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INTRODUCTION |
The apoptotic process is now known to involve the well
orchestrated interactions of cell death receptors, death receptor
adaptors, caspases, and Bcl-2 family members (1-6). Although a number
of stimuli have been reported to result in the up-regulation of the Fas
receptor and its ligand (e.g. UV, c-Myc, and certain
chemotherapeutic drugs), there are many other stimuli for which the
mechanism responsible for their action is still unknown (7-10). An
example of the latter is the ability of cycloheximide
(CHX)1 to either promote or
inhibit apoptosis in divergent cell types and in response to varying
death stimuli (11-16). A large body of evidence has shown that CHX can
potentiate, and in some cases (e.g. TNF
stimulation and
staurosporine) be necessary for, the apoptotic effects of certain death
stimuli (12-14, 16). The studies of Martin et al. (13) and
Tuschida et al. (14) further indicated that CHX,
independently of other stimuli, is also capable of promoting apoptosis
in a number of transformed cell lines and normal cells. Jacobson
et al. (16) have shown that staurosporine- and
staurosporine/cycloheximide-induced death is mediated by a
caspase-3-like activity that is blocked by Z-VAD-FMK, a synthetic
tripeptide inhibitor that demonstrates broad caspase specificity. More
recently, Woo et al. (17) demonstrated that bone marrow
neutrophils from caspase-3
/
mice no longer undergo
CHX-induced apoptosis, indicating that caspase-3 expression is most
likely required for this type of cell death.
The ability of CHX to induce cell death varies considerably from one
cell line to another, suggesting that the continuous synthesis of a
regulatory protein that blocks apoptosis is required for the normal
growth of these CHX-sensitive cell lines (12, 13). Sensitivity to CHX
is not necessarily determined by cell type alone since cell lines from
the same tissue and stage of development (e.g. Jurkat and
CEM C7 T-cells) can be affected in very different ways. Furthermore,
although cell death triggered by cell-surface receptors
(e.g. Fas, DR3, and TNF receptor-1) requires an adaptor
protein such as FADD to promote an apoptotic signal, cell death
triggered by other stimuli (e.g. E1A, c-Myc, and Adriamycin)
may not (18-24). Therefore, it is of interest to determine the basis
of the cellular differences that result in cellular sensitivity or
insensitivity to agents like CHX as well as the mechanism of
CHX-induced apoptosis.
The transformed human T-cell lines Jurkat and CEM C7 are representative
of a similar T-cell developmental stage, and they are equally sensitive
to agonistic anti-Fas mAbs and TNF
(25, 26). However, they exhibit
disparate responses when exposed to CHX. We therefore set out to
determine which apoptotic signaling pathway components are involved in
CHX-induced Jurkat cell death as well as the basis for the CEM C7
cellular resistance to CHX. Here we demonstrate that disruption of
normal FADD function inhibits apoptotic signaling in these cells. This
may indicate that in addition to its role as an adaptor that links
TNF-related receptors to caspase activation, FADD mediates certain
apoptotic signals through a receptor-independent pathway(s). Finally,
we demonstrate that FADD and caspase-8 (FLICE) coalesce into what
appear to be perinuclear death effector filaments (DEFs) (27, 28) in
wild-type Jurkat, Jurkat-FADD-DN, and CEM C7 cells treated
with CHX, even though only the wild-type Jurkat cells apoptose. These
results suggest that the redistribution of FADD and caspase-8 into
these filament structures is necessary, but not sufficient, for cell death to occur.
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EXPERIMENTAL PROCEDURES |
Cell Lines, Expression Constructs, and Transfections--
Human
Jurkat cells and CEM C7 cells were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum. This
particular Jurkat cell line contains a mutation in the bax gene that results in a functionally inactive, truncated protein (29).
