§
* Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and § Cancer Institute
of New Jersey, Rutgers University, Piscataway, New Jersey 08854
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Abstract |
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E1B 19K, the adenovirus Bcl-2 homologue,
is a potent inhibitor of apoptosis induced by various
stimuli including Fas and tumor necrosis factor-. Fas
and TNFR-1 belong to a family of cytokine-activated receptors that share key components in their signaling
pathways, Fas-associating protein with death domain
(FADD) and FADD-like interleukin-1
-converting
enzyme (FLICE), to induce an apoptotic response. We
demonstrate here that E1B 19K and Bcl-xL are able to
inhibit apoptosis induced by FADD, but not FLICE.
Surprisingly, apoptosis was abrogated by E1B 19K and
Bcl-xL when FADD and FLICE were coexpressed. Immunofluorescence studies demonstrated that FADD
expression produced large insoluble death effector filaments that may represent oligomerized FADD. E1B
19K expression disrupted FADD filament formation
causing FADD and FLICE to relocalize to membrane
and cytoskeletal structures where E1B 19K is normally localized. E1B 19K, however, does not detectably bind
to FADD, nor does it inhibit FADD and FLICE from
being recruited to the death-inducing signaling complex
(DISC) when Fas is stimulated. Thus, E1B 19K may inhibit Fas-mediated cell death downstream of FADD recruitment of FLICE but upstream of FLICE activation
by disrupting FADD oligomerization and sequestering
an essential component of the DISC.
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Introduction |
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FAS/APO-1/CD95 (Fas) and tumor necrosis factor receptor-1 (TNFR-1)1 are related receptor molecules
that play a critical role in inducing cells to commit
apoptosis. Fas plays an integral role in the maintenance of
a homeostatic balance within the immune system through
the removal of activated and self-reactive T and B cells
(for review see Nagata, 1997). Cells undergoing Fas- and
TNF-
-mediated apoptosis display classic apoptotic signatures marked by membrane blebbing, chromatin condensation, nuclear degradation, and the formation of apoptotic bodies that are engulfed by neighboring cells (Laster et al., 1988
; Itoh et al., 1991
).
Fas or Fas ligand deficiency in mice causes lymphoproliferative and autoimmune diseases that resemble human
disorders (for review see Nagata, 1997), such as systemic
lupus erythematosus (for review see Thompson, 1995
).
TNF-
has also been implicated in the pathogenesis of
several inflammatory, infectious, and autoimmune diseases. It may play a central role in the development of a
wide range of diseases including diabetes, rheumatoid arthritis, bowel disease, and multiple sclerosis (Klinkert et
al., 1997
; Probert et al., 1997
).
The cytokines Fas ligand or TNF- are the critical components necessary for triggering receptor-mediated cell
death in vivo. For example, in the immune privilege sites
of the eye and testes, constitutive expression of Fas ligand
on its cell surface allows these organs to induce apoptosis
in activated T cells that express Fas before an inflammatory response can be mounted (Bellgrau et al., 1995
; Griffith et al., 1995
). This mechanism for evading an immune
system attack has also been used advantageously by tumor cells. Fas ligand upregulation in melanoma cells induces
Fas-bearing cytotoxic T lymphocytes and natural killer
cells to undergo apoptosis (Hahne et al., 1996
). Thus, cancer cells have subverted the Fas pathway to defend themselves from immune destruction. As a cellular defense
mechanism, cytotoxic T lymphocytes and natural killer cells also express Fas ligand, which contributes to immune
surveillance of virally infected cells that are stimulated to
undergo apoptosis and eliminated through the Fas pathway (for review see Nagata, 1997
). Thus, the Fas and TNF-
apoptosis signaling pathways are required for maintaining
homeostasis in the immune system and for defense against
viral infection.
As premature death of an infected host cell can compromise virus replication, viruses have adapted mechanisms
for escaping apoptosis by expression of antiapoptotic
genes. Examples of viral gene products that interfere with
Fas and TNFR-1 death signaling include: BHRF1 from
Epstein-Barr virus (Henderson et al., 1993; Foghsgaard
and Jäättelä, 1997
); p35 from Autographa californica nuclear polyhedrosis virus (Clem et al., 1991
; Beidler et al., 1995
); MC159 and MC160 from Molluscum contagiosum
virus (Hu et al., 1997
; Thome et al., 1997
); E8 from equine
herpesvirus 2 (Hu et al. 1997
; Thome et al. 1997
); and
CrmA from the cowpox virus (Ray et al., 1992
; Enari et al.,
1995
; Miura et al., 1995
; Tewari and Dixit, 1995
). Adenovirus encodes numerous genes whose products block Fas-
and TNF-
-induced apoptosis. These genes include E3 14.7, 10.4, and 14.5 gene products (Gooding et al., 1988
,
1991a
,b; Shisler et al., 1997
), and the E1B 19K gene product (Gooding et al., 1991a
; Hashimoto et al., 1991
; White
et al., 1992
).
In addition to effectively inhibiting Fas- and TNF--mediated apoptosis, E1B 19K blocks apoptosis induced by apparently unrelated stimuli. During productive adenovirus
infection of human cells, the early gene product E1A stimulates host cell DNA synthesis, thereby causing cells to aberrantly go through the cell cycle (Moran, 1993
). In response to cell cycle deregulation, the host cell undergoes
apoptosis (White et al., 1991
). As a defense mechanism, the E1B 19K protein inhibits this E1A-induced apoptosis
and allows assembly of viral progeny to be completed before the cell commits suicide (White et al., 1991
; Rao et al.,
1992
). Bcl-2 is able to functionally complement E1B 19K
during a permissive infection of human cells (Chiou et al.,
1994b
). In primary baby rat kidney (BRK) cells, E1A also
deregulates cell cycle control and induces apoptosis; however, inhibition of apoptosis in this setting by E1B 19K or
Bcl-2 results in transformation (Rao et al. 1992
; White et
al., 1992
). The induction of apoptosis by E1A in BRK cells is exclusively dependent on wild-type p53 (Debbas and
White, 1993
). E1B 19K, Bcl-2, and Bcl-xL share the ability
to block this p53-mediated apoptosis (Debbas and White,
1993
; Chiou et al., 1994a
; Schott et al., 1995
). Although Bcl-2,
Bcl-xL, and E1B 19K are all capable of conferring a protective effect against apoptosis, the effectiveness of each
varies and may be dependent upon the specific cell type and the death stimuli administered.
In BRK cells, E1A induces accumulation of p53 that
transcriptionally upregulates the death-promoting protein
Bax, leading to activation of downstream caspases and the
final executionary steps in apoptosis (Han et al., 1996a;
Sabbatini et al., 1997
). The biochemical mechanism by
which E1B 19K and Bcl-2 disable p53-mediated apoptosis
is partly a consequence of direct binding to Bax and inhibition of caspase activation (Han et al. 1996a
; Sabbatini et al.
1997
). Despite these insights into the mechanism by which E1B 19K inhibits p53-dependent apoptosis, nothing is
known about how it functions to block Fas- and TNF-
-mediated apoptosis.
