From the Division of Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
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ABSTRACT |
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The death receptor CD95 (APO-1/Fas), the
anticancer drug etoposide, and Cells can undergo apoptosis in response to a broad spectrum of
stimuli, including receptor stimulation, treatment with cytotoxic drugs, and Certain members of the tumor necrosis factor receptor family, including
CD95, directly couple to the caspase cascade via their cytoplasmic
death domain. Upon multimerization by its trimeric ligand, CD95
recruits the FADD adaptor via its death domain (3). Via its death
effector domain (DED),1 FADD,
in turn, recruits caspase-8 (FLICE), which contains two related DEDs in
its amino terminus (4, 5). Recently, several viral proteins, termed
v-FLIP (FLICE inhibitory protein),
were identified that contain two regions of homology to the DEDs of FADD and caspase-8 (6-8). Mammalian cells express FLIP homologues: FLIPS, composed of two DEDs, and FLIPL, which
contains, in addition, a nonfunctional caspase domain (9-11). Both
viral and cellular FLIP proteins bind to FADD and/or caspase-8 and
inhibit death receptor-induced apoptosis, most likely by displacing
DED-containing caspases (caspase-8 and/or -10) from the activated death
receptor complex (8-10).
Whereas it is to some extent understood how death receptors link to the
caspase family, this is less clear for other apoptotic stimuli. In
certain cases, the stimulus indirectly activates death receptors. An
example is the T cell antigen receptor, which induces synthesis of CD95
ligand and therewith activates CD95 in, for example, Jurkat T cells
(12). A similar mechanism has been proposed for apoptosis induction by
anticancer drugs (13, 14). Apart from the death-inducing signaling
complex (DISC) at the death receptor cytoplasmic tail (4, 15, 16), the
mitochondrial membrane is now thought to be a site for initial caspase
activation. In response to various apoptotic stimuli, mitochondria
release cytochrome c (17-19), which, together with Apaf-1
(20) and caspase-9 (21), can activate caspase-3 in vitro.
The Apaf-1·caspase complex is thought to be located at the
mitochondrial membrane since the homologous Caenorhabditis
elegans proteins CED-4 and CED-3 form a complex together with
CED-9, a homologue of mammalian Bcl-2 (22, 23) that resides at this
site (24). Mitochondrial cytochrome c release can be blocked
by the apoptosis inhibitory proteins Bcl-2 and Bcl-xL by an unknown
mechanism (17, 18).
Many apoptosis pathways, including those induced by etoposide and
Several studies suggest that the lipid ceramide is relevant for
apoptosis signaling in response to death receptors, Jurkat cells selected for resistance to CD95-induced apoptosis were
found to be cross-resistant to etoposide and Reagents--
L-[3-14C]Serine (54.0 mCi/mmol) and the enhanced chemiluminescence (ECL) kit were purchased
from Amersham Pharmacia Biotech; etoposide was from Sigma; and
C2-ceramide was from BIOMOL Research Labs Inc. The
anti-CD95 monoclonal antibodies (mAbs) CH-11 and 7C11 were purchased
from Immunotech (Marseille, France). Anti-caspase-8 serum was raised in
rabbits against a synthetic peptide comprising amino acids 2-20 of
human caspase-8. Specificity of the antiserum was confirmed as
described previously (32, 43). Mouse anti-human caspase-3 mAb was
purchased from Transduction Laboratories (Lexington, KY); mouse
anti-human caspase-6 (B93-4) and caspase-7 (B94-1) mAbs and
anti-cytochrome c mAb 7H8.2C12 were from Pharmingen; and
anti-actin mAb C4 was from Roche Molecular Biochemicals. Horseradish peroxidase-conjugated rabbit anti-mouse Ig and swine anti-rabbit Ig
were obtained from Dako A/S (Glostrup, Denmark).
Cells--
The J16 wild-type clone was derived from the human
T-acute lymphoblastic leukemia cell line Jurkat by limiting dilution
and selected for CD95 sensitivity (32, 42). CD95-resistant JA variant
clones were derived by limiting dilution from the Jurkat line, cultured
for 5 weeks in the presence of 1 µg/ml anti-APO-1 mAb (44).