For Fas-induced cell death analysis, cells at a density of 1.5 × 106 were treated with an anti-Fas monoclonal antibody,
CH-11 (Kamiya Biomedical Co.), at 100 ng/ml for 0, 2, 4, 6, and 8 h. CHX-induced cell death was performed by incubating the cells in 20 µg/ml CHX for 6 h, and staurosporine (STS)-induced apoptosis was
accomplished by incubating cells with various concentrations (0.1, 0.3, 0.5, 0.8, and 1.0 µM) of STS for 4 h. Apoptotic
cells were examined with a light microscope for appearance of plasma
membrane blebbing and by TUNEL assay (30), and the percentage of
apoptotic cells was quantitated as described below.
The Jurkat-Neo and Jurkat-FADD-DN cell lines were generated by
resuspending 107 Jurkat cells in 0.3 ml of the cell growth
medium containing 12 µg of either pcDNA3.0 or PCDNA3.0/FADD-DN
(kindly provided by Dr. V. Dixit), which were then electroporated using
a 4-mm gap cuvette at 0.25 kV and 960 microfarads. Cells were cultured
for 48 h before addition of G418 (Geneticin, Life Technologies,
Inc.) at 1 mg/ml. After 4 weeks of selection in the G418 medium, a
subpopulation of non-clonal, transfected cells expressing a moderate to
high level of FADD-DN was selected by exposing cells to an agonistic anti-Fas mAb (CH-11) at 100 ng/ml for 72 h. The surviving cells (~20% of the original population) were then expanded. In contrast to
FADD-DN-transfected Jurkat cells, virtually no pcDNA3.0-transfected Jurkat cells (<0.1%) survived this treatment. FACS analysis
demonstrated that most of the Jurkat-FADD-DN cells (>95%) expressed
levels of cell-surface Fas receptor equivalent to the original Jurkat cell population and the Jurkat-Neo cells. By Western blot analysis, the
Jurkat-FADD-DN cells were found to express levels of wild-type FADD and
caspase-8 equivalent to the levels expressed in the Jurkat-Neo and
Jurkat cells.
Cell Lysis and Immunoblotting--
For Western blotting, cells
were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 10 mM NaF, 10 mM
-glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 mM benzamidine, and 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (Boehringer Mannheim). 50 µg of cell lysate was separated by SDS-polyacrylamide gel
electrophoresis and transferred onto Immobilon-P membrane (Millipore
Corp.). The resulting membrane was blocked with 10% skim milk and
incubated with a designated primary antibody, and the signals were
detected by use of an ECL Western blotting kit (Amersham Pharmacia
Biotech). The anti-caspase-8/FLICE mAb (C15) (31) was kindly provided
by Drs. M. E. Peter and P. H. Krammer, whereas the anti-caspase-3
(Transduction Laboratories), anti-FADD (Transduction Laboratories),
anti-Fas (MBL Co. Ltd.), anti-TNF receptor-1 (Bender MedSystems),
anti-PARP (Upstate Biotechnology, Inc.), anti-cytochrome c
(7H8.2C12; Molecular Probes, Inc.), and anti-cytochrome oxidase subunit
IV (20E8-C12; Molecular Probes, Inc.) antibodies were obtained commercially.
Induction of Caspase Cleavage and Activation, PARP Assays,
Caspase Inhibitors, and Cell Death Assays--
Anti-Fas mAb (CH-11)-,
CHX-, and STS-induced caspase activation in Jurkat and CEM C7 cells was
monitored by cleavage of the endogenous caspase substrate PARP using
Western blot analysis. Cytochrome c release from
mitochondria was determined according to a previously published
protocol (33). Experiments involving the effects of caspase inhibitors
on cytochrome c release in the presence of CHX were
performed as follows. Jurkat cells were incubated in 50 µM Z-FA-FMK, Z-YVAD, Z-IETD, Z-DEVD, or Z-VAD-FMK (Enzyme System Products) before addition of CHX to cells to initiate cell death. Apoptotic cells were detected by TUNEL assay (Boehringer Mannheim) according to the manufacturer. The percentage of apoptotic cells during agonistic CH-11 antibody treatment was quantitated by FACS
(32). The percentage of apoptotic cells during CHX treatment was
quantitated by FACS for annexin V-positive and propidium
iodide-negative cells (34, 35). Briefly, 106 Jurkat cells
were treated with CHX for 6 h, and the cells were then stained
with FITC-conjugated annexin V and propidium iodide.
