m. The effects of these
inhibitors were specific because: 1) the carrier, DMSO, used at the
same concentration, had no effect; and 2) the loss of m induced
by soluble DA44 in Ramos cells was not prevented by IETD-fmk and
AEVD-fmk (data not shown). We further studied the involvement of
caspases sensitive to IETD-fmk and AEVD-fmk by checking by ELISA that
both the AEVD-pNA and IETD-pNA substrates were cleaved in vitro by
lysates from DA44 plus CL-treated BL41 cells (Fig. 3B).
Caspase activity reached a plateau at 36 h, which extended beyond
48 h of activation. The direct involvement of caspase-8 was
demonstrated by the detection on Western blots of active cleaved p20
fragments in DA44 plus CL-stimulated BL41 cells (Fig. 3C). Caspase-8 activation was also assessed in intact
cells by following the cleavage of its natural substrate, Bid. Western
blot analysis showed that DA44 plus CL treatment of BL41 cells led to
the cleavage of Bid, as shown by the disappearance of the p26 uncleaved
form of Bid and production of the p18 fragment (Fig. 3D).
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m
We next wonder whether both caspase-dependent and -independent
pathways of mitochondria could be observed in the same cell. For this,
we screened various subclones of Ramos cells for their capacity to
respond to both soluble and cross-linked DA44 Ab. This was the case for
one of these subclones of Ramos cells, Ramos-NS7. In these cells,
soluble anti-µ Ab (DA44) promoted apoptosis, the level of which
was higher in the presence of cross-linker Abs (DA44 plus CL) (Fig. 4). Cell shrinkage induced by either DA44 Ab or the combination of DA44
Ab plus CL Ab was sensitive to inhibition by zVAD-fmk in all cases. The
loss of m induced by soluble DA44 Ab (Fig. 4A, DA44)
was caspase independent because, as in Ramos cells, it was not
prevented by zVAD-fmk. In contrast, more cells displayed m loss
if cross-linker Ab was added to cultures together with DA44 Ab (Fig. 4A, DA44 plus CL). In the presence of zVAD-fmk, the
proportion of cells exhibiting loss of m was comparable with that
observed in the presence of DA44 Ab alone. This suggested that in
Ramos-NS7 cells, BCR-mediated mitochondrial activation could be
achieved via either caspase independent (DA44) or caspase dependent
(DA44 plus CL) pathways. As in BL41 cells, this caspase-dependent DA44
plus CL-mediated loss of m was associated with caspase-8
activation, assessed by evaluating levels of p20 active fragment of
caspase-8 and cleavage of the caspase-8-specific substrate Bid in
intact cells (Fig. 4, B and C).
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It has been reported previously that CD40 can prevent soluble
anti-µ-induced apoptosis in Burkitt’s cells (1, 24). We next therefore investigated whether a similar regulation
is observed for DA44 plus CL-mediated apoptosis. As shown in Fig. 5, anti-CD40 Ab (G28.5 mAb, 5 µg/ml) was not able to prevent
anti-µ-mediated apoptosis in BL41 cells activated with DA44 plus
CL. In contrast, anti-µ-mediated apoptosis of Ramos cells
activated with soluble DA44 Ab was decreased in the presence of
anti-CD40 Ab. These results strengthen the hypothesis that the BCR
can trigger two different apoptotic pathways.
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We next determined the sequence of events among caspase-8
activation, loss of m, and activation of caspase-9 and caspase-3
in BL41 cells (Fig. 6). Indeed, it is clearly established that caspase-3 can be activated by
caspase-9. This pathway is dependent on the autocleavage of caspase-9
in the apoptosome complex in the presence of mitochondria-derived
cytochrome c and ATP, the release of which is controlled by
the cleaved form of Bid. We therefore tested whether caspase-8
inactivation by specific inhibitors prevented caspase-9 activation. For
this, we first investigated whether AEVD-fmk, which inhibited caspase-8
activation, Bid cleavage, and the loss of m induced by DA44 plus
CL in BL41 cells (Fig. 6, A–C), also modulated the
activation of caspase-9 and caspase-3. Caspase-9 activation was
estimated by monitoring the disappearance of the p47 proform and
production of the cleaved fragment p37 (Fig. 6D) and
caspase-3 activation by monitoring levels of the active fragments p17
and p19 (Fig. 6E) and cleavage of the substrate PhiPhilux
G2D2 in intact cells (data
not shown). Morphological changes (shrinkage) and the activation
of caspase-9 and caspase-3 were prevented by AEVD-fmk (Fig. 6, D and E). These observations are consistent with
a pathway of caspase-3 activation, in DA44 plus CL-stimulated BL41
cells, involving the upstream activation of caspase-8 followed by Bid
cleavage, loss of m, and then the activation of caspase-9 and
caspase-3.