Clonogenic assays indicated that ~2 in 104 cells of the
parental line survive this treatment. JA clones were subcloned in
medium without selecting stimulus and have remained resistant
throughout prolonged culture periods. The expression level of CD95 is
within the wild-type range, and its cytoplasmic tail is wild-type
according to nucleotide
sequencing.2 Jurkat cells
stably transfected with the bcl-2 cDNA and empty vector-transfected control cells were previously described (45). The
Jurkat cell line JFL2, which is stably transfected with
FLIPL cDNA (9), was kindly provided by Dr.
J. Tschopp (Institute of Biochemistry, University of Lausanne,
Epalinges, Switzerland).
Cell Culture and Stimulation--
Cells were cultured in
Iscove's modified Dulbecco's medium supplemented with 10% fetal calf
serum, 2 mM glutamine, and antibiotics at 37 °C and 5%
CO2. bcl-2 cDNA-transfected and empty
vector-transfected Jurkat cells received additional neomycin sulfate
(G418) at 200 µg/ml. Prior to stimulation, cells were incubated
overnight in serum-free Yssel's medium (46) and seeded in the same
medium at 1 × 106/ml (200 µl/well) in round-bottom
96-well plates for apoptosis assays and at 5-10 × 106/ml in 24-well culture plates for caspase processing and
cytochrome c release assays. Cells were irradiated using a
137Cs source (2 × 415 Ci; Von Gahlen Nederland,
B. V.) with the indicated doses, stimulated with anti-CD95 mAb or
etoposide at the indicated concentrations, and incubated at 37 °C
and 5% CO2 for various time periods.
Apoptosis Assay--
For apoptosis measurements, cells were
lysed in 0.1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml
propidium iodide (47). Fluorescence intensity of propidium
iodide-stained DNA was determined on a FACScan (Becton Dickinson, San
Jose, CA), and data were analyzed using Lysys software. Fragmented
apoptotic nuclei are recognized by their subdiploid DNA content.
Immunoblot Analysis for Caspase Processing--
Cells were lysed
in 1% Nonidet P-40 containing lysis buffer (0.01 M
triethanolamine HCl (pH 7.8), 0.15 M NaCl, 5 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml
trypsin inhibitor, and 0.02 mg/ml leupeptin). Lysates were centrifuged
at 13,000 × g for 15 min, and supernatants were mixed
with concentrated reducing SDS sample buffer. Equivalents of
106 cells/lane were separated on 10% SDS-polyacrylamide
minigels. Proteins were transferred to nitrocellulose membranes
(Schleicher & Schüll, Dassel, Germany), which were subsequently
blocked with 5% nonfat dry milk in Tris-buffered saline and 0.05%
Tween 20 (TBST). Blots were incubated with anti-caspase-8 serum or
anti-caspase-6 mAb at a 1:500 dilution or with anti-caspase-3 or
anti-caspase-7 mAb at a 1:1000 dilution in TBST with 1% nonfat dry
milk. After subsequent incubation with a 1:7500 dilution of horseradish
peroxidase-conjugated swine anti-rabbit Ig, proteins were visualized by ECL.
Immunoblot Analysis for Cytochrome c--
After incubation,
cells were washed twice with ice-cold phosphate-buffered saline,
resuspended in 100 µl of extraction buffer (50 mM
PIPES-KOH (pH 7.4), 220 mM mannitol, 68 mM
sucrose, 50 mM KCl, 5 mM EGTA, 2 mM
MgCl2, 1 mM dithiothreitol, and protease inhibitors), and allowed to swell on ice for 30 min (19). Cells were
homogenized by passing the suspension through a 25-gauge needle (10 strokes). Homogenates were centrifuged in a Beckman Airfuge at
100,000 × g for 15 min at 4 °C, and supernatants
were harvested and stored at Ceramide Quantification--
Ceramide levels were determined as
described previously (42, 43). Briefly, cells (1 × 106/ml) were labeled with
L-[3-14C]serine (0.2 µCi/ml) in Yssel's
medium for 20-48 h, washed with Yssel's medium, and resuspended at
5 × 106-ml in 24-well plates. Following stimulation,
total lipids were extracted with chloroform/methanol (1:2, v/v), and
phases were separated using 20 mM HAc. Extracts were
spotted on Silica Gel 60 thin-layer chromatography plates (Merck).
