From the
The baculovirus p35 gene product inhibits virally induced
apoptosis, developmental cell death in Caenorhabditis elegans and Drosophila, and neuronal cell death in mammalian
systems. Therefore, p35 likely inhibits a component of the death
machinery that is both ubiquitous and highly conserved in evolution. We
now show for the first time that p35 also inhibits Fas- and tumor
necrosis factor (TNF)-induced apoptosis. Additionally, p35 blocks TNF-
and Fas-induced proteolytic cleavage of the death substrate
poly(ADP-ribose) polymerase from its native 116-kDa form to the
characteristic 85-kDa form. This cleavage is thought to be catalyzed by
an aspartate-specific protease of the interleukin 1
Two members of the TNF
To circumvent host defense mechanisms, viruses have evolved
mechanisms to antagonize host death signals so that viral propagation
can continue unabated in infected cells(10) . The baculovirus Autographa californica and Bombyx mori nuclear
polyhedrosis viruses encode a 35-kDa protein (p35) that alleviates
apoptosis in virally infected insect cells(11, 12) .
p35-mediated inhibition of apoptosis correlates with increased viral
replication and enhanced yields of viral
progeny(11, 13) . Viral mutants lacking p35 induce
premature death of the host cells with a resultant decline in viral
yields. Surprisingly, p35 can inhibit not only virally induced
apoptosis but also developmentally programmed cell death in the
nematode Caenorhabditis elegans(14) . p35 expression in
transgenic worms, for example, can rescue embryonic lethality resulting
from a mutation in ced-9, an endogenous suppressor of cell
death. Additionally, p35 has been found to suppress developmental cell
death in Drosophila(15) . Ectopic targeted p35
expression to the eyes of transgenic flies eliminates all normally
occurring cell death in this tissue and, furthermore, also inhibits
x-irradiation-induced death. Remarkably, p35 can also inhibit apoptosis
in mammalian neural cells deprived of neurotropic stimulation (16, 17).
These studies suggest that p35 must interrupt a highly conserved and
ubiquitous component of the death machinery. We now show for the first
time that p35 inhibits TNF- and Fas-induced apoptosis and, in addition,
it blocks the cleavage of PARP, a death substrate in the apoptotic
pathway.
For the transient apoptosis assays,
cells transfected with a
To determine if p35 expression was capable of blocking
TNF-induced apoptosis, MCF7, a TNF-sensitive human breast carcinoma
cell line(20) , was stably transfected with a p35 expression
construct, and clonal cell lines were derived. Fig. 1A shows that after an 18-h exposure to TNF (20 ng/ml), only 12% of a
vector-transfected line (V-2) was not apoptotic as recognized by
staining cellular DNA with propidium iodide and assessing nuclear
fragmentation by fluorescence microscopy. However, in two
p35-transfected lines, p35#4 and p35#5, approximately 90% of the cells
were resistant to apoptosis. A third p35-expressing line, p35#2, had
intermediate numbers of apoptotic cells. To confirm that assessing
nuclear fragmentation by fluorescence microscopy corresponded to gross
toxicity, cell survival following an 18-h TNF exposure was determined
by incubating the cells for a further 48 h in TNF-free media. As shown
in the inset to Fig. 1A, p35#4 and p35#5, and
to a lesser extent, p35#2, displayed increased survival rates when
compared with vector controls. The relative lower survival rates
compared with that assessed by staining cellular DNA could either be
due to reversible growth inhibition during the 18-h TNF incubation or
the presence of additional apoptotic events even after the 18-h TNF
exposure. Northern blots of poly(A)
We gratefully acknowledge Dr. David R. Spriggs for
providing the MCF7 cell line, Dr. Fred Wang for providing the BJAB
line, and Marja Jäättelä for producing the MCF-Fas line.
We would like to thank Karen O'Rourke and Arul Chinnaiyan for
their helpful advice and technical assistance, and Ian M. Jones for
help in preparation of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-converting
enzyme family designated prICE (Lazebnik, Y. A., Kaufmann, S. H.,
Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346
-347). Our data suggest that p35 must directly or
indirectly inhibit prICE. Given that p35 inhibits both TNF and Fas
killing, along with previous reports of its ability to block
developmental, viral, and x-irradiation-induced cell death, the present
results indicate that TNF- and Fas-mediated apoptotic pathways must
have components in common with these highly conserved death programs.
(
)receptor family,
TNFR-1 and Fas, are known mediators of host antiviral defense
mechanisms(1, 2) . Activation of the TNF receptor in
virally infected host cells results in their elimination from the host
organism. Similarly, cytotoxic T-cells have been shown to kill infected
host cells by activating the Fas pathway(3) . Activated
cytotoxic T-cells express the Fas ligand, which upon engaging and
activating the Fas receptor on the surface of the virally infected
cells, delivers a death signal. Both elimination events proceed through
a process of active, programmed cell death termed apoptosis(4) ,
which results in the death of virus-bearing host cells in order to
attenuate viral spread. Apoptosis is characterized by a series of
orchestrated intracellular events that lead to the selective removal of
target cells without an accompanying inflammatory response. Apoptotic
events are morphologically distinguishable by cellular shrinkage,
nuclear condensation, cytoplasmic vacuolization, and membrane blebbing.