Over the past several years, the mechanism through
which Fas mediates cell death is beginning to be elucidated. Upon receptor trimerization with Fas ligand or an
anti-APO-1 antibody, Fas is activated and recruits the adaptor molecule Fas-associating protein with death domain
(FAAD)/mediator of receptor-induced toxicity (MORT1)
(FADD) to its cytoplasmic region via their corresponding conserved death domains (DD) (Boldin et al., 1995; Chinnaiyan et al., 1995
; Kischkel et al., 1995
). FADD also contains a death effector domain (DED) at its NH2 terminus
responsible for the recruitment of FADD-like ICE (interleukin-1
-convertase enzyme) (FLICE)/MORT1-associated Ced-3 homologue (MACH)/caspase-8 (FLICE) via its
two DEDs present on its NH2-terminal prodomain (Boldin et al., 1996
; Muzio et al., 1996
). These protein interactions are required for the formation of the death-inducing
signaling complex (DISC) that leads to downstream apoptotic events (Kischkel et al., 1995
). At its COOH terminus,
FLICE is homologous to a cysteine protease that is activated upon cleavage and is responsible for activation of
downstream caspases leading to the amplification of the
cell death signal and the final executionary steps in apoptosis. FADD and FLICE are also activated in the TNFR-1
pathway upon interaction with TNF-
(for review see Nagata, 1997
). Receptor stimulation allows FADD, and thereby
FLICE, to interact with the adaptor molecule TRADD
that is recruited to the cytoplasmic region of TNFR-1 causing FLICE activation and apoptosis (Hsu et al., 1996
).
Here we investigate the ability of E1B 19K to block apoptosis downstream of Fas. The results implicate FADD multimerization through its DED as a novel mechanism for induction of apoptosis. E1B 19K inhibits FADD-induced cell death coincident with FADD filament disruption and relocalization to membranes and cytoskeletal structures. Recruitment of FLICE by FADD allows E1B 19K to block FLICE-induced apoptosis only in the presence of FADD. Since E1B 19K does not directly associate with FADD or FLICE, we conclude another essential DISC component is mediating this protein interaction. Therefore, we propose E1B 19K abrogates Fas-mediated apoptosis by disrupting FADD multimerization and possibly sequestering an integral protein from the DISC.
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Materials and Methods |
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Antibodies
The following antibodies were used for indirect immunofluorescence, immunoprecipitation, and Western blotting analyses: E1B 19K was visualized with the 2F3 monoclonal antibody and a rabbit polyclonal antibody
directed against the E1B 19K protein (Han et al., 1996a; White and Cipriani, 1990
). The monoclonal AU1 antibody directed against an AU1
epitope tag was purchased from Berkeley Antibody Co. (Richmond, CA).
The monoclonal HA.11 and the polyclonal HA.11 antibodies directed
against the hemagglutinin (HA) epitope tag were also purchased from
Berkeley Antibody Co. The monoclonal Myc antibody-1 directed against
a Myc epitope tag was purchased from Oncogene Science (Cambridge,
MA). The monoclonal antibody directed against actin was purchased
from Amersham Corp. (Arlington, IL). The anti-Flag M5 monoclonal antibody was purchased from Scientific Imaging Systems (New Haven, CT).
The monoclonal antibody directed against Apo-1/Fas was purchased from
Alexis Biochemicals Corp. (San Diego, CA). A polyclonal antibody directed against FADD was a gift of V. Dixit (University of Michigan, Ann
Arbor, MI).
Plasmids
pCMV-E1B 19K (White and Cipriani, 1989, 1990
) expresses the wild-type
adenovirus 2 E1B 19K protein from the cytomegalovirus (CMV) promoter and has been previously characterized. pcDNA3-E1B 19K (Han et al.,
1996a
) expresses E1B 19K from a CMV promoter and has been previously described. pCMV-7dl (White et al., 1992
) is identical to pCMV-E1B
19K except that it does not express a functional protein due to a frameshift mutation at codon 7 within the coding region of the E1B 19K gene.
pCMV-7dl was used as a negative control for E1B 19K function.
pcDNA3-AU1-FADD and pcDNA3-AU1-FADD-DN (Muzio et al.,
1996
) are CMV expression vectors for an NH2-terminal AU1-tagged human FADD and an NH2-terminal AU1-tagged truncated form of FADD
lacking 18 NH2-terminal amino acids, respectively. Both expression vectors were generously provided by V. Dixit. pcDNA3-HA-FLICE (Muzio
et al., 1997
) expresses the human FLICE with an influenza virus hemagglutinin (HA) epitope tag at its COOH terminus and driven by a CMV
promoter, and was a gift of V. Dixit. pcDNA3-CrmA (Miura et al., 1993
)
expresses the cowpox virus ICE inhibitor CrmA from a CMV promoter
and was a gift of J. Yuan (Massachusetts General Hospital, Boston, MA).
pcDNA3-Flag-Bcl-xL, (Merino et al., 1995
) a CMV-driven expression plasmid expressing the human Bcl-xL protein with a Flag-epitope tag at its
NH2 terminus was kindly provided by G. Nunez (University of Michigan,
Ann Arbor, MI). pcDNA3-Myc-hNbk expresses a human Nbk/Bik protein, with a Myc-epitope tag at its NH2 terminus, from a T7 promoter for in vitro transcription/translation as previously described (Han et al., 1996b
).
The pcDNA3-Myc-Bcl-2 plasmid expresses human Bcl-2 under the control of the CMV promoter and was cloned from pGEM2-Bcl-2 (Han et al.
1996a
). A specific 5' primer was used to engineer a KpnI site in front of
the Myc tag and ATG start site. The PCR products were digested with
KpnI and XhoI sites and then ligated into pcDNA3. The pCMV-
-galactosidase (
-gal) expression vector expresses
-gal from the CMV promoter and was provided by C. Abate-Shen (Center for Advanced Biotechnology and Medicine and the University of Medicine and Dentistry of
New Jersey, Piscataway, NJ).
Indirect Immunofluorescence
HeLa cells were transfected with 30 µg of the pCMV-7dl control vector, 8 µg of pcDNA3-HA-FLICE, 10 µg of pcDNA3-AU1-FADD, 10 µg of pcDNA3-FADD-DN, 12 µg of pCMV-E1B 19K, 12 µg of pcDNA3-Myc-Bcl-2, and 12 µg of pcDNA3-Flag-Bcl-xL expression plasmids. Cells were also transfected with 12 µg of pcDNA3-CrmA expression plasmid to retain cell viability. Cells were fixed at 48 h posttransfection in a 2% paraformaldehyde solution and then permeabilized with PBS containing 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, MO). Cells were double labeled with the indicated antibodies and visualized with goat anti- mouse rhodamine-conjugated and goat anti-rabbit fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Photography was performed using a microscope equipped with epifluorescence optics (model FXA; Nikon Inc., Garden City, NY).
Western Blotting
Whole cell extracts were prepared for Western blot analysis 48 h posttransfection. 20 µg of protein from each sample were analyzed by SDS-PAGE and semidry-blotted onto nitrocellulose membranes. Proteins were detected by enhanced chemiluminescence and used according to the manufacturer's specifications (Amersham Corp.).