After chromatography, radioactive lipids were visualized and
quantitated using a Fujix BAS 2000 TR PhosphorImager and identified
using external lipid standards. Ceramide was expressed relative to
total radioactivity in phosphatidylserine and phosphatidylethanolamine,
which remained unaltered upon stimulation.
A Common Aspect in Apoptosis Signaling Induced by CD95, Etoposide,
and Apoptosis Signaling Induced by Etoposide and
To determine whether caspase-8 is instrumental in apoptosis induction
by etoposide and IR, we employed Jurkat cells stably transfected with
FLIPL cDNA. It was previously demonstrated
that apoptosis induction by CD95 and TRAIL receptors is inhibited in these transfectants (9). Fig. 2 shows that in these JFL2 cells, the
dramatic caspase-8 processing induced by CD95 was blocked. Also, the
minor caspase-8 processing induced by etoposide and IR was no longer
observed. Whereas FLIPL inhibited CD95-induced apoptosis,
it did not affect the apoptotic response to etoposide and IR (Fig.
3). Apparently, activation of
FLIPL-inhibitable, DED-containing caspases is required for
apoptosis induction by CD95, but not for apoptosis induction
by etoposide or IR.
Apoptosis Signaling Pathways Induced by CD95, Etoposide, and
Cytochrome c release in response to all three stimuli was
severely reduced in the resistant JA1.2 clone (Fig. 4), suggesting that
the apoptosis signaling pathways induced by CD95, etoposide, and IR all
depend on a contribution by the mitochondria. Generally, a possible
contribution of the mitochondrial pathway to apoptosis induction is
assessed by the effects of Bcl-2 family members on the apoptotic
response. Whereas apoptosis induction by DNA-damaging regimens such
as etoposide treatment and IR is consistently modulated by Bcl-2 family
members (25, 26), it has been unclear whether death receptor signaling
is similarly regulated. For instance, in transgenic thymocytes,
CD95-mediated apoptosis is not affected by Bcl-2 or Bax overexpression
(26, 48), whereas in certain cell lines, Bcl-2 and Bcl-xL were found to
inhibit tumor necrosis factor receptor-1- and/or CD95-induced apoptosis
(45, 49, 50). Recently, it was suggested that in certain cells (type I), CD95 employs a Bcl-2-independent pathway, whereas in other cells
(type II), including Jurkat, Bcl-2 and Bcl-xL inhibit CD95 signaling
(51). Using the previously described Bcl-2-overexpressing Jurkat cells
(45, 50), we also found that not only etoposide- and IR-induced
cytochrome c release and apoptosis were inhibited, but also
CD95-induced cytochrome c release (Fig. 4) and apoptosis (Fig. 5). The combined data suggest a
point of convergence in apoptosis signaling induced by CD95, etoposide,
and IR at the mitochondria.
Surprisingly, caspase-8 processing induced by CD95 was inhibited by
Bcl-2 overexpression (Fig. 2). This has also been observed by Scaffidi
et al. (50), who suggested that CD95 signaling in Jurkat
cells involves rapid, low level caspase-8 activation at the DISC,
followed by a slow, more pronounced response, which is under
mitochondrial control. Rapid caspase-8 activation within minutes could
not be observed in total lysates of Jurkat cells (data not shown) (50).
Since FLIPL inhibits caspase-8 activation at the DISC (9),
we cannot assess whether the late, Bcl-2-controlled caspase-8 response
contributes to apoptosis induction by CD95. Bcl-2 also
inhibited caspase-8 processing as induced by etoposide and IR (Fig.
2).
Regulation of Effector Caspase Processing in Response to CD95,
Etoposide, and Ceramide and Apoptosis Signaling Induced by CD95, Etoposide, and
Exogenous C2-ceramide induced effector caspase processing
(Fig. 7B) and apoptosis (Fig. 7C) in wild-type
Jurkat cells as well as in the JA1.2 clone. More important, whereas
C2-ceramide bypassed apoptosis resistance in JA1.2, the
Bcl-2 transfectant was resistant to C2-ceramide (Fig. 7,
B and C), indicating that apoptosis resistance of
JA1.2 is not due to overexpression of Bcl-2 (-related) proteins.