Biochemical events include the activation of specific proteases
resulting in the cleavage of certain recently identified protein
substrates(5, 6) . There is now overwhelming genetic and
biochemical evidence implicating interleukin 1
-converting enzyme
(ICE)-like proteases as components of the cell death
pathway(7, 8) . This unusual class of cysteine proteases
cleaves only following certain specific aspartic acid residues, and
until of late, the only known substrate was prointerleukin-1
,
which is converted to the active cytokine by ICE. Now, however, the
116-kDa DNA repair enzyme poly(ADP-ribose) polymerase (PARP) has been
shown to be proteolytically processed to a signature 85-kDa fragment by
an aspartate-specific cleavage mediated by an ICE-like protease (termed
prICE) that appears to be distinct from ICE itself(6) .
Importantly, PARP cleavage has been observed in a variety of apoptotic
responses(5) , including TNF- and Fas-mediated cell death (9).
Cell Lines and Plasmids
The TNF-sensitive MCF7
breast carcinoma cell line was provided by Dr. David R. Spriggs
(University of Wisconsin). The Fas-sensitive MCF-Fas subclone was
obtained by transfecting a Fas receptor expression construct into the
MCF7 cells (18). The BJAB cell line was kindly provided by Dr. Fred
Wang (Harvard Medical School, Cambridge, MA). The plasmid pPRM-35K-ORF
containing the entire p35 open reading has been described previously
(19). The entire p35 open reading frame obtained as a 0.9-kilobase pair EcoRI/XbaI fragment was cloned into the mammalian
expression vector pcDNA3 (Invitrogen).
Transfections
Transfection by electroporation of
p35/pcDNA3 and pcDNA3 (vector alone) into MCF7 cells and selection of
stable lines was carried out as described previously(20) . For
transient transfection of MCF-Fas lines, 10 cells were
plated onto 6-well plates and incubated for 18 h. Transfections were by
the Lipofectamine (Life Technologies, Inc.) procedure according to the
manufacturer's instructions. Cells were exposed to agonist
anti-Fas antibody (Upstate Biotechnology Inc.) 28 h following
transfection with the indicated plasmids. Transient transfection of
BJAB cells was by electroporation, followed by anti-Fas exposure 48 h
later.
Assessment of TNF- and anti-Fas-induced
Apoptosis/Toxicity
To quantitate TNF-induced apoptosis in stably
transfected MCF7 lines, a previously described quantitative apoptosis
assay using propidium iodide was used(20) . For measurement of
TNF toxicity, 2 10
cells were plated in 24-well
plates, incubated for 24 h, exposed to 20 ng/ml TNF (1.2
10
units/mg, Genentech) for 18 h, washed, and incubated for
a further 48 h in TNF-free media. Wells were fixed and stained by
adding 0.5% methylene blue in 50% ethanol for 30 min, and after
washing, the incorporated methylene blue was solubilized in 1% Sarkosyl
in phosphate buffered saline. The resulting color intensities were
quantitated on a microplate reader (BioTek Instruments) at an
absorbance of 630 nM.
-galactosidase expression vector together
with either a p35 expression construct or vector control were exposed
to anti-Fas (250 ng/ml) for 16 h, fixed with 0.5% glutaraldehyde (EM
Sciences) or methanol (for MCF-Fas and BJAB cells, respectively), and
stained with 1 mg/ml 5-bromo-4-chloro-3-indoyl
-D-galactoside (Boehringer Mannheim) to detect
-galactosidase expression. The resulting
-galactosidase
positive (green) cells were examined for apoptotic morphology including
cellular shrinkage, nuclear condensation, and cytoplasmic blebbing, by
phase contrast microscopy (MCF-Fas), or by propidium iodide staining
and fluorescence microscopy (BJAB).
RNA Isolation and Northern Analysis
Total RNA was
isolated (21) and enriched for the poly(A) fraction
using a kit (Qiagen) according to the manufacturer's
instructions. RNA was transferred to nylon membranes and probed with a
P-labeled 1-kilobase pair DNA fragment corresponding to
the entire p35 open reading frame according to established procedures
(22).
PARP Western Blot
The preparation of cell lysates
and immunoblotting of TNF- and anti-Fas-treated MCF7 lines was
performed as described previously(6) . The anti-PARP monoclonal
antibody (clone C2-10, IgG1(23) ) was used at a dilution
of 1:10,000, and the secondary antibody (anti-mouse Ig/horseradish
peroxidase; Sigma) was used at a dilution of 1:1,000. The peroxidase
signal was visualized by chemiluminescence (ECL, Amersham Corp.).
-enriched RNA from
these lines verified p35 expression in the p35 stable transfectants (Fig. 1B). The p35#2 line had considerably less
transcript when compared with p35#4 and p35#5, which would explain the
intermediate levels of protection observed in p35#2. Therefore, there
exists a dose-response relationship between the level of p35 expression
and the degree of protection conferred from TNF-induced cell death.
