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Journal of Virology, August 2000, p. 7470-7477, Vol. 74, No. 16
Department of Biological Sciences, Columbia
University, New York, New York 10027
Received 6 April 2000/Accepted 12 May 2000
Induction of apoptotic cell death generally requires the
participation of cysteine proteases belonging to the caspase
family. However, and similar to most cell types, mouse fibroblasts are normally resistant to tumor necrosis factor alpha (TNF- Tumor necrosis factor alpha
(TNF- Induction of cell death by members of the TNFR superfamily typically
occurs by apoptosis (1, 29). A unique feature of apoptotic cells is that they retain cell membrane integrity
even after they have disintegrated into characteristic
apoptotic bodies (43). Apoptotic cells and bodies
are phagocytosed by macrophages, thus preventing an inflammatory
reaction that can result from cell lysis (31, 32). In
contrast, cells dying by necrosis can readily lose membrane integrity,
which can lead to inflammation (43). Induction of
apoptotic cell death by TNFR1 requires the activation of
cysteine proteases belonging to the caspase family. In particular,
activation of caspase 8 is a critical step in induction of
TNF-induced apoptosis (6, 28). Consistent with the
pivotal role of caspases in apoptotic cell death,
TNF- TNF- Cells, viruses, and materials.
Fibroblasts were cultured in
high-glucose Dulbecco's modified Eagle's medium (DMEM) containing
L-glutamate (2 mM), penicillin (100 U/ml), streptomycin
(100 µg/ml), and calf serum (10%). Mouse 3T3 fibroblasts were
derived as described previously (2). Western reserve (WR)
strain wild-type vaccinia virus and B13R mutant and revertant viruses
were a generous gift from G. L. Smith (Oxford University, Oxford,
United Kingdom) (19). DMEM plus 2.5% fetal bovine serum was
used for viral infections. Human TNF- Vaccinia virus infection of mouse fibroblasts.
3T3
fibroblasts (2 × 105) were plated on each well of a
six-well plate 1 day before infection. Viruses were then used to infect cells at 2 PFU per cell for 16 h, after which the cells were left untreated or treated with TNF- Analysis of cell death. (i) Cell morphology.
For
morphological observations, cells in six-well plates were washed with
phosphate-buffered saline (PBS) twice before being treated with trypan
blue (Gibco-BRL) for 1 min. The cells were washed with PBS once and
then examined under a 20× objective. Representative fields were
photographed. Cells which excluded trypan blue and showed membrane
blebbing and apoptotic bodies were considered
apoptotic, while cells that were trypan blue permeable and
exhibited a "balloon-like" morphology were considered necrotic.
(ii) Nucleus morphology.
Cells in plates were rinsed with
PBS, fixed with 3.7% formaldehyde, and permeabilized with 0.2% Triton
X-100 for 5 min. They were then washed and incubated with DAPI labeling
solution (2 µg/ml in PBS) for 5 min and examined under a fluorescence microscope.
(iii) Cell viability experiments.
Approximately 2 × 105 cells were plated on each well of a six-well plate 1 day before the experiments. Caspase inhibitors or mitochondrial
inhibitors were added to the indicated concentrations 1 h before
the addition of TNF- Detection of superoxide production.
Cells were trypsinized
after treatments and resuspended in DMEM plus 10 µM DHE at
106/ml. The cells were incubated at 37°C for 30 min and
then washed and resuspended in PBS before being subjected to
fluorescence-activated cell sorter analysis.
EMSA, affinity blots, and Western blots.
Electrophoretic
mobility shift assay (EMSA) was carried out as described previously
(3). Affinity blotting was performed essentially as
described previously (9). Briefly, approximately 5 × 106 cells were harvested after the treatments indicated.