In Vitro Protein Interaction Assay
Expression vectors encoding E1B 19K (pcDNA3-E1B 19K), FADD
(pcDNA3-AU1-FADD), FADD-DN (pcDNA3-AU1-FADD-DN), FLICE
(pcDNA3-HA-FLICE), and Nbk/Bik (pcDNA3-Myc-hNbk), were in
vitro transcribed and translated according to the manufacturer's specifications (Promega Corp., Madison, WI). The in vitro translated 35S-labeled
proteins were titrated to equal concentrations and incubated with an anti-FADD polyclonal antibody or an anti-E1B 19K polyclonal antibody (see
figure legend) in the presence of 0.5 ml NETN lysis buffer (20 mM Tris,
pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.2% NP-40) (Han et al., 1996a).
Protein complexes were collected with protein A-Sepharose (Pharmacia
Biotech Inc., Piscataway, NJ) and washed three times with NETN buffer.
Proteins were resolved on a 17% SDS-PAGE gel and then subjected to
autoradiography.
Immunoprecipitation
HeLa cells were transiently transfected with 3 µg of pcDNA3-HA-FLICE,
3 of µg pcDNA3-AU1-FADD, 12 µg of pCMV-E1B 19K, and 12 µg of
pcDNA3-CrmA expression plasmids as indicated in Fig. 8, and then subjected to immunoprecipitation analysis at 48 h posttransfection (Kischkel
et al. 1995). Briefly, a 10-cm plate of cells was trypsinized, centrifuged at
1,000 g, and then resuspended in 37°C DME. Fas receptor was stimulated
by preincubation with 2 µg/ml of anti-APO-1 antibody for 15 min and
then immediately placed on ice. Cells were washed in PBS and lysed in 1 ml
of lysis buffer (as stated above) containing 1% Triton X-100 and 10%
glycerol for 30 min at 4°C. Lysates were centrifuged and protein A-Sepharose (Pharmacia Biotech Inc.) was added to the supernatant. As a negative control, 2 µg/ml of anti-APO-1 antibody and protein A-Sepharose were added to the lysates of unstimulated cells. Lysates were incubated at
4°C on a rotator (Labquake Shaker model 400110; Barnstead/Thermolyne Inc., Dubuque, IA) for 3 h. The beads were washed five times in lysis
buffer, boiled for five minutes in Laemmli buffer (Laemmli, 1970
), and then
resolved by SDS-PAGE analysis. Immunoprecipitated FADD and FLICE
were detected using Western blot analysis by probing with anti-FADD
polyclonal and anti-HA polyclonal antibodies, respectively.
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Viability Assays
Induction of cell death by FADD and its inhibition by Bcl-2 family members was assessed by a -gal viability assay (Han et al., 1996b
). HeLa cells
(6-cm plates) were electroporated, as previously described (Chiou et al.,
1994a
), with 18 µg of the pCMV-7dl control vector, 6 µg of pcDNA3-AU1-FADD, 12 µg of pCMV-E1B 19K, 12 µg of pcDNA3-Myc-Bcl-2, and
12 µg of pcDNA3-Flag-Bcl-xL expression plasmids, as indicated in the
text. All samples were also transfected with 6 µg of pCMV-
-gal DNA.
Total DNA transfected was kept constant at 18 µg. 48 h posttransfection,
cells were fixed and the percentage of viable blue cells assessed by
5-bromo-4-chloro-3-indoxyl-
-D-galactopyranoside staining and examined
by phase-contrast microscopy as previously described (Han et al., 1996b
).
FLICE functional assays were performed by transfecting HeLa cells
with 30 µg of the pCMV-7dl control vector, 8 µg of pcDNA3-HA-FLICE, 10 of µg pcDNA3-AU1-FADD, 12 µg of pCMV-E1B 19K, 12 µg of
pcDNA3-Myc-Bcl-2, and 12 µg of pcDNA3-Flag-Bcl-xL expression constructs as indicated in Fig. 1 b. 10 µg of pCMV--gal was added to all the
samples and the total DNA concentration was kept constant at 30 µg. Viability was assessed as stated above.
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Results |
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E1B 19K and Bcl-xL Inhibit FADD but Not FLICE-induced Apoptosis
The adenoviral E1B 19K protein is a potent inhibitor of
Fas- and TNF--induced apoptosis (Gooding et al. 1991a
;
Hashimoto et al. 1991
; White et al. 1992
). Other Bcl-2 family members, however, vary in their ability to block death
receptor-mediated apoptosis (Chiou et al. 1994b
; Armstrong et al., 1996
; Chinnaiyan et al., 1996a
; Nagata 1997
).
We sought to identify at which point in the signaling cascade E1B 19K, Bcl-2, and Bcl-xL interfere with apoptosis induction by Fas and TNF-
, and whether they act by a
similar mode to inhibit cell death.
E1B 19K, and to a lesser extent Bcl-2, have been shown
to abrogate Fas- and TNF--induced apoptosis in HeLa
cells (Gooding et al., 1991a
; White et al., 1992
; Chiou et al.,
1994b
; Armstrong et al., 1996
). Since E1B 19K does not affect the presence of the TNF receptors at the cell surface
of adenovirus infected cells, we have postulated E1B 19K
must be acting at, or downstream of, the receptor to block
cell death (White et al., 1992
). To determine at which
point downstream of death receptors the survival signal is
exerted, we examined the ability of E1B 19K, Bcl-2, and
Bcl-xL to inhibit FADD- and FLICE-induced death.
Transient expression of FADD in HeLa cells caused
only 10% of the transfected cells to remain viable compared with transfection with a plasmid control pm7dl
(7dl), that does not express a functional protein (Fig. 1 a).
CrmA acts as a competitive inhibitor for the ICE-related
proteases, and has been shown to abrogate both Fas- and
TNF--mediated apoptosis (Enari et al., 1995
; Miura et al., 1995
; Tewari and Dixit, 1995
). As a positive control for apoptosis inhibition, FADD was coexpressed with the ICE inhibitor CrmA, which restored cell viability, as expected
(Fig. 1 a). FADD was then coexpressed with E1B 19K, to
determine if apoptosis could be abrogated (Fig. 1 a). Approximately 90% of the cells survived when E1B 19K protein was coexpressed with FADD. Thus, E1B 19K potently inhibits both Fas- and FADD-induced apoptosis.
Bcl-2 and Bcl-xL were similarly examined for suppression of FADD-induced death. Hela cells were cotransfected with FADD, and either Bcl-2 or Bcl-xL. Although
both Bcl-2 and Bcl-xL were highly expressed, Bcl-2 was not
able to block FADD-induced death. In fact, overexpression of Bcl-2 in HeLa cells appears to be partially toxic as
indicated by the 50% reduction in viability (Fig. 1 a). In
contrast, Bcl-xL restored viability to FADD-transfected
cells to a similar extent as E1B 19K expression (Fig. 1 a).
These results correlate with the ability of E1B 19K, and
not Bcl-2, to nearly completely abrogate Fas-induced cell
death (Gooding et al., 1991a; White et al., 1992
; Chiou et al.,
1994b
). Therefore, we conclude Bcl-xL and E1B 19K, but
not Bcl-2, can ablate the apoptotic function of FADD
downstream of Fas.
FLICE activation is induced upon its recruitment to the
DISC that is mediated through DEDs in the prodomain of
FLICE interacting with FADD (Boldin et al., 1996; Muzio
et al., 1996
). We addressed whether inhibition of cell death
was acting at FADD or further downstream at FLICE.