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Various studies have demonstrated that caspase-8 activation
is dependent on the oligomerization of this enzyme via its association
with the adaptor protein FADD, involving the DED of the two molecules.
This requires the interaction of FADD either directly or indirectly
with surface receptors, such as Fas, that possess death domains
(13, 25). As activated B cells (including various
Burkitt’s cell lines) are highly sensitive to Fas-mediated apoptosis
(3, 26, 27, 28, 29), we investigated the possible involvement of
Fas in DA44 plus CL-mediated apoptosis in BL41 cells (Fig. 7). Under our experimental conditions, BL41 cells were insensitive to
Fas-mediated apoptosis, as assessed using the agonist Ab ligand CH11 Ab
(0.1 µg/ml) in the presence or absence of DA44 Ab and CL Ab (Fig. 7A). As a control, we verified that the same concentration
of CH11 Ab induced a strong apoptotic response in Jurkat cells (Fig. 7B). Furthermore, CH11 Ab, at a final concentration of up to
10 µg/ml, did not induce apoptosis in BL41 cells (data not shown). In
addition, the Fas antagonist ZB4 Ab, which inhibited CH11-mediated
Jurkat apoptosis, did not affect the extent of DA44 plus CL-apoptosis
in BL41 cells. Thus, caspase-8 activation triggered by DA44 plus CL is
independent of the Fas pathway. To determine the precise pathway
leading to caspase-8 activation on BCR stimulation, we next
investigated the requirement for FADD in caspase-8 activation in DA44
plus CL-stimulated BL41 cells. For this, we established stably
transfected clones of BL41 cells expressing a dominant negative mutant
of FADD lacking the DED domain, which were therefore unable to
associate with caspase-8 (30). We selected various clones
of BL41, with either endogenous FADD only (Fig. 8A, clone 1) or with various amounts of the truncated FADD,
which was detected as a band that migrated faster than the wild-type
band (Fig. 8A, clones 9 and 23). We found that DA44 plus CL
stimulation induced apoptosis (as assessed by cell shrinkage and
decreases in DiOC6 staining) in all clones. We also observed that
BCR-induced caspase-8 cleavage into the active fragment p20 occurred to
a similar extent in cells producing or not producing FADD-DN (Fig. 8B). We also verified that transfection with FADD-DN was
able to prevent death receptor-mediated apoptosis. Indeed, BL41 cells
are sensitive to the apoptotic signal provided by TRAIL (Fig. 8C): whereas recombinant TRAIL induced 55% cell shrinkage
in clone 1, and 13 and 8% cell shrinkage was observed in the two
clones (9 and 23) expressing the truncated form of FADD. Thus,
altogether our data show that caspase-8 activation observed in BL41
cells on BCR cross-linking is independent of the presence of the DED
domain of FADD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m and subsequent
activation of caspase-9 and caspase-3. One of these pathways, detected
in the Burkitt’s cell line Ramos activated with soluble anti-µ
Ab DA44, leads to caspase-independent mitochondrial activation. The
other, detected in the Burkitt cell line BL41 stimulated with
cross-linked anti-µ Ab, is caspase dependent and associated with
caspase-8 activation. This apoptotic response of BL41 cells, assessed
in terms of cell shrinkage and loss of m, was also observed with
anti-µ Ab cross-linked to Sepharose beads (data not shown). We
have previously reported that the apoptotic response mediated by
surface Ig or BCR-associated molecules such as CD19 and CD22 varies
with the degree of cross-linking (23). Indeed, although
soluble anti-µ Ab promotes apoptosis in Ramos cells, only
cross-linked anti-µ Ab induces apoptosis in the EBV-positive
Ramos AW cells. In contrast, anti-CD19 and anti-CD22 Ab are
able to mediate apoptosis in Ramos, Ramos AW, or resting tonsillar B
cells only if these Ab are cross-linked with a secondary Ab. Our data
show that the requirement for cross-linking for anti-µ
Ab-mediated apoptosis in Burkitt’s cells is not restricted to
EBV-infected cells; we observed the same pattern with the BL41 cell
line, which is EBV negative. We selected a subclone of Ramos,
Ramos-NS7, that displayed both Ramos and BL41 patterns. Indeed, soluble
DA44 Ab directly triggers apoptosis and loss of m in Ramos-NS7
cells. Both cell shrinkage and loss of m were up-regulated if
anti-µ Ab were cross-linked by either adding a secondary Ab (Fig. 4
) or using anti-µ Ab-coated Sepharose beads (data not
shown). The soluble DA44-mediated loss of m in Ramos-NS7 cells
did not decrease in the presence of the broad spectrum caspase
inhibitor zVAD-fmk. This suggests that, as in Ramos cells,
DA44-mediated mitochondrial activation in Ramos-NS7 cells is caspase
independent. However, increase in m loss, observed in the
presence of DA44 cross-linking, was prevented by zVAD-fmk, suggesting
that cross-linking of the DA44 Ab promotes a supplementary pathway of
mitochondrial activation in Ramos-NS7 cells that is caspase dependent.