The cells were then washed once with PBS and pelleted, and the pellet
was snap-frozen on dry ice. An equal volume of 1 µM biot-VAD-fmk in MDB buffer (50 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES,
1 mM dithiothreitol [pH 7]) was added to the cell pellet, and the cells were lysed by three cycles of freezing and thawing. The lysates
were incubated at 37°C for 15 min and centrifuged. Then 20-µg
samples of protein lysates from the supernatant were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked
for 30 min with TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween 20)
supplemented with 2% nonfat dry milk (NFDM) and then incubated for
1 h in avidin-Neutralite (Molecular Probes) at 1 µg/ml in TBST
supplemented with 1% NFDM. The membrane was then washed and incubated
in biotinylated horseradish peroxidase (Molecular Probes) at 25 ng/ml
in TBST for 1 h. The labeled protein was visualized by enhanced
chemiluminescence (Amersham). For Western blots, whole-cell extracts
were made by boiling the cell pellet in SDS-PAGE sample buffer. The
lysates were then sonicated before being subjected to separation by
SDS-PAGE. The C2.10 poly(ADP-ribose) polymerase (PARP) antibody (Enzyme
System Products) was used at a 1:10,000 dilution in subsequent steps.
The caspase inhibitor B13R sensitizes vaccinia
virus-infected fibroblasts to TNF-
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Induction of Necrotic-Like Cell Death by Tumor Necrosis Factor
Alpha and Caspase Inhibitors: Novel Mechanism for Killing
Virus-Infected Cells
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)-induced apoptosis. Surprisingly, TNF- treatment of vaccinia virus-infected mouse fibroblasts resulted in necrotic-like cell death, which was
significantly reduced in cells infected with a vaccinia virus mutant
lacking the caspase inhibitor B13R. Furthermore, TNF- also
induced necrotic-like cell death of fibroblasts in the presence of
peptidyl caspase inhibitors. In both cases, necrosis was
accompanied by generation of superoxide species. Caspase inhibitors
also sensitized fibroblasts to killing by double-stranded RNA and gamma
interferon. In all cases, cell death was efficiently blocked by
antioxidants or mitochondrial respiratory chain inhibitors. These
results define a new mitochondrion-dependent mechanism which may be
important in the killing of cells infected with viruses encoding
caspase inhibitors.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) is a pleiotropic cytokine which was originally identified as
a consequence of its ability to cause tumor cell killing
(5). Later studies demonstrated a key role for this cytokine
in inflammation and in responses to infectious agents including
bacteria and many types of viruses (38). Two TNF-
receptors called TNFR1 and TNFR2 have been identified (10,
22). Both receptors belong to a large family of related molecules
(TNFR superfamily) which also includes the death-inducing Fas protein
(30). The cytotoxic effects of TNF- are generally mediated by TNFR1, which, like Fas but unlike TNFR2, contains a
"death domain." Recent studies have shown that TNFR1 and Fas bind
related downstream signaling proteins that function as mediators of
cell death signals (1, 29). However, different from Fas, TNFR1 cytotoxicity to most cell types is evident only if RNA or protein
synthesis is inhibited, indicating the existence of survival mechanisms
dependent on de novo RNA and protein synthesis (38). Such a
survival mechanism is dependent on the NF-
B transcription factor and
activation of survival genes that are regulated by this factor (2,
23, 37, 41).
-induced apoptosis was shown to be blocked by
caspase inhibitors in tumor cell lines such as HeLa and MCF7
(27, 35).