Indirect Immunofluorescence Analysis--
Jurkat,
Jurkat-FADD-DN, Jurkat-Neo, or CEM C7 cells were prepared for
immunofluorescence by cytospin. These cells were either left untreated
or treated with an agonistic anti-Fas mAb for 4 h, with 20 µg/ml
CHX for 6 h, or with 20 µg/ml CHX for 6 h in the presence
of a 50 µM concentration of the caspase inhibitor Z-VAD-FMK before being prepared for analysis (32). Cells were then
fixed and stained with an anti-FADD mAb and anti-caspase-8 polyclonal
antiserum (goat anti-human IgG, SC-6134, Santa Cruz) as described
previously (36). The anti-FADD mAb was detected using a
rhodamine-conjugated anti-mouse secondary antibody (Southern Biotechnology, Birmingham, AL), and the anti-caspase-8 polyclonal antibody was detected using an FITC-conjugated anti-goat secondary antibody (Southern Biotechnology) with an Olympus BX-50 fluorescent microscope as described (36).
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RESULTS |
Ability of Cycloheximide to Induce Cell Death in Jurkat and CEM C7
T-cell Lines--
It has been shown that CHX can augment many
different death signals, presumably through its ability to inhibit the
synthesis of a protective factor, as well as induce apoptosis on its
own (12-14, 16). In fact, cell lines derived from the same source (e.g. lymphoid cells) respond quite differently when exposed
to CHX; some (e.g. Jurkat T-cells) rapidly undergo
apoptosis, whereas others (e.g. CEM C7 T-cells) do not (25,
26). To explore the possible molecular mechanisms involved in this
response, we examined the ability of CHX to induce cell death in Jurkat
and CEM C7 cells. As little as 5 µg/ml CHX was capable of inducing
apoptosis in Jurkat cells (Fig. 1) within
4-6 h, whereas even 20 µg/ml did not induce CEM C7 cell death over a
48-h time period (data not shown). For these initial experiments,
cleavage of the nuclear caspase substrate PARP in CHX-treated Jurkat
cells was used as a biochemical marker of apoptosis (Fig.
1A). Annexin V staining of cell-surface phosphatidylserine
was used as a measure to determine the percentage of apoptotic cells
present (Fig. 1A). Furthermore, a much higher level of
cytochrome c was released from the mitochondria into the
cytosol of CHX-treated Jurkat cells compared with CHX-treated CEM C7
cells (Fig. 2). To demonstrate that
cytochrome c release was not due to physical disruption of
the mitochondria, proteins corresponding to the cytoplasmic and
mitochondrial fractions of the CHX-treated Jurkat and CEM C7 cells were
Western-blotted with an antibody to cytochrome oxidase (subunit II)
(Fig. 2). Cytochrome oxidase is a protein that is not released from the
mitochondria during apoptosis (37). If the cytoplasmic cytochrome
c in the Jurkat cells were generated by physical disruption
of the mitochondria or mitochondrial contamination, one would expect to
see cytochrome oxidase present in this cell fraction as well. No
cytochrome oxidase was detected in the cytoplasmic fraction from the
CHX-treated Jurkat cells, whereas a small amount was detected in the
CEM C7 cells. The former result demonstrates that the mitochondria from the Jurkat cells were intact, whereas the latter result suggests that
the CEM C7 cell mitochondria were slightly damaged, and/or the cytosol
was contaminated with a low level of mitochondria. This explains the
small amount of cytochrome c in the cytoplasmic fraction
from the CHX-treated CEM C7 cells (Fig. 2).

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Fig. 1.
Characterization of apoptotic events
associated with CHX-mediated cell death. A, cleavage of
PARP by caspases in Jurkat cells exposed to varying amounts of CHX. The
p85 band represents the cleaved product. The percentage of apoptotic
cells quantitated by FITC-conjugated annexin V staining is indicated
below the panel. B, cleavage of PARP (left panel)
and the release of cytochrome c (right panel) in
Jurkat cells treated with 20 µg/ml CHX for increasing amounts of
time. The percentage of apoptotic cells quantitated by FITC-conjugated
annexin V staining is shown below the panel. C, cleavage and
activation of caspase-3 (left panel), caspase-2
(middle panel), and caspase-8 (right panel) in
Jurkat cells treated with 20 µg/ml CHX for increasing amounts of
time. Detection of the smallest caspase subunit, released when caspases
are fully activated, by the corresponding caspase antibody is indicated
by the arrow.