Figure 1:
Inhibition of TNF-induced apoptosis in
MCF7 cells by p35. A, sensitivity of stable transfectants to
TNF-induced apoptosis (20 ng/ml for 18 h) as determined by staining
cellular DNA with propidium iodide and assessing nuclear fragmentation
by fluorescence microscopy. V-2, control, vector-transfected
clone; p35#2, p35#4, and p35#5, p35-transfected
clones. Inset, TNF-induced cell death (20 ng/ml for 18 h)
assessed by the methylene blue toxicity assay and expressed as the
relative percentage of live cells compared with untreated control
cultures. Data represent the mean ± S.D. from duplicate samples
of two separate experiments. B, p35 transcript levels in the
corresponding clones. C, photograph of ethidium
bromide-stained gel to assess equivalency of loading as judged by the
intensity of the 18 and 28 S rRNA bands.
It had previously been found that anti-Fas killing in the parental
MCF7 line required the concomitant presence of cycloheximide, a protein
synthesis inhibitor, to enhance Fas toxicity(20) . To allow a
comparison of TNF- and Fas-induced killing in the same line without
cycloheximide coincubation, thereby avoiding complications resulting
from the toxicity of cycloheximide itself, the level of Fas expression
was augmented by transfecting with a Fas expression construct and
deriving a stable transfectant designated MCF-Fas(18) . This
cell line was susceptible to Fas-induced death in the absence of a
protein synthesis inhibitor. MCF-Fas cells were transiently
cotransfected with a -galactosidase reporter and a p35 expression
or vector control construct followed by Fas activation by cross-linking
with an agonistic antibody. Assessment of nuclear morphology of
-galactosidase-positive cells revealed that p35 markedly protected
cells from Fas-induced apoptosis (Fig. 2A). Additional
transient transfections performed in BJAB cells, a B cell line that is
sensitive to Fas-induced apoptosis(20) , also demonstrated that
p35 protected cells from Fas-induced apoptosis (Fig. 2B).
Figure 2:
Inhibition of Fas-induced apoptosis in
MCF-Fas and BJAB cells by p35. Transient cotransfections with a
-galactosidase reporter construct plus either vector control or a
p35 expression construct were carried out in MCF-Fas (A) or
BJAB (B) cells. Cultures were incubated in the presence (diagonalbars) or absence (solidbars) of an agonist anti-Fas antibody ((250 ng/ml) for 16
h). Morphology of
-galactosidase-positive cells was assessed by
phase contrast microscopy (MCF-Fas) or propidium iodide staining and
fluorescence microscopy (BJAB). In each experiment, two separate counts
of at least 200 cells were performed. Data represent the mean ±
S.D. from three experiments (MCF-Fas) or two experiments
(BJAB).
Analysis of the integrity of the death
substrate PARP in the vector-transfected MCF7 line revealed complete
cleavage of the native 116-kDa PARP to the signature 85-kDa proteolytic
fragment within 16 h of exposure to TNF (Fig. 3A).
However, in the p35#4 and p35#5 lines, only low levels of the 85-kDa
proteolytic product were seen even after 24-48 h of TNF exposure.
The underexpressing p35#2 line displayed a PARP cleavage rate that was
only marginally slower than the vector-transfected control, V-2.
Similar results were seen when MCF7 cells were treated with anti-Fas (Fig. 3B). While PARP cleavage was slower and not as
complete as in TNF-treated cultures, p35 still significantly inhibited
its cleavage in the p35#4 and p35#5 clones and to a lesser extent in
the intermediate expressing p35#2 line. Therefore, as with TNF-induced
killing, there exists a dose-response relationship between the level of
p35 expression and the degree of inhibition of PARP cleavage.
Figure 3:
p35
inhibits TNF- and Fas-induced PARP cleavage. MCF7 stable transfectants
were exposed to TNF (A) or anti-Fas (B) for the
indicated time periods after which cell lysates were prepared for
immunoblotting with the anti-PARP monoclonal antibody as described
under ``Materials and Methods.'' The 116-kDa band represents
the intact protein, and the 85-kDa band denotes the signature cleavage
product.
In
summary, the baculovirus p35 protein was able to block not only Fas-
and TNF-induced apoptosis but also Fas- and TNF-induced PARP cleavage.
Given that p35 inhibits both TNF and Fas killing, along with previous
reports of its ability to block developmental, viral, and
x-irradiation-induced cell death, the present results indicate that
TNF- and Fas-mediated apoptotic pathways must have components in common
with these highly conserved death programs. Inhibition of cleavage of
the death substrate PARP has also been observed with the anti-apoptotic
cowpox virus CrmA gene product(9) . However, CrmA is a serpin
capable of inhibiting ICE family members whereas p35 possesses no
homology to any protease inhibitor. This suggests that p35 must inhibit
cleavage of the death substrate PARP and ultimately cell death at a
site unique in the cell death pathway. Thus, p35 protein will be an
invaluable tool in helping identify components of this highly conserved
cell death pathway.
-converting enzyme; PARP,
poly(ADP-ribose) polymerase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.