FLICE was expressed alone or in the presence of E1B
19K, Bcl-2, and Bcl-xL (Fig. 1 b). FLICE overexpression caused a 50% reduction in cell viability compared with the
plasmid control 7dl. Coexpression of E1B 19K, Bcl-2, or
Bcl-xL did not significantly increase cell survival upon
FLICE overexpression. In conclusion, E1B 19K and Bcl-xL, but not Bcl-2, can inhibit FADD-induced cell death,
but neither E1B 19K or Bcl-xL can block cell death upon
FLICE overexpression.
E1B 19K and Bcl-xL Inhibit FLICE-induced Apoptosis in the Presence of FADD
As implicated in the FADD and FLICE functional assays, inhibition of apoptosis was upstream of FLICE and thus possibly acting at FADD. To further address where the Bcl-2 family of inhibitors exert their protective effects, FADD and FLICE were coexpressed alone or in the presence of E1B 19K, Bcl-xL, or Bcl-2. When FADD was coexpressed with FLICE, apoptosis was increased significantly compared with FLICE overexpression (Fig. 1 b). E1B 19K expression nearly completely abrogated cell death when FADD and FLICE were coexpressed (Fig. 1 b). This inhibition in cell death by E1B 19K was just as efficient as that of the CrmA control. Thus, E1B 19K, despite being unable to block cell death upon FLICE activation, was able to block FLICE-induced apoptosis in the presence of overexpressed FADD.
Similarly, Bcl-xL and Bcl-2 were coexpressed with FADD and FLICE to determine if they possessed a similar activity to that of the E1B 19K protein. A dramatic increase in viable cell number was observed when Bcl-xL, and less profoundly when Bcl-2, were coexpressed with FADD and FLICE (Fig. 1 b). Taken together, these results suggest that Bcl-2 is not very proficient at blocking Fas-mediated apoptosis, or may be acting differently from E1B 19K and Bcl-xL.
E1B 19K Disrupts FADD Filament Formation
E1B 19K is found to associate with the nuclear lamina as
well as the cytoplasmic and nuclear membranes (White et
al., 1984; White and Cipriani, 1990
). Previous experiments
have shown E1B 19K partially colocalizes with Bax in cytoplasmic membranes and inhibits cell death through direct interaction with Bax during p53-mediated apoptosis
(Han et al., 1996). To gain insight into the mechanism
through which E1B 19K abrogated FADD-induced, as well as FLICE-induced apoptosis in the presence of
FADD, indirect immunofluorescence was performed.
HeLa cells were transfected with either an AU1-tagged
FADD, or an HA-tagged FLICE, alone or in the presence
of E1B 19K. Double-label indirect immunofluorescence at
48 h posttransfection was performed to ascertain whether
the subcellular localization of FADD or FLICE could be
altered in the presence of E1B 19K.
HeLa cells were transfected with HA-tagged FLICE
and protein expression was visualized with an anti-HA
polyclonal antibody. Cells transiently transfected with
FLICE demonstrated a diffuse cytoplasmic and nuclear
staining that was not observed in vector control transfected cells (Fig. 2, A and B). Although diffuse staining
patterns as we have observed for FLICE are less obvious by indirect immunofluorescence than proteins displaying a
discrete localization pattern, FLICE-positive cells were
clearly visible. E1B 19K localized to the cytoplasmic and
nuclear membranes and the cytoskeletal structures as previously reported (Fig. 2 D) (White and Cipriani, 1989,
1990
) Co-expression of E1B 19K with FLICE did not alter
the subcellular localization of FLICE, nor was any colocalization of the two proteins observed (Fig. 2, C and D).
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HeLa cells transiently expressing AU1-tagged FADD
were stained with an anti-FADD polyclonal or anti-AU1
monoclonal antibodies. The staining pattern of FADD
was strikingly different from FLICE, with FADD overexpression causing the formation of large filamentous structures in the nucleus and cytoplasm that react with anti-FADD or anti-FADD epitope tag antibodies (Fig. 3 B).
These structures were not present in the 7dl vector control-transfected cells (Fig. 3 A). Yeast two-hybrid experiments have shown FADD self-associates and this oligomerization process may be essential for the induction of
cell death (Boldin et al., 1995). This is similar to the manner in which Fas receptor trimerization is required for
transduction of the death signal (Kischkel et al., 1995
).
Thus, filament formation due to FADD overexpression
may result from FADD multimerization.
|
To address whether E1B 19K can alter the subcellular localization of FADD, immunofluorescence studies with E1B 19K coexpression were performed. As shown in Fig. 3 C, coexpression of FADD with E1B 19K dramatically altered the localization pattern of FADD, with complete disruption of the filaments into large aggregates in the cytoplasm. However, FADD filaments within the nucleus did not appear affected as indicated by phase-contrast and electron microscopy (data not shown). E1B 19K does not localize within the nucleus and, therefore, may not be able to disrupt nuclear FADD filaments. Double labeling with an anti-E1B 19K monoclonal antibody and an anti-FADD polyclonal antibody revealed complete colocalization of the two proteins, with FADD displaying the typical localization of the E1B 19K protein (cytoplasmic and nuclear membranes as well as the cytoskeletal structures) (Fig. 3, C and D).
An AU1-tagged FADD-DN, a death effector domain
deletion mutant that does not self-associate or induce
death, was visualized with an anti-AU1 monoclonal antibody (Boldin et al., 1995; Chinnaiyan et al., 1996b
). Its expression pattern was diffuse within the cytoplasm and nucleus similar to that of FLICE (Fig. 3 F). This result
suggests that FADD filament formation, and thus oligomerization, may play an integral role in mediating apoptosis. As seen in Fig. 3, G and H, addition of E1B 19K did
not change FADD-DN subcellular localization, suggesting
that colocalization of E1B 19K with FADD requires either
full-length or the DED of FADD.
FADD Recruits FLICE into Filaments That Are Disrupted by E1B 19K
To ascertain whether FADD could alter the localization of FLICE, Hela cells transiently coexpressing AU1-tagged FADD and HA-tagged FLICE were examined by indirect immunofluorescence with anti-FADD and anti-HA antibodies, respectively. The intracellular staining pattern of FADD remained as large filaments, however, FLICE no longer had a diffuse cytosolic pattern (Fig. 4 A) but was recruited by FADD into the large filamentous structures as evidenced by the colocalization of FADD and FLICE (Fig. 4 B). Thus, FADD was able to alter the localization pattern of FLICE most likely through a direct protein- protein interaction with FLICE. We were, however, not able to detect endogenous FADD or FLICE filaments because antibodies sufficient to detect the endogenous proteins by indirect immunofluorescence were not available.
|
To test if E1B 19K could alter the localization of both FADD and FLICE, E1B 19K was coexpressed with both, and the transfected cells were examined by indirect immunofluorescence for E1B 19K, FADD, and FLICE. Fig. 4, C-E demonstrates that coexpression of FLICE and FADD in the presence of E1B 19K significantly altered the diffuse cytosolic distribution of FLICE such that FLICE colocalized with both FADD and E1B 19K (Fig. 4 D). FADD directly binds to FLICE and by E1B 19K altering the localization of FADD, E1B 19K apparently alters the localization of FLICE (Fig. 4, C-E). The alteration in localization of FADD and FLICE by E1B 19K suggests that the ability of E1B 19K to protect the cells from apoptosis may be through functional sequestration of both FADD and FLICE.