also possesses significant antiviral activity that can be
manifested by either direct killing of infected cells or induction of a
state of resistance in uninfected cells, which may prevent further
infection (26, 42). These antiviral properties are also
shared by the gamma interferon (IFN-
) and double-stranded RNA
(dsRNA) signaling pathways (4, 14). The physiological significance of these mechanisms is evident by the fact that many viruses encode proteins capable of inhibiting the TNF-, dsRNA, and
IFN- signaling pathways (17, 21). Many viruses also
encode proteins which can inhibit caspase proteases and may thus
prevent or delay apoptotic cell death by TNF- or by other
inducers of apoptosis (15). Here we report a new
mechanism by which TNF- may kill virus-infected cells by induction
of necrotic-like cell death. This mechanism is dependent on the
presence of caspase inhibitors in viruses and is efficiently
duplicated in uninfected cells by peptidyl caspase inhibitors. We
also show that induction of necrotic-like cell death by TNF- and
caspase inhibitors appears to be dependent on the production of
superoxides by the mitochondrial respiratory chain. Similarly to
TNF-, dsRNA and IFN- also induced cytotoxicity to
caspase inhibitor-treated cells. These results provide
evidence for a novel function of caspase inhibitors in inducing
cell death by agents which by themselves are noncytotoxic. Such a
mechanism may be important in killing of cells infected with viruses
encoding caspase inhibitors.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and mouse IFN- was obtained
from R&D Systems and used at a final concentration of 10 ng/ml each in
all experiments if not specified otherwise. dsRNA [poly(I-C)] was
obtained from Sigma and used at a final concentration of 100 µg/ml.
Caspase inhibitors (Enzyme System Products) were dissolved in dimethyl
sulfoxide at 20 mM and used at 100 µM. The mitochondrial inhibitors
amytal, thenoyltrifluoroacetone (TTFA), and the antioxidant butylated
hydroxyanisole (BHA) were obtained from Sigma. Actinomycin D and
cycloheximide were used 2 and 10 µg/ml, respectively. The nuclear dye
4',6-diamidino-2-phenylindole (DAPI) and the superoxide-specific dye
dihydroethidium (DHE) were obtained from Molecular Probes.
(1 ng/ml) for 3 h.
or TNF- plus actinomycin D. After the
indicated periods, the cells were trypsinized and viable cells were
counted by trypan blue exclusion. Four independent readings within a
single experiment were used to calculate the standard deviation.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
cytotoxicity.
Virus-encoded caspase inhibitors are thought to protect
infected cells from apoptotic cell death and may thus represent
a strategy to escape from host defense mechanisms (15).
Based on these results, we wanted to determine whether TNF--induced cell death in susceptible cell types was inhibited upon infection with
viruses encoding caspase inhibitors. We tested this by using vaccinia virus, which encodes two proteins that are highly similar to
the cowpox virus caspase inhibitor CrmA. These putative caspase inhibitors are B13R (92% identical to CrmA) and B22R (19,
33). We first tested the effect of vaccinia virus infection on a
TNF--resistant 3T3 fibroblast line derived from mouse embryonic
fibroblasts (RelA+/+ 3T3, subsequently referred to as 3T3
fibroblasts) (2). These 3T3 fibroblasts were first infected
with vaccinia virus for 16 h, after which TNF- was added for an
additional 3 h. Unexpectedly, infection of these cells with
vaccinia virus greatly sensitized them to TNF--induced killing (Fig.
1A). More surprisingly, infection of 3T3
fibroblasts with a B13R mutant virus significantly attenuated TNF--induced cytotoxicity, while a B13R revertant virus was as cytotoxic as the wild-type virus (Fig. 1A). These results suggest that
the putative caspase inhibitor B13R can enhance TNF--induced cytotoxicity.
View larger version (59K):
[in a new window]
FIG. 1.
Caspase inhibitors sensitize mouse fibroblasts to
TNF- -mediated cytotoxicity. (A) 3T3 fibroblasts were infected with
either WR strain wild-type vaccinia virus or B13R deletion (DEL) or
B13R revertant (REV) vaccinia virus before being treated with TNF-
(1 ng/ml) for 3 h. Viable cells after treatment are shown as a
percentage of viable untreated cells (UT). (B) 3T3 fibroblasts were
treated with TNF- (
), z-VAD-fmk (
), or TNF- plus z-VAD-fmk
(
) for 3 or 6 h. (C) 3T3 fibroblasts were treated with TNF-
alone or with z-VAD-fmk, BD-fmk, or z-FA-fmk for 6 h.
Peptidyl caspase inhibitors can sensitize multiple cell types
to TNF--cytotoxicity.