Our finding that cells selected for resistance to CD95-induced
apoptosis were cross-resistant to apoptosis induction by etoposide and
IR suggested that apoptosis signaling induced by these three stimuli
has a common aspect. Since CD95 signaling involves FADD-mediated recruitment of caspase-8 and its subsequent proteolytic activation, we
examined whether etoposide and IR also induced caspase-8 processing. Caspase-8 was cleaved to a minor extent in etoposide-treated and irradiated cells, but this proved to be dispensable for effector caspase processing and the cell death response. This is an important finding since it was suggested previously that anticancer drugs (13,
14) and Such a mechanism would obviously account for the observed
cross-resistance. However, involvement of the CD95 receptor-ligand system in anticancer drug- and Caspase-8 cleavage induced by etoposide and Our study suggests that, in Jurkat cells, apoptosis signaling induced
by etoposide and Concomitant with its failure to display cytochrome c
release, effector caspase processing, and apoptosis in response to
CD95, etoposide, and IR, the JA1.2 clone (and other clones from the same resistant culture) fails to mount a ceramide response. Whether ceramide participates in the apoptosis signaling pathway is unknown, but this possibility is suggested by correlations between lack of a
ceramide response and a failure to undergo apoptosis (34, 37, 40, 41)
as well as by the finding that exogenous ceramide can activate caspases
and induce apoptosis. We have recently found that ceramide production
in response to CD95, etoposide, and IR occurs in the absence of
effector caspase activation, but depends on inducer caspase
activity.3 The failure to
mount a ceramide response in the resistant JA clones is therefore not a
consequence of defective effector caspase activation, but rather
reflects a defect at or downstream from inducer caspase activation.
Apoptosis resistance in the JA clones is most likely not due to
overexpression of inhibitory Bcl-2 family members since the variant
cells were sensitive to apoptosis induction by exogenous ceramide,
whereas Bcl-2-overexpressing cells were resistant (Fig. 7). Moreover,
immunoblotting for various Bcl-2 family members did not reveal altered
expression in JA cells as compared with wild-type Jurkat cells (data
not shown). It is also unlikely that the inhibitor of
apoptosis proteins (IAPs) mediate the
resistance, since these do not interfere with the release of cytochrome
c (58). Therefore, it will be of great interest to define
the cause of common apoptosis resistance in these variant clones.
-radiation induce apoptosis in the
human T cell line Jurkat. Variant clones selected for resistance to
CD95-induced apoptosis proved cross-resistant to etoposide- and
radiation-induced apoptosis, suggesting that the apoptosis pathways
induced by these distinct stimuli have critical component(s) in common.
The pathways do not converge at the level of CD95 ligation or caspase-8
signaling. Whereas caspase-8 function was required for CD95-mediated
cytochrome c release, effector caspase activation, and
apoptosis, these responses were unaffected in etoposide-treated and
irradiated cells when caspase-8 was inhibited by FLIPL.
Both effector caspase processing and cytochrome c release
were inhibited in the resistant variant cells as well as in Bcl-2
transfectants, suggesting that, in Jurkat cells, the apoptosis
signaling pathways activated by CD95, etoposide, and
-radiation are
under common mitochondrial control. All three stimuli induced ceramide
production in wild-type cells, but not in resistant variant cells.
Exogenous ceramide bypassed apoptosis resistance in the variant cells,
but not in Bcl-2-transfected cells, suggesting that apoptosis signaling
induced by CD95, etoposide, and
-radiation is subject to common
regulation at a level different from that targeted by Bcl-2.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-radiation. Although a given stimulus may activate unique
signaling molecules, the current model states that the molecular events
in the execution phase of apoptotic cell death are shared. Members of
the caspase family, which are aspartate-specific cysteine proteases,
are key initiators of this execution phase (1, 2). Their proteolytic
action on specific cellular components, including other caspases,
structural proteins, and enzymes, leads to the ordered degradation of
the cell into apoptotic bodies.
-radiation, are controlled by the Bcl-2 family, suggesting that, in
these cases, mitochondrial participation is essential for the cell
death response (25, 26). Etoposide inhibits topoisomerase II and
therewith induces double strand DNA breaks (27), whereas
-radiation
also induces DNA damage. Both stimuli activate caspases (28-30), but
it is unknown how the signal is transmitted to these enzymes. In
certain cell types, DNA damage induces p53-directed de novo
synthesis of the Bcl-2 antagonist Bax (31), indicating a mechanism for
regulation of the mitochondrial caspase pathway. It has also been shown
that anticancer drugs can induce expression of CD95 and/or its ligand
and therewith activate the CD95 pathway (13, 14). However, this is not
a general mechanism since etoposide-induced apoptosis is
CD95-independent in murine thymocytes and Jurkat cells (32).