Bcl-xL Expression Attenuates FADD Filament Formation
To address whether other Bcl-2 homologues may also implement a similar mechanism for inhibition of FADD-
induced apoptosis, immunofluorescence studies were executed with Bcl-2 and Bcl-xL. Bcl-2 and Bcl-xL localize at
the mitochondrial, endoplasmic reticulum, and nuclear
membranes (Figs. 5 B and 6 B) (for review see White, 1996). In HeLa cells, transient FLICE expression remained diffuse throughout the cytoplasm and nucleus
even when FLICE was coexpressed with Bcl-2 or Bcl-xL,
(Figs. 5 A and 6 A, respectively).
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|
When FADD was coexpressed with Bcl-2, FADD filaments were still apparent but reduced in length or in number (Fig. 5 D) compared with that of FADD overexpression alone (Fig. 5 C). In contrast, overexpression of Bcl-xL substantially attenuated the degree of FADD filament formation. Bcl-xL expression caused FADD filaments to be highly disrupted (Fig. 6 D). Occasionally, FADD-expressing cells displayed filament formation in the presence of Bcl-xL. These cells, however, routinely expressed unusually high levels of Bcl-xL, suggesting a concentration dependence of the ability of Bcl-xL to disrupt FADD filaments. However, in contrast to E1B 19K, Bcl-xL does not colocalize with FADD. These results suggest that Bcl-2 and Bcl-xL have a differential effect on FADD filament formation and that Bcl-xL inhibits Fas-mediated apoptosis differently than E1B 19K.
When HeLa cells were transiently transfected with FADD, FLICE, and either Bcl-2 or Bcl-xL, FLICE remained diffuse throughout the cell (Figs. 5 G and 6 G, respectively). When FADD, FLICE, and Bcl-2 were coexpressed, the appearance of FADD filaments remained consistent with that of FADD and Bcl-2 coexpression alone. Together, these results suggest that FADD failed to recruit FLICE into these filaments in the presence of Bcl-2 (Fig. 5, G and F). Coexpression of Bcl-xL with FADD and FLICE profoundly disrupted FADD filaments in almost all transfected cells (Fig. 6 F). In contrast to the results in E1B 19K-expressing cells, there was no colocalization of FLICE and FADD when Bcl-2 or Bcl-xL was coexpressed (Figs. 5 and 6). Therefore, we conclude, Bcl-xL alters the ability of FADD to polymerize and recruit FLICE, whereas E1B 19K apparently disrupts FADD organization without affecting FLICE recruitment.
E1B 19K Does Not Interact with FADD or FLICE In Vitro
The E1B 19K protein is predominantly insoluble and
bound to the membranes and cytoskeleton in adenovirus
infected and transformed cells (White et al., 1984; White
and Cipriani, 1989
, 1990
). Alteration in FADD and
FLICE subcellular localization by E1B 19K and colocalization with E1B 19K suggested that E1B 19K, FADD,
and FLICE may directly associate to form an insoluble
protein complex. To examine the ability of E1B 19K to associate with FADD or FLICE in vitro, E1B 19K, FADD,
and FLICE were in vitro transcribed/translated and analyzed by coimmunoprecipitation. As expected, an anti-FADD antibody was able to coimmunoprecipitate FADD
and FLICE but there was no detectable interaction between FADD-DN and FLICE (Fig. 7, lanes 1 and 2). Interestingly, coincubation of FADD and FLICE caused the
appearance of a 21-kD band that was not detectable in individually in vitro-translated FADD or FLICE. Whether
this band represents a FADD or FLICE cleavage product
remains to be determined. An E1B 19K-specific antibody
was not able to coimmunoprecipitate E1B 19K with either
FADD or FLICE individually or when cotranslated (Fig.
8, lanes 3-5). The E1B 19K interacting protein Nbk/Bik
served as a positive control for the presence of a direct
protein-protein interaction with E1B 19K (Fig. 7, lane 6).
|
Yeast two-hybrid analysis has also confirmed no detectable direct association between FADD or the prodomain of FLICE with E1B 19K (data not shown). Thus, we can conclude E1B 19K does not directly interact with FADD or FLICE, or that FADD and/or FLICE require modification that does not take place in vitro or in yeast to interact with E1B 19K.
E1B 19K Does Not Inhibit FADD and FLICE from Being Recruited to the DISC
Coimmunoprecipitation experiments revealed that engagement of Fas receptor with anti-APO-1 antibody recruits FADD and FLICE to the cytoplasmic domain of
Fas, and this protein complex collectively forms the DISC
(Kischkel et al., 1995). This multiprotein interaction may
allow FLICE to undergo autocatalytic activation and to induce apoptosis. To address whether E1B 19K was able to
abrogate DISC formation, HeLa cells overexpressing
FADD, FLICE, and CrmA, with and without E1B 19K,
were subjected to Fas activation using an anti-APO-1
monoclonal antibody (Fig. 8 a). HeLa cells were first preincubated with an anti-APO-1 antibody for 15 min before
cell lysis (Fig. 8 a, lanes 1, 3, and 5). Immune complexes
were collected with protein A and subjected to immunoblot analysis using an anti-FADD polyclonal antibody. As
a negative control for Fas-interacting proteins, the cell lysates were generated without receptor engagement with
the addition of an anti-APO-1 antibody after cell lysis
(Fig. 8 a, lanes 2, 4, and 6). FADD and FLICE were able
to coimmunoprecipitate with Fas even in the presence of
E1B 19K (Fig. 8 a, lanes 3 and 5). This data suggests the
mechanism for protection by E1B 19K is not solely the sequestration of FADD and FLICE to an insoluble intracellular compartment, since both proteins were able to interact with Fas at the plasma membrane.
E1B 19K Inhibits FLICE Activation
To address whether overexpression of FADD and FLICE autocatalyzes FLICE activation in the presence of E1B 19K, HeLa cells were transiently transfected with FLICE or FADD plus FLICE alone or in the presence of E1B 19K (Fig. 8 b). 7dl vector control was overexpressed as a negative control for FLICE expression. Whole cell lysates were produced 20 (data not shown) and 48 h posttransfection and then probed with an anti-HA polyclonal antibody. Full-length proFLICE appeared as a 66-kD band in cells that overexpressed FLICE or FLICE plus E1B 19K (Fig. 8 b, lanes 2 and 3). Due to ~40% of the transfected cells remaining viable, the inactive zymogen form of FLICE was still detectable. Unprocessed full-length proFLICE, however, disappeared when FADD and FLICE were coexpressed, indicating that increased processing and activation of FLICE had taken place (Fig. 8 b, lane 4). We were not able to detect the 10-kD COOH-terminal cleavage product, which suggested that the activated forms of cytosolic FLICE may have a short half-life. Coexpression of E1B 19K, FADD, and FLICE inhibited FLICE activation as indicated by the abundance of unprocessed proFLICE (Fig. 8 b, lane 5). These results suggest E1B 19K blocks FLICE-mediated apoptosis, in a FADD-dependent fashion, upstream of FLICE activation.