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Fig. 2.
Release of cytochrome c into
the cytosol of CEM C7 and Jurkat T-cells in the absence
( ) and presence (+) of CHX. 40 µg of
cytosolic protein was Western-blotted with an anti-cytochrome
c antibody in the top panels to determine whether
this protein was released from mitochondria in response to CHX
treatment. In the bottom panels, cytosolic (cyto)
and mitochondrial (mito) proteins from CHX-treated CEM C7 or
Jurkat T-cells were Western-blotted with an antibody to mitochondrial
cytochrome oxidase to determine whether the cytosolic fractions were
contaminated with mitochondria.
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The effects of treatment of Jurkat cells with 20 µg/ml CHX on PARP
cleavage, release of cytochrome c, and annexin V staining in
Jurkat cells during 8 h of exposure were assessed (Fig.
1B). All three were induced with similar kinetics. Since
these results suggested that caspase-dependent apoptosis
was being triggered, we examined the effect of CHX treatment on the
processing of several caspases (Fig. 1C). Caspase-3
(CPP32/YAMA) activation, as judged by the appearance of the 17-kDa
subunit, started to occur within 2 h. The processing of caspase-2
and caspase-8, as judged by the appearance of their 17-18-kDa
subunits, started to occur within 2-4 h of CHX treatment. This
suggests either that our ability to detect activation of caspase-8 was
limited by the sensitivity of the reagents we used or that caspase-3 is
responsible for the processing and activation of caspase-8 during
CHX-induced Jurkat cell death.
FADD-DN Expression Inhibits Cycloheximide- and
Staurosporine-Induced T-cell Death--
Since caspase-8 is
activated in CHX-treated Jurkat cells, and it appears that this
activity might be linked to a cellular commitment to death, we decided
to examine the effects of a dominant-negative form of FADD on this
process. Others have shown that expression of a FADD-DN protein,
containing the death domain but not the death effector domain, is
capable of effectively inhibiting a number of different
receptor-mediated apoptotic signals (18, 21, 38). The same FADD-DN
expression construct was stably introduced into Jurkat cells,
and the truncated protein was constitutively expressed along with
endogenous FADD (Fig. 3A).
Jurkat cells transfected with the empty expression vector (Neo) were
used as a control. Since it has been shown that expression of the
FADD-DN protein effectively inhibits Fas-mediated cell death, we
examined the ability of these cells to survive Fas receptor
oligomerization. As measured by cell viability (FITC-conjugated annexin
V staining) and two different biochemical markers (i.e. PARP
cleavage and caspase-8 processing), these Jurkat-FADD-DN cells were
resistant to Fas-mediated apoptosis induced by an agonistic anti-Fas
mAb (Fig. 3A).

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Fig. 3.