-radiation, and
anticancer drugs. Tumor necrosis factor receptor-1 (33); CD95 (34);
-radiation (35); and daunorubicin, vincristine, and cytosine
arabinoside (36-39) can induce accumulation of ceramide. Moreover,
failure to generate ceramide has been associated with apoptosis
resistance (34, 37, 40, 41). Exogenous short-chain ceramide can induce
apoptosis in certain cell types, but it is not clear whether it is
equivalent in its action to naturally generated ceramide. The ceramide
response induced by CD95 lies upstream from DEVD-inhibitable caspases
and nuclear segmentation (42), allowing for the possibility that
ceramide contributes to generation of the apoptotic phenotype.
-radiation. Therefore,
we have examined possible convergence of the apoptosis signaling
pathways induced by these three stimuli. We find that the pathways
share features downstream from DED-containing caspases and are subject
to common mitochondrial control. In addition, comparing responses in
Bcl-2 transfectants and the variant clones, we find evidence for a
Bcl-2-independent mechanism that commonly regulates apoptosis signaling
induced by CD95 and the DNA-damaging regimens.
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ABSTRACT
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70 °C until analysis by gel
electrophoresis. Ten µg of cytosolic protein, as determined by the
Bio-Rad protein assay, were loaded onto a 12% SDS-polyacrylamide gel.
Proteins were transferred to nitrocellulose sheets, which were blocked as described above and probed in TBST with anti-cytochrome c
mAb (1:2000) and anti-actin mAb (1:3000) to confirm equal loading. After incubation with a 1:7500 dilution of horseradish
peroxidase-conjugated rabbit anti-mouse Ig, blots were developed by ECL.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Radiation--
The human T-acute lymphoblastic leukemia cell
line Jurkat is sensitive to multiple apoptotic stimuli, including CD95
triggering, exposure to certain anticancer drugs, and
-radiation
(IR). We have selected variant clones from the wild-type Jurkat line
for resistance to CD95-mediated apoptosis. These clones proved
cross-resistant to apoptosis induction by etoposide and IR, as
shown in Fig. 1A. Whereas the
wild-type clone J16 undergoes dose- and time-dependent apoptosis in response to anti-CD95 mAb, etoposide, or IR, apoptosis incidence in the variant clone JA1.2 does not exceed background levels,
even at high dose stimulation. Yet, the resistant JA1.2 variant cells
sense the DNA damage as induced by etoposide and
-radiation since
they arrest in the G2 phase of the cell cycle upon
treatment, as wild-type Jurkat cells do. However, in wild-type cells,
the G2 arrest is followed rapidly by apoptotic cell death, whereas this does not occur in JA1.2 cells (Fig. 1B).
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Fig. 1.
CD95-resistant clone JA1.2 senses DNA damage,
but fails to undergo apoptosis in response to etoposide or IR.
A, apoptosis incidence in the wild-type Jurkat clone J16 and
the representative variant clone JA1.2 as read out by nuclear
fragmentation (47) at 8 or 14 h after addition of anti-CD95 mAb
CH-11 or etoposide or after exposure to IR at the indicated doses. Data
are representative of multiple independent experiments. Means ± S.D. from duplicate samples in one experiment are shown. B,
DNA profiles of nuclei from J16 and JA1.2 cells analyzed 16 h
after addition of etoposide (E; 2.5 µg/ml) or after IR (10 Gy). Fluorescence intensity of propidium iodide (PI)-stained
nuclei (47) indicates DNA content and allowed identification of nuclei
in the G1 and G2 phases of the cell cycle as
well as subdiploid fragmented nuclei derived from apoptotic cells.
Percentage apoptosis is indicated for each sample.