We then tested whether the
broad-specificity peptidyl caspase inhibitor
Cbz-Val-Ala-Asp(OMe)-fmk (z-VAD-fmk), which covalently associates with
activated caspases, could also enhance TNF- killing of 3T3
fibroblasts. 3T3 fibroblasts readily lost viability after TNF-
treatment in the presence of z-VAD-fmk, while TNF- or z-VAD-fmk alone had no significant effect (Fig. 1B). A different caspase inhibitor, Boc-Asp(OMe)-fmk (BD-fmk), also sensitized these cells to
TNF- (Fig. 1C). Importantly, a cysteine protease inhibitor, Cbz-Phe-Ala-fmk (z-FA-fmk), which does not have a critical aspartate residue and is thus not specific for caspases, showed no
significant effect on cell viability after TNF- treatment (Fig. 1C).
These results demonstrate that both virus-encoded and peptidyl
caspase inhibitors can enhance the killing of cells that are
normally resistant to TNF-.
-z-VAD-fmk treatment on other
mouse and human cell types. Mouse BALB/c 3T3 mouse fibroblasts also
underwent cell death in the presence of TNF- plus z-VAD-fmk (data
not shown). Similarly, vaccinia virus-infected BALB/c fibroblasts were
readily killed in the presence of TNF-, while cell death was
significantly attenuated when these cells were infected with B13R
mutant virus (data not shown). Similar results were also obtained in
human promonocytic U937 cells treated with TNF- plus z-VAD-fmk (data
not shown). Thus, both fibroblast and monocytic cell types can be
sensitized to TNF- killing by caspase inhibitors.
Effect of caspase inhibitors on caspase activation and
TNF--induced apoptosis.
Like most cell types,
fibroblasts are resistant to TNF- but may be sensitized to
TNF--induced apoptosis in the presence of RNA or protein
synthesis inhibitors (38). As expected, significant cell
death was observed in 3T3 fibroblasts after treatment with TNF- and
the RNA synthesis inhibitor actinomycin D (Fig.
2A) or with TNF- and the protein
synthesis inhibitor cycloheximide (data not shown). Dying cells
displayed typical changes associated with apoptotic cell death,
such as cytoplasmic shrinkage, membrane blebbing, and appearance of
apoptotic bodies (Fig. 2A). An important feature of
apoptotic cell death is retention of cell membrane integrity,
sometimes even after disintegration of cells into apoptotic bodies (43). 3T3 fibroblasts undergoing cell death were
impermeable to trypan blue, indicating that membrane integrity was
retained (Fig. 2A). These results suggest that TNF- induces
apoptotic cell death of fibroblasts in the presence of
macromolecule synthesis inhibitors (also see below).
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plus actinomycin D, we used
biotinylated Val-Ala-Asp(OMe)-fmk (biot-VAD-fmk) (9).
TNF--actinomycin D treatment of fibroblasts resulted in increased
binding to biot-VAD-fmk of a protein (P1) which corresponds in
molecular mass to the activated form of caspase proteases
(approximately 18 kDa) (7) (Fig. 2B). The ability of P1 to
associate with a caspase inhibitor and its molecular mass suggest
that it represents a caspase protease activated by TNF--actinomycin D treatment (also see Discussion). As expected, z-VAD-fmk strongly competes with biot-VAD-fmk for binding to P1 while
z-FA-fmk competes to a significantly lesser extent (Fig. 2B).
Furthermore, the caspase substrate PARP was also processed after
TNF--actinomycin D treatment (Fig. 2C). Importantly, z-VAD-fmk, but
not z-FA-fmk, inhibited PARP cleavage, suggesting that z-VAD-fmk can
inhibit caspase activation in vivo (Fig. 2C). Similar to z-VAD-fmk treatment, vaccinia virus infection completely inhibited P1 activation in 3T3 fibroblasts induced by TNF--cycloheximide treatment (Fig. 2D). In contrast, activation of P1 occurred in cells infected with B13R
mutant virus but not B13R revertant virus following TNF--cycloheximide treatment (Fig. 2D). However, P1 activation in
B13R mutant virus-infected cells was significantly lower than in
uninfected cells, perhaps as a consequence of the presence of the B22R
putative caspase inhibitor. In conclusion, these results indicate
that both z-VAD-fmk and B13R can function as caspase inhibitors
during apoptosis.