Demonstration that the expression of the
FADD-DN protein in Jurkat cells substantially inhibits the ability of
CHX to induce apoptosis. In A, the left
panel shows the expression of the FADD-DN protein in stably
selected Jurkat cells compared with the Neo vector control as detected
by Western blot analysis with an anti-FADD mAb. The middle
panel demonstrates that PARP cleavage resulting from Fas receptor
oligomerization induced by treatment with an agonistic mAb is inhibited
in Jurkat cells expressing the FADD-DN protein as compared with Jurkat
cells containing the Neo vector only. Below this panel, the percentage
of apoptotic cells quantitated by FITC-conjugated annexin V staining is
indicated. The right panel demonstrates that caspase-8/FLICE
processing induced by Fas receptor oligomerization is also inhibited in
Jurkat cells expressing the FADD-DN protein as compared with the Neo
vector control cells. FLICE was detected with the C15 mAb previously
described (31). B shows the comparison of PARP cleavage, and
cytochrome c release induced by treatment of Jurkat cells
expressing the FADD-DN protein or containing the Neo control vector
with increasing amounts of either CHX (0, 5, 10, and 20 µg/ml) or STS
(0, 0.1, 0.3, and 0.5 µM). The percentage of apoptotic
cells was quantitated by FITC-conjugated annexin V staining and is
indicated below the panels demonstrating PARP cleavage. C
shows the cleavage of caspase-8, resulting in the production of its
characteristic partial products and the p18 subunit, by increasing
amounts of either CHX (0, 5, 10, and 20 µg/ml) or STS (0, 0.1, 0.3, and 0.5 µM) in Jurkat-Neo control cells or Jurkat-FADD-DN
cells. 40 µg of cellular protein was Western-blotted with a mAb to
caspase-8/FLICE (C15) (31). D shows the cleavage of
caspase-3, resulting in the production of its p17 subunit, by
increasing amounts of either CHX (0, 5, 10, and 20 µg/ml) or STS (0, 0.1, 0.3, and 0.5 µM) in Jurkat-Neo control cells or
Jurkat-FADD-DN cells. 40 µg of cellular protein was Western-blotted
with anti-caspase-3 polyclonal antiserum.
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We next examined the ability of CHX and STS, a broad spectrum protein
kinase inhibitor that induces caspase-dependent apoptosis independently of cell-surface death receptors (16, 39), to trigger cell
death in the Jurkat-FADD-DN cells. PARP cleavage and cell death, as
measured by the percentage of annexin V-positive cells, were
significantly inhibited in the FADD-DN cells as compared with the Neo
controls as the cells were exposed to increasing amounts of CHX (Fig.
3B). However, the ability of STS to induce PARP cleavage and
apoptosis was not significantly diminished. The effect of FADD-DN
expression on the ability of CHX and STS to induce the release of
cytochrome c from mitochondria, which is involved in the
propagation of several apoptotic signals (40), was examined next.
FADD-DN expression did not significantly inhibit the release of
cytochrome c in the CHX- or STS-treated cells (Fig. 3B). In addition, although the FADD-DN protein inhibited
CHX-induced cell death as measured by the use of an FITC-conjugated
anti-annexin V antibody, it did not have a similar effect on
STS-induced apoptosis (Fig. 3B).
Delineation of a FADD-dependent Apoptotic Pathway in
CHX-treated Cells--
There are at least two possibilities to explain
how FADD-DN expression can interfere with CHX-induced Jurkat cell
death. One is to block CHX-induced cytochrome c release; the
other is to block caspase activation, downstream of cytochrome
c release. To distinguish between these two possibilities,
we first examined CHX-induced cytochrome c release in both
Jurkat-Neo and Jurkat-FADD-DN cells. Fig. 3B shows that
expression of the FADD-DN protein did not prevent cytochrome
c release induced by CHX or STS. Conversely, FADD-DN
expression inhibited caspase-8 activation in CHX- and STS-treated cells
(Fig. 3C). Surprisingly, FADD-DN expression also efficiently
inhibited caspase-3 activation induced by CHX treatment, but not by
higher doses of STS (Fig. 3D). Since FADD-DN expression had
no apparent effect on cytochrome c release in both CHX- and
STS-treated cells, the inhibition of caspase-3 processing in
CHX-treated Jurkat-FADD-DN cells may be the result of caspase-8 inhibition. This possibility is further supported by the fact that at
higher doses of STS (>0.3 µM), caspase-8 processing
continued to be inhibited by FADD-DN expression (Fig. 3C),
whereas caspase-3 processing was not (Fig. 3D). Taken
together, these results suggest that expression of the FADD-DN protein
directly inhibits caspase-8 processing, whereas it may indirectly
inhibit caspase-3 processing through its effects on caspase-8. The
ability of FADD-DN expression to inhibit caspase processing is
reflected by its ability to inhibit cell death, as judged by PARP
cleavage and the percentage of FITC-conjugated annexin V-positive cells
(Fig. 3B).