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Discussion |
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---|
We have established E1B 19K is highly efficient at inhibiting FADD- but not FLICE-induced apoptosis. Coexpression of FADD and FLICE profoundly diminishes cell viability and E1B 19K dramatically rescues cells from this
apoptosis. This mechanism for inhibition of cell death may
be functionally homologous to the C. elegans model for
abrogation of apoptosis. C. elegans encodes three integral genes necessary for the regulation of programmed cell
death (Hengartner, 1996). The ced-3 gene encodes an effector protein that is homologous to the interleukin-1
-converting enzyme ICE-like family members (Yuan et al.,
1993
). Its activation is inhibited by the Ced-9 gene product
that is able to sequester Ced-3 through protein-protein interactions (Chinnaiyan et al., 1997a
,b; Seshagiri and
Miller, 1997
; Spector et al., 1997
; Wu et al., 1997
). The Ced-3-Ced-9 interaction is mediated by a third bridging protein
encoded by the ced-4 gene. This model for protection of cell
death can be applied to mammalian cells through protein
substitution. Several labs have shown Bcl-xL can inhibit
FLICE activation and, subsequently, cell death when in the
presence of the Ced-4 gene product (Chinnaiyan et al.,
1997b
; Seshagiri and Miller, 1997
; Wu et al., 1997
).
The adenoviral E1B 19K protein is able to abrogate Fas-mediated apoptosis likely through protein-protein interactions with key DISC components. The FADD adaptor molecule is required for E1B 19K to block FLICE-induced cell death. However, another protein(s), posttranslational modification and/or a conformational change of FADD appears to be required for E1B 19K to interact with FADD and for disruption of FADD filament formation.
Immunofluorescence studies reveal overexpression of
FADD causes FADD to form filaments within the cell (refer to Fig. 3 C) (Siegel et al., 1998). Electron microscopy
indicates FADD forms higher order filamentous structures arranged in bundles (data not shown). These filaments are localized in the cytosol as well as in the nucleus.
Furthermore, they do not associate with cytoskeletal structures such as vimentin (data not shown) or tubulin (Siegel et al., 1998). Full-length FADD is known to self-associate
(Boldin et al., 1995), suggesting the filaments result from
FADD multimerization.
A FADD-DN expression vector containing a deletion
within the death effector domain does not self-associate
nor does it induce cell death (Boldin et al., 1995; Chinnaiyan et al., 1996b
). Overexpression of FADD-DN does
not cause filament formation but is rather localized diffusely throughout the cell. This result implies that FADD
oligomerization may be required for mediating cell death.
This is similar to other death domain-containing molecules such as Fas, whereby trimerization is necessary for
transduction of the death signal (Kischkel et al., 1995
).
The DED, but not the DD, of FADD is able to directly bind to full length FADD (Boldin et al., 1995
). This suggests the DED is essential for both multimerization and
for inducing cell death.
E1B 19K does not alter the subcellular localization of FLICE but is able to strikingly disrupt FADD filament formation and relocalize FADD into the membranous and cytoskeletal elements of the cell where the E1B 19K protein is normally found. The disruption of FADD filaments is concurrent with the ability of E1B 19K to block FADD-induced apoptosis. E1B 19K also blocks FLICE-induced cell death in the presence of FADD but apparently does not block FLICE from associating with FADD in the cytoplasm or the DISC.
Indirect immunofluorescence (refer to Fig. 4) and subcellular fractionation (data not shown) studies demonstrate that E1B 19K is also able to sequester FLICE to an
insoluble intracellular compartment only in the presence
of FADD. These results further indicate FADD is acting
as an adaptor molecule bridging FLICE with E1B 19K.
Other viruses such as Molluscum contagiosum and equine
herpes-virus 2 are capable of blocking FADD-mediated
cell death, but not FLICE-induced apoptosis, through expression of viral inhibitory proteins MC159 and E8, respectively (Hu et al., 1997; Thome et al., 1997
). The mechanism used by these viral proteins is by direct interaction
with the inducers of cell death, FADD and FLICE, which
abrogates formation of a complete DISC and results in inhibition of apoptosis. Therefore, we propose E1B 19K
may act analogously and form a probably indirect physical
interaction with FADD, thereby causing an alteration in
its subcellular localization and ability to oligomerize. Since
we have been unable to detect binding of E1B 19K to
FADD, another protein, posttranslational modification,
and/or a conformational change of FADD may be required for E1B 19K to interact with the FADD-FLICE
complex and disrupt its function.
The current model of Fas activation proposes that heterooligomerization of FADD and FLICE at the DISC results in FLICE activation. Coimmunoprecipitation of
FADD and FLICE with Fas demonstrates FADD and
FLICE are recruited to the DISC upon Fas activation by anti-APO-1 antibody (Kischkel et al., 1995; Muzio et al.,
1996
). Thus, sequestration of FADD/FLICE into an insoluble intracellular compartment by E1B 19K may abrogate
activation of FLICE by preventing recruitment to the receptor complex upon Fas receptor stimulation. However,
FADD and FLICE recruitment occurs even in the presence of E1B 19K. Thus, we can conclude FADD and
FLICE are present at the DISC, but E1B 19K is still able
to abrogate cell death. Recent data has shown that FLICE
needs to be clustered for full activation and induction of
apoptosis (Muzio et al., 1998
). Thus, we propose a mechanism in which FADD needs to be multimerized at the
DISC, similar to Fas, to activate FLICE. E1B 19K may sequester a portion of the FADD molecules, thereby depleting the number of FADD molecules recruited to the DISC
and subsequently inhibiting its oligomerization and activation of FLICE.
Conflicting data has been reported by many labs as to
whether other Bcl-2 family members, particularly Bcl-2
and Bcl-xL, are able to block Fas-mediated apoptosis.
Even within the same cell line, Bcl-2 has been shown to be
either nonfunctional or act as a potent inhibitor of Fas-
mediated death (Armstrong et al., 1996; Chinnaiyan et al.,
1996a
). These discrepancies may be due to differences in
cell type specificity, strength of the death signal, and levels of expression of the inhibitory proteins. Our results demonstrate Bcl-xL and E1B 19K efficiently block Fas-induced
cell death, whereas Bcl-2 functions poorly in the same assay. Bcl-xL inhibits FADD- but not FLICE-induced death
and is also able to block FLICE-mediated death in the
presence of FADD. These results are coincident with Bcl-xL disrupting the formation of FADD filaments. Bcl-2,
however, appears to be only marginally effective in inhibiting Fas-mediated cell death and does not substantially alter FADD filament formation. Recent evidence has shown
Bcl-2 is cleaved subsequent to anti-Fas antibody treatment, generating a cleavage product that has proapoptotic activity similar to that of Bax and Bak (Cheng et al., 1997
). E1B 19K, however, is not cleaved during the apoptotic
process to generate a proapoptotic derivative (Han et al.,
1996). The difference between the potent inhibitory activity of E1B 19K, in contrast to the weak effect of Bcl-2, in
Fas-mediated apoptosis may reside within this intrinsic
difference between E1B 19K and Bcl-2.
Coexpression of FADD and FLICE without E1B 19K caused increased FLICE activation as indicated by the disappearance of full-length FLICE. In contrast, the presence of FLICE in the unprocessed pro form when coexpressed with FADD and E1B 19K, indicates that E1B 19K expression inhibits FLICE activation. This result, however, does not rule out the possibility that undetectable levels of processed FLICE are present.