The involvement of these two pathways in Ramos-NS7 cells can also be
monitored by following caspase-8 activation. Thus, our data show that
BCR can trigger two different pathways leading to mitochondrial
activation and apoptosis. Further evidence for the existence of these
two different BCR-mediated apoptotic pathways is provided by the
observation that DA44 plus CL-mediated apoptosis in BL41 cells was not
prevented by CD40 costimulation, although, as previously described,
soluble anti-µ Ab-mediated apoptosis was sensitive to the
protective effect of CD40 (1, 24). This suggests that
these two pathways are probably regulated differently and raises
questions concerning the biological role and pertinence of these two
pathways in vivo. A down-regulation of the immune response at
supraoptimal doses of stimuli has also been proposed by Newell et al.
(31), who described the death-promoting effect of
anti-MHC class II Ab on murine splenic B cells. Such a mechanism
may account for the results of previous studies, reporting that
exposure to "too much Ag" leads to hyporesponsiveness. Thus, the
suicide response of tonsillar B cells may involve the binding of a
threshold amount of Ag, with a given valency potential and affinity for
its clonotypic receptor. For many receptors, dimers are sufficient to induce a signal. However, depending on the cell type, for these same receptor systems, dimers may be much less effective than trimers or higher oligomers at inducing a signal (32). Fas-mediated apoptosis and caspase-8 activation are also dependent on multimerization of the surface Fas molecule (33, 34). In our Burkitt’s lymphoma model, dimerization of the BCR with soluble anti-µ Ab is sufficient to induce apoptosis in Ramos cells, whereas extensive cross-linking of anti-µ Ab is necessary to induce the death of BL41 cells. In BL41 cells, artificial cross-linking of the Ag receptor may induce the formation of clustered lipid rafts, which may recruit cross-linking proteins that bind to the proteins of other rafts or oligomerize individual BCR with weak affinity leading to an increase in the time of residence in rafts (35, 36). The formation of clustered rafts is likely to lead to amplification via the concentration of signaling molecules or the initiation of signal transduction via an associated protein coreceptor activated during raft clustering.
The two pathways merge at the mitochondrial level, leading to the loss
of m and the subsequent activation of caspase-3 via the
apoptosome complex. Indeed, caspase-3 activation observed during
DA44-mediated (Ramos cells) or DA44 plus CL-mediated (BL41) apoptosis
was associated with caspase-9 activation, which for its autocleavage
must be complexed with the cytoplasmic protein Apaf1 and
mitochondria-derived cytochrome c and ATP (19, 37, 38). Although we cannot exclude the possibility that caspase-3
activation may be partly due to the direct effect of caspase-8 on the
proform of caspase-3, our data suggest that most of the caspase-3
activation observed with DA44 plus CL stimulation is dependent on
mitochondrial activation. Indeed, the inhibition of caspase-8
activation by AEVD-fmk or IETD-fmk in BL41 and NS7 cells prevents the
activation of both caspase-9 and caspase-3 in BL41 cells, suggesting
that there is a direct link between these caspases, as reported in many
other systems.
Strikingly, we found that the caspase-dependent mitochondrial
activation observed after cell treatment with cross-linked anti-µ
Ab was associated with and dependent on caspase-8 activation.
This involvement of caspase-8 was demonstrated by the detection
of the active cleaved fragment p20 and the ability of cell lysates from
DA44 plus CL-activated BL41 cells to cleave in vitro colorimetric
substrates specific for caspase-8 (IETD-pNA and AEVD-pNA). Caspase-8
activation was correlated with Bid cleavage, which directly controls
the opening of the permeability transition pore and the release of
cytochrome c into the cytoplasm, in turn controlling the
activation of caspase-9 (39). All these events were
inhibited by AEVD-fmk and IETD-fmk, strongly suggesting that DA44 plus
CL-induced mitochondrial depolarization was mediated by Bid, after its
cleavage by caspase-8. It has been suggested that caspase-10, which is
very similar to caspase-8, is recruited with caspase-8 into
apoptosis-signaling complexes associated with the death receptor and
that caspase-10 plays a functional role in death receptor-mediated
apoptosis (40, 41). However, cleavage of this caspase was
not demonstrated due to a lack of reliable Abs. AEVD-fmk and IETD-fmk
both inhibit caspase-10, although to a lesser extent than caspase-8
(42, 43). Thus, the involvement of caspase-10 in DA44 plus
CL-activated pathways cannot be ruled out. Our data are consistent with
the notion that, in BL41 cells, the DA44 plus CL-induced apoptotic
response is based on the sequential activation of caspase-8, cleavage
of Bid, and loss of m, followed by the activation of caspase-9
and caspase-3.