Based on these results, we decided to examine the effects of various
caspase inhibitors on cytochrome c release in Jurkat cells
treated with CHX. Fig. 4 shows that none
of these caspase inhibitors prevented the release of cytochrome
c from mitochondria during CHX-induced Jurkat cell death,
whereas cell death was inhibited (data not shown). Others have shown
that, significantly, caspase inhibitors do not prevent cytochrome
c release induced by a number of non-receptor apoptotic
stimuli, including STS (6). These data, coupled with that from the
Jurkat cells expressing the FADD-DN protein, suggest that cytochrome
c release is upstream of caspase activation in Jurkat cells
undergoing apoptosis in response to CHX. We discuss the possible
mechanisms and implications of these observations below.

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Fig. 4.
Effect of various caspase peptide inhibitors
on CHX-mediated apoptosis in Jurkat cells. A, the
effect of the caspase inhibitors Z-VAD, DEVD, and YVAD, as well as the
control compound ZFA and the carrier Me2SO
(DMSO), on cytochrome c release induced by CHX
treatment was assessed.
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Changes in the Subcellular Localization of FADD Occur during
Fas-mediated and Cycloheximide-induced T-cell Death--
How does
FADD, a death receptor adaptor protein, mediate receptor-independent
apoptotic signals? Recently, it has been demonstrated that
overexpression of either FADD or caspase-8 induces apoptosis through
the formation of unique cellular filament structures that contain the
death effector domains of these proteins (27, 28). Accordingly, these
structures have been termed "death effector filaments," and their
formation is believed to be important for the execution phase of
apoptosis. We reasoned that CHX might be inducing cell death through
the formation of DEFs. To examine this possibility, we prepared
untreated and CHX-treated Jurkat and CEM C7 cells for indirect
immunofluorescence analysis using an anti-FADD mAb and
anti-caspase-8 polyclonal antiserum. In the untreated Jurkat,
Jurkat-FADD-DN, and CEM C7 cells, FADD and caspase-8 were dispersed
throughout the cytoplasm, as expected (Fig.
5A). Conversely, in Jurkat
cells undergoing CHX-induced apoptosis, FADD formed perinuclear
filaments (Fig. 5B) that were very similar in appearance to
what has been reported by others when FADD, its death effector domain,
or caspase-8 is overexpressed (27). Caspase-8 was colocalized to these
same filaments, consistent with the results of these investigators (27,
28). In addition, these aggregate structures did not colocalize with a
known cytoskeletal element, tubulin, in these cells (Fig.
5D). In fact, tubulin remained equally distributed
throughout the cytoplasm of CHX-treated Jurkat cells. This demonstrates
that these perinuclear aggregate structures containing FADD and
caspase-8 are not the result of the cytoplasm collapsing due to cell
shrinkage during apoptosis. Somewhat surprisingly, CHX-treated CEM C7
T-cells and the Jurkat-FADD-DN cells also formed the FADD/caspase-8
perinuclear DEFs (Fig. 5, C and D). Finally, when
we examined the distribution of FADD and caspase-8 in Jurkat cells
treated with CHX in the presence of Z-VAD-FMK, we found that the
perinuclear DEF-like structures were present (Fig. 5E). This
suggests that active caspases are not necessary for the formation of
these structures in response to CHX. When the CHX was removed from the
CEM C7 cell media, the perinuclear DEFs disappeared, and the cells
resumed their normal growth within a few hours (data not shown). This
did not happen with the Jurkat cells. Thus, whereas CHX treatment
resulted in the formation of DEFs in all three of these cell lines,
only the wild-type Jurkat cells underwent apoptosis. This suggests that
FADD/caspase-8 DEF formation may be necessary, but not sufficient, for
cell death signals and that as yet unidentified "factors" may need
to be recruited to this filament complex to initiate other apoptotic
events (e.g. the caspase cascade).

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Fig. 5.