-Radiation Does Not
Require DED-containing Caspases--
The cross-resistance suggested
that CD95 and the DNA-damaging stimuli etoposide and
-radiation
require common molecular events to induce apoptosis. To find a possible
point of convergence in apoptosis signaling induced by these three
inputs, we first tested whether the anticancer treatments induced
caspase-8 processing, as does CD95 (15). Immunoblotting of whole cell
lysates with an antiserum directed against the amino terminus of
caspase-8 allowed detection of two proforms, of ~54 and 50 kDa, prior
to stimulation. CD95 stimulation gave rise to a caspase-8 doublet at
~40 and 36 kDa within ~1 h, which increased in intensity in the
following hours, whereas the procaspase-8 signal decreased concomitantly (Fig. 2). This is
consistent with the release of the carboxyl-terminal caspase fragment
(15). In addition, a 23-kDa species was detected, which most likely
represents the amino-terminal region of caspase-8 after release of the
second caspase subunit. Etoposide and IR also induced caspase-8
processing, but to a minor extent. In these cases, only the 40/36-kDa
digestion products could be detected, even upon long-term stimulation
(Fig. 2). All three stimuli failed to induce caspase-8 processing in the resistant clone JA1.2 (Fig. 2).
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Fig. 2.
Regulation of caspase-8 processing in
response to CD95, etoposide, and IR. Cells were stimulated with
anti-CD95 mAb 7C11 at 200 ng/ml, etoposide at 10 µg/ml, or IR at 30 Gy and harvested after the indicated periods of incubation. J
neo is the control Jurkat clone. JA1.2 is the representative
resistant variant clone. JFL2 denotes Jurkat cells transfected with
FLIPL cDNA as described (9). J
Bcl-2 indicates Jurkat cells transfected with Bcl-2
cDNA (45). Total cell lysates were separated by SDS-polyacrylamide
gel electrophoresis, and caspase-8 processing was monitored by
immunoblotting with an antiserum directed at amino acids 2-20 of human
procaspase-8 (pro-casp-8) (43). The procaspase-8 forms at 54 and 50 kDa are indicated by bars. The caspase-8 digestion
products at ~40/36 and 23 kDa are indicated by
arrowheads.
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Fig. 3.
FLIPL inhibits apoptosis
induction by CD95, but not by etoposide and IR. Control Jurkat
cells (J16) or Jurkat cells transfected with the human
FLIPL cDNA (JFL2) were stimulated with
anti-CD95 mAb CH-11, treated with etoposide (E), or
irradiated (IR), and apoptosis was read out as nuclear
fragmentation (47) 6 h after CD95 stimulation and 14 h after
addition of etoposide or after irradiation. The medium controls were
measured after 14 h of incubation. Data are representative of
three independent experiments and show means ± S.D. from
duplicate samples in one experiment.
-Radiation Are under Common Mitochondrial Control--
Since
apoptosis signaling induced by CD95, etoposide, and
-radiation did
not converge at the level of DED-containing caspases, we investigated
whether the presumed mitochondrial caspase activation complex might be
involved in these three pathways. To directly examine mitochondrial
involvement, release of cytochrome c into the cytoplasm was
determined. Fig. 4 shows that CD95,
etoposide, and IR induced release of cytochrome c in control
Jurkat cells. CD95-induced cytochrome c release was
inhibited by FLIPL overexpression, indicating that CD95
communicates to the mitochondria via caspase-8. FLIPL did
not inhibit cytochrome c release in response to etoposide or
IR, substantiating the lack of caspase-8 involvement in the DNA damage
pathways (Fig. 4).
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Fig. 4.
CD95, etoposide, and IR induce cytochrome
c release into the cytoplasm. Control J16 cells,
FLIPL-overexpressing JFL2 cells, Jurkat cells transfected
with empty vector (J neo) or bcl-2 cDNA
(J Bcl-2), and JA1.2 cells were stimulated with anti-CD95
mAb 7C11 at 200 ng/ml, etoposide at 10 µg/ml, or IR at 30 Gy for the
indicated time periods. Cells were lysed, and mitochondrion-free
cytosol was prepared as described (19). Equal amounts of protein were
loaded after quantification, and immunoblotting was performed for both
cytochrome c (cyt c) and actin as an additional
loading control. The experiment is representative of three.
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Fig. 5.