Treatment of cells with caspase inhibitors can prevent
apoptosis by death-inducing agents (7). We therefore
tested the effect of z-VAD-fmk on apoptosis induced by TNF-
plus actinomycin D in 3T3 fibroblasts. However, z-VAD-fmk significantly
enhanced cell death induced by TNF- plus actinomycin D (Fig.
2E). These results thus demonstrate that z-VAD-fmk enhances the
cytotoxicity of TNF- plus actinomycin D even though it inhibits
caspase activation.
TNF--caspase inhibitor treatment results in necrotic-like
cell death.
Recent studies have demonstrated an important
role for the NF-B transcription factor in protecting cells
from TNF--induced apoptosis (2, 23, 37, 41). To
investigate whether TNF--caspase inhibitor cytotoxicity
was a result of inhibition of NF-B activation, an EMSA analysis was
performed. No significant difference was seen in NF-B activation by
TNF- in the presence of z-VAD-fmk (Fig.
3A). Thus, TNF--caspase inhibitor
cytotoxicity was not due to inhibition of NF-B.
|
-caspase inhibitor induced cell
death was accompanied by morphological transformations similar to those
seen during apoptotic cell death (43). TNF- plus
actinomycin D treatment of 3T3 fibroblasts resulted in chromatin
condensation and nuclear fragmentation characteristic of
apoptosis (Fig. 3B). In contrast,
TNF--z-VAD-fmk-treated cells did not reveal such changes in
nuclear morphology (Fig. 3B). Furthermore, DNA cleavage occurred in
apoptotic but not in TNF--z-VAD-fmk-treated cells (data not
shown). Importantly, TNF--z-VAD-fmk-treated 3T3 fibroblasts appeared very different from the TNF--actinomycin D-treated cells. The first visible effect in these dying cells was a moderate degree of
cellular edema, which progressed to significant cellular swelling so
that eventually the cells assumed a "balloon-like" morphology (Fig.
3C). Such effects are typically associated with cells undergoing necrotic death (43). Perhaps the most physiologically
relevant difference between apoptotic and necrotic cell death
is that apoptotic cells retain membrane integrity while
necrotic cells do not (43). Unlike apoptotic cells,
TNF--z-VAD-fmk-treated fibroblasts lost membrane integrity rapidly
and before detachment from the culture dish (Fig. 3C). In addition,
TNF--z-VAD-fmk treatment of BALB/c 3T3 fibroblasts and U937
cells or TNF- treatment of vaccinia virus-infected 3T3 fibroblasts
also resulted in similar morphological transformations and cell
lysis (data not shown). These results indicate that
TNF--caspase inhibitor-treated cells do not undergo cell death
by apoptosis but, rather, by a mechanism that appears similar
to necrosis.
Superoxide production by mitochondrial respiratory-chain complexes
during necrotic cell death.
Reactive oxygen species (ROSs) have
previously been implicated in the induction of necrotic cell death
(11). To investigate whether ROSs were generated after
treatment with TNF- plus caspase inhibitors, we first used DHE,
a probe that specifically binds superoxides (45). TNF- or
z-VAD-fmk alone did not significantly affect superoxide levels in 3T3
fibroblasts, while combined treatment with TNF- plus z-VAD-fmk
significantly increased superoxide levels (Fig.