Bcl-xL appears to affect FADD and FLICE differently
than E1B 19K. Rather than diverting FADD and FLICE
into large aggregates, Bcl-xL appears to prevent FADD filament formation and FLICE recruitment into the filaments. This may be a mechanism for inhibiting FADD from promoting FLICE activation. However, Bcl-xL has
been reported to act downstream of FLICE activation
(Boise and Thompson, 1997). Whether dispersed FLICE,
in FADD- and Bcl-xL-expressing cells, is activated, has yet
to be determined. Nonetheless, E1B 19K appears to function differently than Bcl-xL to inhibit the Fas and TNFR-1 death-signaling pathway by blocking FLICE activation
subsequent to FADD recruitment.
![]() |
Footnotes |
---|
Received for publication 4 February 1998 and in revised form 28 April 1998.
Address all correspondence to Eileen White, Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: (732) 235-5329. Fax: (732) 235-5795. E-mail: ewhite{at}mbcl.rutgers.eduWe wish to thank V. Dixit, G. Nunez, and J. Yuan for expression plasmids and antibodies. We would also like to thank K. Degenhart and G. Kasoff (both from Center for Advanced Biotechnology and Medicine, Piscataway, NJ) for advice and critical reading of the manuscript.
This work has been supported by a grant from the National Institutes of Health (CA53370) to E. White.
![]() |
Abbreviations used in this paper |
---|
-gal,
-galactosidase;
BRK, baby rat
kidney;
CMV, cytomegalovirus;
DD, death domain;
DED, death effector
domain;
DISC, death-inducing signaling complex;
HA, hemagglutinin;
ICE, interleukin-1
-convertase enzyme;
TNFR-1, tumor necrosis factor
receptor-1.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Armstrong, R.C., T. Aja, J. Xioang, S. Gaur, J.F. Krebs, K. Hoang, X. Bai, S.J. Korsmeyer, D.S. Karanewsky, L.C. Fritz, and K.J. Tomaselli. 1996. Fas- induced activation of the cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors. J. Biol. Chem. 27128: 16850-16855 . |
2. |
Beidler, D.R.,
M. Tewari,
P.D. Friesen,
G. Poirier, and
V.M. Dixit.
1995.
The
baculovirus p35 protein inhibits Fas- and tumor necrosis factor-induced apoptosis.
J. Biol. Chem.
270:
16526-16528
|
3. | Bellgrau, D., D. Gold, H. Selawry, J. Moore, A. Franzusoff, and R.C. Duke. 1995. A role for CD95 ligand in preventing graft rejection. Nature. 377: 630-632 |
4. |
Boise, L.H., and
C.B. Thompson.
1997.
Bcl-xL can inhibit apoptosis in cells that
have undergone Fas-induced protease activation.
Proc. Natl. Acad. Sci.
USA.
94:
3759-3764
|
5. |
Boldin, M.P.,
E.E. Varfolomeev,
Z. Pancer,
I.L. Mett,
J.H. Camonis, and
D. Wallach.
1995.
A novel protein that interacts with the death domain of Fas/
APO1 contains a sequence motif related to the death domain.
J. Biol. Chem.
270:
7795-7798
|
6. | Boldin, M.P., T.M. Goncharov, Y.V. Goltsev, and D. Wallach. 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815 |
7. |
Cheng, E.H.-Y.,
D.G. Kirsch,
R.J. Clem,
F. Ravi,
M.B. Kastan,
A. Bedi,
K. Ueno, and
J.M. Hardwick.
1997.
Conversion of Bcl-2 to a Bax-like death effector by caspases.
Science.
278:
1966-1968
|
8. | Chinnaiyan, A.M., K. O'Rourke, M. Tewari, and V.M. Dixit. 1995. FADD, a novel death domain-constraining protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81: 505-512 |
9. |
Chinnaiyan, A.M.,
K. Orth,
K. O'Rourke,
H. Duan,
G.G. Poirier, and
V.M. Dixit.
1996a.
Molecular ordering of the cell death pathway.
J. Biol. Chem.
271:
4573-4576
|
10. |
Chinnaiyan, A.M.,
C.G. Tepper,
M.F. Seldin,
K. O'Rourke,
F.C. Kischkel,
S. Hellbardt,
P.H. Krammer,
M.E. Peter, and
V.M. Dixit.
1996b.
FADD/
MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis
factor receptor-induced apoptosis.
J. Biol. Chem.
271:
4961-4965
|
11. | Chinnaiyan, A.M., D. Chaudhary, K. O'Rourke, E.V. Koonin, and V.M. Dixit. 1997a. Role of CED-4 in the activation of CED-3. Nature. 388: 728-729 |
12. |
Chinnaiyan, A.M.,
K. O'Rourke,
B.R. Lane, and
V.M. Dixit.
1997b.
Interaction
of CED-4 with CED-3 and CED-9: a molecular framework for cell death.
Science
275:
1122-1126
|
13. | Chiou, S.-K., L. Rao, and E. White. 1994a. Bcl-2 blocks p53-dependent apoptosis. Mol. Cell. Biol. 14: 2556-2563 [Abstract]. |
14. | Chiou, S.-K., C.C. Tseng, L. Rao, and E. White. 1994b. Functional complementation of the adenovirus E1B 19K protein with Bcl-2 in the inhibition of apoptosis in infected cells. J. Virol. 68: 6553-6566 [Abstract]. |
15. | Clem, R.J., M. Fechheimer, and L.K. Miller. 1991. Prevention of apoptosis by a Baculovirus gene during infection of insect cells. Science. 254: 1388-1390 |
16. | Debbas, M., and E. White. 1993. Wild-type p53 mediates apoptosis by E1A which is inhibited by E1B. Genes Dev. 7: 546-554 [Abstract]. |
17. | Enari, M., H. Hug, and S. Nagata. 1995. Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature. 375: 78-81 |
18. | Foghsgaard, L., and M. Jäättelä. 1997. The ability of BHRF1 to inhibit apoptosis is dependent on stimulus and cell type. J. Virol. 171: 7509-7571 . |
19. | Gooding, L.R., L.W. Elmore, A.E. Tollefson, H.A. Brady, and W.S.M. Wold. 1988. A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell. 53: 341-346 |
20. | Gooding, L.R., L. Aquino, P.J. Duerksen-Hughes, D. Day, T.M. Horton, S. Yei, and W.S.M. Wold. 1991a. The E1B-19K protein of group C adenoviruses prevents cytolysis by tumor necrosis factor of human cells but not mouse cells. J. Virol. 65: 3083-3094 |
21. | Gooding, L.R., R. Ranheim, A.E. Tollefson, L. Aquino, P. Duerksen-Hughes, T.M. Horton, and W.S.M. Wold. 1991b. The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor. J. Virol. 65: 4114-4123 |
22. | Griffith, T.S., T. Brunner, S.M. Fletcher, D.R. Green, and T.A. Ferguson. 1995. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science. 270: 1189-1192 [Abstract]. |
23. |
Hahne, M.,
D. Rimoldi,
M. Schröter,
P. Romero,
M. Schreier,
L.E. French,
P. Schneider,
T. Bornand,
A. Fontana,
D. Lienard, et al
.
1996.
Melanoma cell
expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape.
Science.