Caspase-8 activation is mostly associated with apoptosis mediated by
members of the TNFR family, which possess a death domain in their
cytoplasmic region (9). On ligand binding, these receptors
may directly or indirectly recruit the FADD adaptor protein. Caspase-8
activation results in autocleavage of the oligomerized proforms after
association with FADD via interactions between the DEDs of the two
molecules (10, 11). Because B cells are highly sensitive
to Fas activation and, following specific activation such as
anti-class II MHC stimulation, B cells can express Fas-L surface
molecules (3, 26, 27, 28, 29), we investigated the role of
Fas/FasL interactions in our experimental conditions. We found that the
DA44 plus CL-induced activation of caspase-8: 1) did not induce the
up-regulation of FasL (data not shown); and 2) did not decrease in the
presence of the blocking antagonist anti-Fas Ab ZB4. This strongly
suggests that DA44 plus CL-mediated apoptosis in independent of Fas. To
determine the molecular basis of the DA44 plus CL-induced caspase-8
activation, we therefore investigated more directly the role of FADD,
using a dominant negative FADD mutant lacking the DED domain
(30). Various clones producing only the endogenous FADD
molecule or various amounts of the DED-truncated FADD-DN displayed
similar patterns of apoptotic response and caspase-8 cleavage after
DA44 plus CL treatment. This suggests that DA44 plus CL-mediated
caspase-8 activation and apoptosis are independent of FADD. However, it
is not yet clear whether BCR-induced caspase-8 activation is completely
independent of FADD or whether it could be mediated by FADD molecules
devoid of DED domains. Various studies have recently suggested that
caspase-8 could be activated independently of its association with the
FADD molecule. Anticancer drugs can induce apoptosis and caspase-8
cleavage in a FADD-independent manner (15), and Ding et
al. (44) have reported that in a cell-free system,
p53-triggered caspase-8 activation takes place in a 600-kDa complex in
the absence of detectable FADD. In Burkitt’s cells, TGF--mediated
apoptosis has also been reported to be associated with caspase-8
activation independent of death domain receptors (14), and
we also found that this caspase-8 activation was also
FADD-independent.5
The nature of the adaptor molecules mediating caspase-8
oligomerization and cleavage is unknown. Nevertheless, our data are
consistent with the emerging hypothesis that caspase-8 activation is
not restricted to death receptors.
In conclusion, we show that depending on the extent to which it is cross-linked, BCR can mediate either caspase-dependent or caspase-independent mitochondrial activation leading to apoptosis. Moreover, cross-linked anti-µ Ab can also promote a novel pathway for caspase-8 activation in BL41 cells: this pathway is independent of the DED of FADD.
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Footnotes |
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2 L.B. and N.S. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. Aimé Vazquez at the current address: Institut National de la Santé et de la Recherche Médicale Unité 542, Bâtiment Lavoisier, Hôpital Paul Brousse, 14 Avenue Paul Vaillant Couturier, 94807 Villejuif cedex, France. E-mail address: vazquez{at}infobiogen.fr
4 Abbreviations used in this paper: FADD, Fas-associated death domain protein; DED, death effector domain; CL, cross-linker Ab; zVAD-fmk, Z-Val-Ala-DL-Asp-fluoromethyl ketone; zIETD-fmk, Z-Ile-Glu-Thr-Asp-fluoromethyl ketone; zAEVD-fmk, Z-Ala-Glu-Val-Asp-fluoromethyl ketone; m, mitochondrial transmembrane potential; DiOC6, 3,3'-dihexylocarbocyanin iodide; BCR, B cell receptor; TRAIL, TNF-related apoptosis-inducing ligand; pNA, p-nitroanaline.
5 N. Schrantz, M. F. Bourgeade, S. Mouhamad, G. Leca, S. Sharma, and A. Vazquez. p38-mediated regulation of a FADD-independent pathway leading to caspase-8 activation during TGF-induced apoptosis in the human Burkitt lymphoma B cells BL41. Submitted for publication.
Received for publication January 31, 2001. Accepted for publication May 8, 2001.
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1 in Burkitt’s lymphoma cells is caspase 8 dependent but is death receptor independent. J. Immunol. 165:2500.-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J. Biol. Chem. 272:6578.This article has been cited by other articles:
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