Localization of FADD and caspase-8/FLICE in
control and CHX-treated cells. Jurkat cells (A), Jurkat
cells expressing the FADD-DN protein (B), or CEM C7 cells
(C) were either left untreated (control) or
treated with CHX for 6 h. Jurkat cells treated with CHX in the
presence of the caspase inhibitor Z-VAD-FMK (CHX + zVAD)
were stained with either an anti-FADD mAb or an anti-caspase-8 mAb
(E). In D, Jurkat cells were either left
untreated (control) or treated with CHX for 6 h and
stained with a mAb to tubulin. The arrows in D
point out the 4,6-diamidino-2-phenylindole (DAPI)-stained
apoptotic nuclei and the more evenly distributed tubulin staining in
the corresponding cell. In all panels, the blue
4,6-diamidino-2-phenylindole counterstain indicates nuclei. The
arrows in the panels visualized with the anti-FADD and
anti-caspase-8 antibodies point out their perinuclear localization
CHX-treated cells as compared with their diffuse cytoplasmic staining
pattern in the top panels (control). The
arrows in the
4,6-diamidino-2-phenylindole-counterstained nuclei point
out fragmented, apoptotic nuclei (Jurkat cells; A) and
normal nuclei (Jurkat-FADD-DN and CEM C7 cells; B and
C). The arrows in the top panels of
B point out the enhanced staining of the overexpressed
FADD-DN protein in these cells, creating the appearance of a rim around
the nucleus since these suspension cells represent cytospin
preparations.
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DISCUSSION |
Apoptosis can be initiated by a number of different stimuli.
Although the mechanism of action for many of the cell-surface death
receptors has been established (2, 42), the signaling mechanism(s)
responsible for the action of many other apoptotic agents is less
clear. Examples of the latter include cycloheximide, which can induce
cell death either on its own or in concert with another signaling
molecule (e.g. TNF
) (11-15), and STS, a broad spectrum
protein kinase inhibitor that is a potent inducer of apoptosis (39). In
addition, Jacobson et al. (16) have shown that both CHX and
STS induce apoptosis in a number of different cell lines/types by a
caspase-3-dependent pathway. Furthermore, not all cells,
both in vivo and in vitro, are equally sensitive to the death-inducing effects of CHX (12-15, 17). Understanding the
molecular mechanisms responsible for these differential effects may
provide clues to how certain cells are "sensitized" to selected death agents while others are not as well as provide valuable information regarding novel receptor- and/or non-receptor-mediated apoptotic signaling pathways.
In this study, we have demonstrated that CHX signals cell death through
a FADD-dependent mechanism that does not involve the action
of the cell-surface Fas death receptor. This is a rather surprising
result since it has been assumed that FADD functions primarily as a
death receptor adaptor molecule essential for the recruitment and
activation of specific procaspases, such as caspase-8 and caspase-10
(18, 19, 21, 43-46). Using both FADD gene knockout and
FADD-DN-overexpressing transgenic mice, it has recently been shown that
in addition to its well known role in apoptotic signaling, FADD also
contributes to certain aspects of cellular proliferation (24, 38).
Although these studies suggest that FADD does not play a role in
apoptosis induced by viral oncoproteins (e.g. E1A), c-Myc,
or certain chemotherapeutic agents (e.g. Adriamycin), data
from several other groups suggest that FADD might be important (9, 10,
46). Thus, FADD may function in a number of different contexts relevant
to cellular proliferation as well as apoptotic signaling. Here we have
shown that agents such as CHX mediate cell death in certain cells
through a FADD-dependent mechanism. It should be noted,
however, that the effect of the FADD-DN protein on this process in
these cells does not need to be due to FADD itself. This phenotype
could be the result of FADD-DN disruption of the binding of another
protein to FADD. In addition, it appears unlikely that Fas or TNF
receptor-1 and, most likely, DR3 are involved in the CHX-mediated
Jurkat cell death we have studied here. Since the TNF-related receptors
DR4 and DR5 induce apoptosis independently of FADD (47), it is
improbable that they are involved in mediating CHX-induced cell death
in these cells.