In Jurkat cells, Bcl-2 inhibits apoptosis
induction by CD95, etoposide, and IR. Jurkat cells transfected
with empty vector (J neo) or Bcl-2 cDNA
(J Bcl-2) were exposed to anti-CD95 mAb CH-11 (250 ng/ml),
etoposide (3 µg/ml), or IR (20 Gy). Apoptosis was read out as nuclear
fragmentation (47) at the indicated times after stimulation. Data are
representative of two independent experiments and show means ± S.D. from duplicate samples in one experiment.
-Radiation--
To further delineate the convergence
in apoptosis signaling, we set out to identify effector caspases that
were under the control of CD95, etoposide, and IR. CD95 effectively
induced caspase-3, -6, and -7 processing in control Jurkat cells (Fig.
6A). In response to etoposide
treatment and IR, caspase-3 and caspase-6 were cleaved to a minor
degree, whereas caspase-7 processing was readily detectable (Fig. 6).
FLIPL inhibited CD95-induced caspase-3, -6, and -7 processing (Fig. 6B), indicating that a DED-containing
caspase (most likely caspase-8) is upstream from these effector
caspases in the CD95 pathway. In contrast, effector caspase cleavage in
response to etoposide and IR occurred independent of caspase-8
activation (Fig. 6B). CD95-, etoposide-, and IR-induced
effector caspase processing did not occur in the resistant JA1.2 clone
or in Bcl-2-overexpressing Jurkat cells (Fig. 6B). We
conclude that CD95 and the DNA-damaging regimens can signal to the same
effector caspases, but with different efficiencies. CD95 signals to
effector caspases via caspase-8, whereas etoposide and IR use a
caspase-8-independent route. Downstream from caspase-8, CD95-,
etoposide-, and IR-induced effector caspase activation is subject to
common mitochondrial regulation.
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Fig. 6.
Regulation of effector caspase processing in
response to CD95, etoposide, and IR in Jurkat cells. Control
Jurkat cells (A) and JFL2, JA1.2, and
Bcl-2 cDNA-transfected Jurkat cells
(B) were stimulated with anti-CD95 mAb 7C11
at 200 ng/ml, etoposide at 10 µg/ml, or IR at 30 Gy and harvested
after the indicated periods of incubation. Total cell lysates were
separated by SDS-polyacrylamide gel electrophoresis, and caspase
(casp)-3, -6, and -7 processing was monitored by
immunoblotting with specific antibodies. The procaspase forms are
indicated by dashes, and the degradation products are shown
by arrowheads.
-Radiation--
Reported common signaling events induced by CD95,
certain anticancer drugs, and IR include generation of ceramide, which
has been implicated in apoptosis induction. In Jurkat cells, CD95 ligation gives rise to a slow and sustained elevation of ceramide levels upstream from DEVD-inhibitable caspases (42). Fig.
7A shows that CD95, as well as
etoposide and IR, induced significant ceramide accumulation in
wild-type Jurkat cells, but not in the apoptosis-resistant JA1.2
clone.
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Fig. 7.
Ceramide and apoptosis signaling induced by
CD95, etoposide, and IR. A, J16 wild-type cells and the
JA1.2 clone were treated with anti-CD95 mAb CH-11 at 500 ng/ml,
etoposide at 5 µg/ml, or IR at 15 Gy, and ceramide (Cer)
levels were determined at the indicated time points and are expressed
as -fold increase relative to time-matched controls. Means ± S.D.
from duplicate samples in one experiment are shown. B, empty
vector-transfected (J neo) and bcl-2
cDNA-transfected (J Bcl-2) Jurkat cells and JA1.2 were
treated with vehicle (dimethyl sulfoxide (DMSO)) or 75 µM C2-ceramide
(C2-Cer) for the indicated time periods.
Proteolytic processing of caspase-6 was determined by immunoblotting of
total cell lysates separated by SDS-polyacrylamide gel electrophoresis.
Procaspase-6 (pro-casp-6) at 35 kDa is indicated.
C, cells as indicated were treated with vehicle or
C2-ceramide for 24 h. Apoptosis was read out as
nuclear fragmentation (47). Means ± S.D. from duplicate
measurements in one experiment are shown.
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ABSTRACT
INTRODUCTION
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REFERENCES
-radiation (51) can induce apoptosis by activating the CD95
receptor system. Anticancer drugs would do so by inducing synthesis of
CD95 ligand (13, 14) and, in certain cases, also the receptor (14).