4A). Similarly, TNF- treatment of
vaccinia virus-infected 3T3 fibroblasts was also accompanied by the
production of superoxides (Fig. 4B). In contrast, production of the
peroxide ROSs was not affected after the treatment with TNF plus
z-VAD-fmk (data not shown), indicating the generation of superoxides
but not peroxides during necrosis. Mitochondrial
respiratory-chain complexes are an important source of
intracellular superoxides (36). We therefore tested the
possible involvement of superoxides generated by mitochondrial respiratory-chain complexes in the induction of necrotic cell death by
TNF- plus caspase inhibitors. Remarkably,
TNF--z-VAD-fmk-induced superoxide production and necrosis were both
significantly blocked by either mitochondrial complex I and II
inhibitors (amytal and TTFA) or by the anti-oxidant BHA (Fig. 4C and
D). Similarly, TNF--induced killing of vaccinia virus-infected 3T3
fibroblasts was also significantly blocked by complex I and II
inhibitors or BHA (Fig. 4E). These results indicate a unique role for
mitochondrial respiratory-chain complexes in TNF--induced necrotic
cell death that appears to be dependent on their ability to generate
superoxide species.
|
Caspase inhibitors can also sensitize fibroblasts to dsRNA- and
IFN--induced cytotoxicity.
We then tested whether killing by
other cytotoxic agents could also be potentiated in the presence of
caspase inhibitors. However, killing by two well-known inducers of
apoptosis, staurosporine and ceramide, was not potentiated in
the presence of z-VAD-fmk in 3T3 fibroblasts (data not shown). We then
tested whether treatment with dsRNA or IFN-, two cytotoxic agents
that play an important role in the antiviral response (4,
14), could induce the killing of 3T3 fibroblasts in the presence
of caspase inhibitors. Neither dsRNA nor IFN- treatment had any
cytotoxic effect on 3T3 fibroblasts (Fig.
5). However, in the presence of
z-VAD-fmk, dsRNA and IFN- induced dramatic necrotic-like killing
(Fig. 5). In both cases, BHA or mitochondrial respiratory-chain complex inhibitors significantly blocked cell death (Fig. 5). These results demonstrate that caspase inhibitors can sensitize fibroblasts to
cytotoxicity induced by multiple agents.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results presented here provide evidence for a novel function
of caspase inhibitors in induction of necrotic-like cell death.
Thus, both the vaccinia virus-encoded B13R protein and peptidyl
caspase inhibitors can mediate necrotic killing of cells by
TNF-. Previous studies have demonstrated a key role for NF-B in
inhibiting TNF--induced apoptosis. Here we show that the
caspase inhibitor z-VAD-fmk allows necrotic killing of fibroblasts
by TNF- but does not inhibit the activation of NF-B. Thus,
TNF- cytotoxicity can be mediated by at least two mechanisms:
inhibition of NF-B results in death by apoptosis, while
inhibition of caspase proteases results in death by necrosis. Our
results demonstrate that TNF--induced necrotic killing can be used
to eliminate cells infected with viruses encoding caspase
inhibitors. Thus, the very presence of virus-encoded caspase
inhibitors may render infected cells susceptible to TNF--induced
necrotic cell death.
We have also shown that necrotic killing appears to be dependent on the
generation of superoxides. Interestingly, superoxide production
was not elevated during TNF--induced apoptosis of fibroblasts (data not shown), suggesting that superoxide production accompanies necrotic but not apoptotic cell death. In addition, TNF- and z-VAD-fmk did not induce superoxide production in cells which are resistant to necrotic killing, such as HeLa (data not shown).
In fact, vaccinia virus infection of HeLa cells can render them
resistant to killing by TNF- plus cycloheximide in a B13R-dependent manner (references 8 and 19 and
data not shown). These results suggest that induction of necrosis by
TNF- and caspase inhibitors may occur only when superoxide
generation takes place. Our results also demonstrate the involvement of
the mitochondrial respiratory chain in superoxide production and
induction of necrotic cell death. Thus, mitochondria may also play an
important role in the regulation of necrotic cell death, in addition to
their established function during apoptosis (12).