Why would FADD function in an apparently receptor-independent manner
during apoptosis induced by agents such as CHX? Several recent reports
suggest one possibility, namely, the formation of
perinuclear filament structures that also recruit caspase-8 (27, 28),
which may play a role in transducing cell death signals in the absence
of an appropriate death-inducing signaling complex (DISC). Furthermore,
Scaffidi et al. (48) have shown that so-called "type I"
cells form the DISC and activate caspase-8 and caspase-3 very rapidly,
whereas "type II" cells do not efficiently form the DISC and
therefore activate these caspases over a much longer time. These
investigators demonstrated that in type II (but not type I) cells, the
overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and
caspase-3 activation as well as apoptosis. In separate studies, it was
shown that Bcl-xL overexpression and, to a lesser extent,
Bcl-2 substantially impaired the formation of FADD-containing DEFs in
HeLa cells (28). Both Jurkat and CEM C7 cells have been classified as
type II DISC-forming cells (HeLa cells have not been classified), and
Bcl-2/Bcl-xL inhibit Fas-mediated apoptosis in these
cell lines (48). The marked similarities between the ability of these
cells to rapidly form a functional DISC and the ability of
Bcl-2/Bcl-xL to inhibit cell death might suggest that these
processes are related to the formation of DEFs in the absence of an
appropriate DISC. Such an interpretation would also be consistent with
the data presented here concerning CHX-mediated cell death. Additional
study of the similarities and differences between type I and II cells
as well as their ability/inability to form DEFs in response to
different apoptotic stimuli may help to answer this question.
Perhaps even more surprising is the ability of the FADD-DN protein to
inhibit apoptosis, but not the formation of the DEF-like structures or
the release of cytochrome c, in response to CHX. Furthermore, caspase inhibitors do not block the release of cytochrome c from mitochondria or prevent the formation of DEF-like
structures in cells treated with CHX. Although these results suggest
that the release of cytochrome c is not sufficient to cause
apoptosis under these circumstances, they do not rule out the
possibility that its release is required for cell death. Salvesen and
co-workers (49) have recently shown that procaspase-3 is the major
physiological substrate of caspase-8 and that the processing of
procaspase-9 in death receptor-mediated apoptosis requires the presence
and activation of caspase-3. As these authors suggested, the
mitochondrion-mediated death signal may function as an accelerator of
the death signal mediated by these receptors. They also suggested that
cells that are programmed to undergo cell death during their
development, such as those associated with the immune response
(e.g. Jurkat T-cells), have developed the death
receptor·caspase-8·caspase-3 activator complex to allow rapid
apoptotic signaling. Finally, it has recently been demonstrated that
the processing of procaspase-9 is blocked in cells by Akt protein
kinase phosphorylation of Ser-196 of procaspase-9 (41). Therefore, it
is possible that the phosphorylated form of procaspase-9 prevents cell
death in the presence of cytochrome c released after
treatment with CHX. This possibility is consistent with the ability of
high levels of STS to induce apoptosis, even in the presence of the
FADD-DN protein (Fig. 3B). These issues definitely warrant
further study.
Finally, we have shown that the formation of FADD- and
caspase-8-containing DEFs occurs in response to CHX in cells that
apoptose as a result of this treatment as well as in cells that do not. The recent studies of Siegel et al. (27) and Perez and White (28) concerning these DEFs dealt with their formation as a result of
the overexpression of FADD and/or caspase-8 rather than their induction
by exogenously administered apoptotic stimuli. Here we demonstrate
that such DEFs are also formed in response to exogenous stimuli,
possibly independently of cell-surface death receptors, consistent with
the interpretation of Siegel et al. (27). However, we have
also shown that these FADD- and caspase-8-containing DEFs are generated
in cells that do not undergo apoptosis as a result of CHX treatment
(i.e. CEM C7) as well as in Jurkat cells protected from this
apoptotic signal by the expression of a FADD-DN protein or treatment
with the caspase inhibitor Z-VAD-FMK. This suggests that although the
formation of DEFs may be necessary for apoptosis induced by certain
stimuli, it may not be sufficient for the propagation of a death
signal. Since both FADD and caspase-8 were found to be in the DEFs of
the non-apoptosing cells, we also suggest that the recruitment of an
additional factor and/or a specific modification may be necessary for
execution of cell death.