CD95 was implicated in apoptosis induction by
-radiation based on
diminished radiation sensitivity of CD95-deficient splenocytes from
lpr mice (51).
-radiation-induced apoptosis has been contradicted by several reports (32, 52-54). For instance, in
Jurkat cells, we could not detect CD95 ligand synthesis in response to
etoposide or doxorubicin, and drug-induced apoptosis could not be
inhibited by blocking interaction of CD95 with its ligand (32). In
addition, CD95-deficient lpr thymocytes are sensitive to
etoposide-induced (32) and
-radiation-induced (52) apoptosis to the
same degree as wild-type cells. Based on our experiments using
FLIPL-overexpressing cells, we can exclude a role for
caspase-8 and therefore for CD95 in apoptosis signaling induced by
etoposide and
-radiation in Jurkat cells, in contrast to an earlier
suggestion (55). Moreover, since FLIPL also inhibits TRAIL
receptor-induced apoptosis (9), these death receptors can also be
excluded from playing a role in the DNA damage pathways in the cell
type examined here.
-radiation was inhibited
by Bcl-2 overexpression, suggesting a dependence on mitochondrial
components such as cytochrome c. Most likely, caspase-8 is
processed as a result of the activation of other caspases that are
instrumental in etoposide- and radiation-induced apoptosis. Caspase-9
would be a good candidate to initiate the apoptosis signaling pathway
induced by etoposide and
-radiation since it can take part in the
Apaf-1 complex (21). However, available antibodies against caspase-9
did not allow detection of this enzyme in Jurkat cells (data not shown).
-radiation converges with a CD95-induced pathway
downstream from caspase-8, at or upstream from the
mitochondrion-dependent caspase complex. It has been
debated whether the CD95 pathway can be regulated by the mitochondria
as assessed by effects of Bcl-2, Bcl-xL, or Bax overexpression in
transgenic or transfected cells (25, 26, 48-50). The discrepant
results obtained in these systems were recently reconciled by the
suggestion that, in some cell types (e.g. SKW6.4 cells),
CD95 effectively activates caspase-8 at the DISC and induces activity
of effector caspases independent of a mitochondrial contribution. In
other cell types (e.g. Jurkat cells), caspase-8 activation
at the DISC is inefficient, and a contribution of the mitochondrial
pathway is required for effective induction of apoptosis (50). Our
findings in Jurkat cells are consistent with those of Scaffidi et
al. (50): detectable CD95-induced caspase-8 activation is slow and
inhibited by Bcl-2 overexpression. However, we find that CD95-induced
cytochrome c release is inhibited by FLIPL
overexpression, indicating that caspase-8 signals to the mitochondria.
Most likely, undetectable amounts of activated caspase-8 generated in
the DISC are sufficient to initiate the apoptosis pathway. Recently,
caspase-8 was shown to induce cytochrome c release from the
mitochondria via its substrate Bid, a Bcl-2-interacting protein (56,
57). Apparently, CD95 signaling can diverge at caspase-8, with one
branch of the pathway leading directly to effector caspase activation
and the other branch communicating with the mitochondria (50, 56,
57).
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Tschopp and D. W. Nicholson for gifts of reagents.
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Note Added in Proof |
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Another study recently reported that FLIP does not inhibit apoptosis induced by DNA damaging regimens in Jurkat cells (Kataoka, T., Schroter, M., Hahne, M., Irmler, M., Thome, M., Froehlich, C. J., and Tschopp, J. (1998) J. Immunol. 161, 3936-3942).
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FOOTNOTES |
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* This work was supported by the Dutch Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 31-20-5121972;
Fax: 31-20-5121989; E-mail: jborst{at}nki.nl.
2 J. G. R. Boesen-de Cock and U. Oettinger, unpublished data.
3 Tepper, A. D., de Vries, E., van Blitterswijk, W. J., and Borst, J. (1999) J. Clin. Invest. 103, 971-978.
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ABBREVIATIONS |
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The abbreviations used are:
DED, death effector
domain;
DISC, death-inducing signaling complex;
mAb, monoclonal
antibody;
FLIP, FLICE inhibitory protein;
PIPES, 1,4-piperazinediethanesulfonic acid;
IR, -radiation;
Gy, gray.
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REFERENCES |
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