Interestingly, recent studies have shown that caspase proteases are
also present in mitochondria (25, 34). It is intriguing to
speculate that inhibition of these caspases may be responsible for
increased production of ROSs by the mitochondrial respiratory chain and
induction of necrotic killing by TNF-.
Previous studies have demonstrated a key role for caspase proteases
in the induction of apoptotic cell death. However, our results
provide evidence for a novel function of caspase proteases in
inhibition of necrotic cell death by TNF-. Although caspases involved in inhibiting necrosis are not known, we have recently identified caspase 3 as a major caspase activated in
RelA
/ 3T3 fibroblasts treated with TNF- or
RelA+/+ 3T3 fibroblasts treated with TNF- plus
actinomycin D (corresponding to P1 [unpublished results]). However,
since both z-VAD-fmk and B13R (based on similarity to CrmA) are capable
of interaction with many distinct caspase proteases (including
caspase 3), their necrotic effects could be mediated by inhibition
of multiple caspases. These may also include constitutively active
caspases present at low levels in nonapoptotic cells, such
as TNF--treated 3T3 fibroblasts.
A recent study has also demonstrated increased necrosis in L929 cells
after caspase inhibition (39). However, L929 cells also
undergo necrosis in the presence of TNF- alone (13),
while our results indicate that inhibition of necrosis by caspases
is likely to be a more general phenomenon which can efficiently kill cells that are normally resistant to TNF-. In this regard, our results are also distinct from those of previous studies, which have
shown a switch from apoptosis to necrosis in the presence of
caspase inhibitors but without affecting total killing of cells (16, 44). During the preparation of this paper, Khwaja and Tatton also reported that NIH 3T3 and U937 cells undergo necrotic killing in the presence of TNF- and peptidyl caspase inhibitors (20). These findings, and the results presented here,
demonstrate a role for caspase inhibitors in killing of cells that
are normally resistant to TNF-.
We also show that caspase inhibitors can mediate cytotoxicity in
dsRNA- and IFN--treated cells. Similar to TNF-, dsRNA and IFN-
cytotoxicity was also significantly inhibited by antioxidant or
mitochondrial respiratory-chain inhibitor treatment. These results
suggest that a similar cytotoxic pathway is induced by TNF-, dsRNA,
and IFN- in the presence of caspase inhibitors. In this respect,
it is interesting that viruses have devised strategies to subvert
signaling by all three of these agents (17, 21). Thus,
inhibition of these signaling pathways may not only inhibit the
activation of antiviral gene expression (e.g., alpha/beta IFNs, PKR,
adenylate synthase) but may also inhibit the killing of virus-infected
cells. Similar to TNF-, dsRNA, and IFN-, the Fas death receptor
is an important mediator of host defense. More specifically, Fas
expression on virus-infected cells mediates cytotoxicity through
interaction with Fas ligand-expressing cytotoxic T lymphocytes
(24). Two studies have recently demonstrated the induction
of necrotic killing by the Fas death receptor (18, 40).
Vercammen et al. have shown that Fas induces the necrotic killing of
L929 cells in the presence of caspase inhibitors (40), while Kawahara et al. have demonstrated the induction of necrosis by Fas in cells devoid of caspase 8 (18). These studies
and the results presented here suggest that caspase
inhibitor-mediated killing may be a general mechanism shared by
multiple antiviral agents. Such killing may play an important
role in host defense strategies against viral infections.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge G. L. Smith for providing vaccinia virus mutants. We also thank J. Manley and Carol Prives for comments on the manuscript, P. Bruzzo for technical assistance, and J. Shanley for help with photography.
This work was supported in part by NIH grant R01 CA074892 to A.A.B.
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FOOTNOTES |
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* Corresponding author. Mailing address: 1110 Fairchild Center, Department of Biological Sciences, 1212 Amsterdam Ave., Columbia University, New York, NY 10027. Phone: (212) 854-5939. Fax: (212) 854-5945. E-mail: aab41{at}columbia.edu.
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Discussion
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