274:
1363-1366
|
24. | Han, J., P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White. 1996a. The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein. Genes Dev. 10: 461-477 [Abstract]. |
25. | Han, J., P. Sabbatini, and E. White. 1996b. Induction of apoptosis by human Nbk/Bik, a BH3 containing E1B 19K interacting protein. Mol. Cell. Biol. 16: 5857-5864 [Abstract]. |
26. | Hashimoto, S., A. Ishii, and S. Yonehara. 1991. The E1B oncogene of adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody. Int. Immunol 3: 343-351 [Abstract]. |
27. |
Henderson, S.,
D. Huen,
M. Rowe,
C. Dawson,
G. Johnson, and
A. Rickinson.
1993.
Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2,
protects human B cells from programmed cell death.
Proc. Natl. Acad. Sci.
USA.
90:
8479-8483
|
28. | Hengartner, M.O.. 1996. Programmed cell death in invertebrates. Curr. Opin. Genet. Dev. 6: 34-38 |
29. | Hsu, H., H.-B. Shu, M.G. Pan, and D.V. Goeddel. 1996. TRADD-TRAF2 and TRADD-FADD interaction define two distinct TNF receptor 1 signal transduction pathways. Cell. 84: 299-308 |
30. |
Hu, S.,
C. Vincenz,
M. Buller, and
V.M. Dixit.
1997.
A novel family of viral
death effector domain-containing molecules that inhibit both CD-95- and tumor necrosis factor receptor-1-induced apoptosis.
J. Biol. Chem.
272:
9621-9624
|
31. | Itoh, N., S. Yonehara, A. Ishii, M. Yonehara, S. Mizushima, M. Sameshima, A. Hase, Y. Seto, and S. Nagata. 1991. The polypeptides encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 66: 233-243 |
32. | Kischkel, F.C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P.H. Krammer, and M.E. Peter. 1995. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO (Eur. Mol. Biol. Organ.) J. 14: 5579-5588 [Abstract]. |
33. |
Klinkert, W.E.,
K. Kojima,
W. Lesslauer,
W. Rinner,
H. Lassmann, and
H. Wekerle.
1997.
TNF-![]() |
34. | Laemmli, U.K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685 |
35. |
Laster, S.M.,
J.G. Good, and
L.R. Gooding.
1988.
Tumor necrosis factor can induced both apoptic and necrotic forms of cell lysis.
J. Immunol.
141:
2629-2634
|
36. | Merino, R., D.A. Grillot, P.L. Simonian, S. Muthukkumar, W.C. Fanslow, S. Bondada, and G. Nunez. 1995. Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction. J Immunol. 155: 3830-3838 [Abstract]. |
37. |
Miura, M.,
H. Zhu,
R. Rotello,
E.A. Hartwieg, and
J. Yuan.
1993.
Induction of
apoptosis in fibroblasts by IL-1![]() |
38. | Miura, M., R.M. Friedlander, and J. Yuan. 1995. Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway. Proc. Natl. Acad. Sci. USA. 92: 8318-8322 [Abstract]. |
39. | Moran, E.. 1993. DNA tumor virus transforming proteins and the cell cycle. Cur. Opin. Gen. Dev. 3: 63-70 . |
40. | Muzio, M., A.M. Chinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Bretz, M. Zhang, R. Gentz, M. Mann, P.H. Krammer, M.E. Peter, and V.M. Dixit. 1996. FLICE, a novel FADD-homologous ICE/ CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85: 817-827 |
41. |
Muzio, M.,
G.S. Salvesen, and
V.M. Dixit.
1997.
FLICE-induced apoptosis in a
cell-free system.
J. Biol. Chem.
272:
2952-2956
|
42. |
Muzio, M.,
B.R. Stockwell,
H.R. Stennicke,
G.S. Salvesen, and
V.M. Dixit.
1998.
An induced proximity model for caspase-8 activation.
J. Biol. Chem.
273:
2926-2930
|
43. | Nagata, S.. 1997. Apoptosis by death factor. Cell 88: 355-365 |
44. | Probert, L., K. Akassoglou, G. Kassiotis, M. Pasparakis, L. Alexopoulou, and G. Kollias. 1997. TNF-a transgenic and knockout models of CNS inflammation and degeneration. J. Neuroimmunol 72: 137-141 |
45. | Rao, L., M. Debbas, P. Sabbatini, D. Hockenberry, S. Korsmeyer, and E. White. 1992. The adenovirus E1A proteins induce apoptosis which is inhibited by the E1B 19K and Bcl-2 proteins. Proc. Natl. Acad. Sci. USA. 89: 7742-7746 [Abstract]. |
46. |
Ray, C.A.,
R.A. Black,
S.R. Kronheim,
T.A. Greenstreet,
P.R. Sleath,
G.S. Salvesen, and
D.J. Pickup.
1992.
Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1![]() |
47. |
Sabbatini, P.,
J.H. Han,
S.-K. Chiou,
D. Nicholson, and
E. White.
1997.
Interleukin 1![]() |
48. | Schott, A.F., I.J. Apel, G. Nuñez, and M.F. Clarke. 1995. Bcl-XL protects cancer cells from p53-mediated apoptosis. Oncogene 11: 1389-1394 |
49. | Seshagiri, S., and L.K. Miller. 1997. Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis. Curr. Biol. 7: 455-460 |
50. | Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis. J. Virol. 71: 8299-8306 [Abstract]. |
51. | Spector, M.S., S. Desnoyers, D.J. Hoeppner, and M.O. Hengartner. 1997. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature. 385: 653-656 |
52. |
Tewari, M., and
V.M. Dixit.
1995.
Fas- and TNF-induced apoptosis is inhibited
by the poxvirus crmA gene product.
J. Biol. Chem.
270:
3255-3260
|
53. | Thome, M., P. Schneider, K. Hofmann, H. Fickenscher, E. Meinl, F. Neipel, C. Mattmann, K. Burns, J.-L. Bodmer, M. Schröter, C. Scaffidl, P.H. Krammer, M.E. Peter, and J. Tschopp. 1997. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature. 386: 517-521 |
54. | Thompson, C.B.. 1995. Apoptosis in the pathogenesis and treatment of disease. Science. 267: 1456-1462 |
55. | White, E.. 1996. Life, death, and the pursuit of apoptosis. Genes Dev. 10: 1-15 |
56. | White, E., and R. Cipriani. 1989. Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the adenovirus E1B oncogene. Proc. Natl. Acad. Sci. USA. 86: 9886-9890 [Abstract]. |
57. | White, E., and R. Cipriani. 1990. Role of adenovirus E1B proteins in transformation: altered organization of intermediate filaments in transformed cells that express the 19-kilodalton protein. Mol. Cell. Biol. 10: 120-130 |
58. | White, E., S.H. Blose, and B. Stillman. 1984. Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of cellular DNA. Mol. Cell. Biol. 4: 2865-2875 |
59. | White, E., R. Cipriani, P. Sabbatini, and A. Denton. 1991. The adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. J. Virol. 65: 2968-2978 |
60. | White, E., P. Sabbatini, M. Debbas, W.S.M. Wold, D.I. Kusher, and L. Gooding. 1992. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor a. Mol. Cell. Biol. 12: 2570-2580 [Abstract]. |
61. |
Wu, D.,
H.D. Wallen, and
G. Nuñez.
1997.
Interaction and regulation of subcellular localization of CED-4 by CED-9.
Science.
275:
1126-1128
|
62. |
Yuan, J.,
S. Shaham,
S. Ledoux,
H.M. Ellis, and
H.R. Horvitz.
1993.
The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1![]() |