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Journal of Virology, November 1998, p. 8893-8903, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Induction of Programmed Cell Death by Parvovirus
H-1 in U937 Cells: Connection with the Tumor Necrosis Factor Alpha
Signalling Pathway
Béatrice
Rayet,
José-Antonio
Lopez-Guerrero,
Jean
Rommelaere,* and
Christiane
Dinsart
Angewandte Tumorvirologie, Abteilung F0100,
Deutsches Krebsforschungszentrum, and Virologie Appliquée à
l'Oncologie (Unité INSERM 375), D-69009 Heidelberg, Germany
Received 20 March 1998/Accepted 24 July 1998
 |
ABSTRACT |
The human promonocytic cell line U937 undergoes apoptosis upon
treatment with tumor necrosis factor alpha (TNF-
). This cell line
has previously been shown to be very sensitive to the lytic effect of
the autonomous parvovirus H-1. Parvovirus infection leads to the
activation of the CPP32 ICE-like cysteine protease which cleaves the
enzyme poly(ADP-ribose)polymerase and induces morphologic changes that
are characteristic of apoptosis in a way that is similar to TNF-
treatment. This effect is also observed when the U937 cells are
infected with a recombinant H-1 virus which expresses the nonstructural
(NS) proteins but in which the capsid genes are replaced by a reporter
gene, indicating that the induction of apoptosis can be assigned to the
cytotoxic nonstructural proteins in this cell system. The c-Myc
protein, which is overexpressed in U937 cells, is rapidly downregulated
during infection, in keeping with a possible role of this product in
mediating the apoptotic cell death induced by H-1 virus infection.
Interestingly, four clones (designated RU) derived from the U937 cell
line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642-1653, 1997) failed to decrease
c-Myc expression upon treatment with differentiation agents and also
resisted the induction of cell death after TNF- treatment. Our data
suggest that the RU clones have developed defense strategies against
apoptosis, either by their failure to downregulate c-Myc and/or by
activating antiapoptotic factors.
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INTRODUCTION |
Parvoviruses are small,
single-stranded DNA viruses that infect a wide variety of animal
species, including humans (80). The H-1 virus belongs to the
subgroup of autonomous, replicating parvoviruses and contains a linear
DNA genome of about 5 kb (15, 74). The low genetic
complexity of parvoviruses renders them tightly dependent on cellular
factors that are expressed as a function of cell proliferation (S phase
of the cell cycle) and differentiation to achieve their lytic cycle
(15). Parvoviruses are unable to force quiescent cells into
the S phase. Cancer cells seem to provide parvoviruses with an
environment favorable to their multiplication. Indeed, several
parvoviruses exhibit a remarkable oncotropism (75). In
agreement with this, it has been shown previously that many in
vitro-transformed cells are sensitized to the parvovirus-induced
killing compared to their untransformed counterparts, which correlates
with an increased capacity of the transformants to sustain certain
steps of the viral life cycle (10, 13, 77). In particular,
the production and toxic activity of the nonstructural (NS) protein
NS-1, which is a key effector of parvovirus replication and
cytopathogenicity, can be stimulated in oncogene-transformed cells
(61). This may account, at least partially, for the fact
that parvoviruses can exert an oncosuppressive activity in vivo
(75).
In order to investigate the mechanisms involved in parvovirus
anticancer surveillance, we have recently isolated and characterized rare variants that derive from the human myeloid leukemia cell line
U937 (83), are designated RU, and differ from the parental cell line in that they are resistant to H-1 virus infection
(52). Of the four RU clones analyzed, three showed both a
significant decrease, compared with the parental U937 cells, of the
steady-state level of the c-Myc oncoprotein and a reduced tumorigenic
capacity when implanted in scid mice. Moreover, all of the RU cells
exhibited a constitutive production of nitric oxide (NO) and superoxide anion (O2
). Deregulated c-Myc expression in
various tumor cells has been involved in their susceptibility to
undergo apoptosis in response to several inducers (9),
including tumor necrosis factor alpha (TNF-) (35, 36).
Furthermore, NO was reported to inhibit apoptosis in mononuclear cells
(45) and B-lymphocytes (53), and superoxide anion
was found to suppress Fas-mediated cell death (12).
Altogether, the data prompted us to investigate whether parvovirus H-1
was able to induce apoptosis in U937 cells and, if so, whether the H-1
virus-resistant RU cells also resist known apoptotic inducers, TNF-
in particular, that efficiently lead U937 cells to apoptosis (6,
31, 64, 96, 97).
Apoptosis can be induced in response to various stimuli (92,
93), including such viruses as chicken anemia virus
(38), measles virus (20), human immunodeficiency
virus (58), influenza virus (85), or murine
cytomegalovirus (102). Some ultrastructural features of
erythroid precursors infected with parvovirus B19 suggest that this
virus also triggers apoptosis in these cells (60). Other
viruses have developed strategies to block the commitment of cells into
the cell death program that can be viewed as host defense mechanisms
against infection (5, 72), and some viral products can even
have antagonistic effects on apoptosis, such as the adenovirus E1a and
E1b gene products (7, 16).
Programmed cell death or apoptosis represents a complex phenomenon that
is characterized morphologically by chromatin condensation, plasma
membrane blebbing, and cell fragmentation into membrane-enclosed vesicles (apoptotic bodies). The underlying molecular mechanisms of
apoptosis involve the expression of an increasing number of genes
conserved from nematodes to insects and mammals (29, 79). Cellular genes such as p53 (71), members of the bcl-2 family (23, 73, 84, 101), or certain oncogenes modulate the
commitment of cells into the apoptotic program in response to various
stimuli (92, 93). Recent evidence shows the importance of
the ICE/CED-3 family of cysteine proteases, now renamed caspases, in
the apoptotic process (24, 55). Members of this family, for
which several genes have already been identified (63), have
common features and are all related to the ced-3 product of
Caenorhabditis elegans (103). ICE was first
described as the cysteine protease cleaving pro-interleukin-1
to
generate active interleukin-1 (87). All the caspases
contain the highly conserved QACRG sequence which comprises the
active-site cysteine. Active cysteine proteases are produced by
cleavage of a proenzyme at key aspartate residues, generating two
subunits of approximately 20 and 10 kDa that can assemble into a
heterotetrameric protein with two active sites (26). Several
substrates for proteolytic cleavage by ICE-like proteins have been
identified (for a review, see reference 55), in
particular the poly(ADP-ribose) polymerase (PARP) (40, 46) that catalyzes the poly(ADP-ribosylation) of nuclear proteins when
activated by DNA strand breaks (48), and, recently, Nagata and coworkers (19, 76) identified a caspase-activated DNase (CAD) that degrades DNA during apoptosis and its interacting protein inhibitor (ICAD), which is cleaved by caspase-3 and releases CAD.
Members of the TNF receptor (TNF-R) superfamily have been shown to play
an important role in the activation of the apoptotic execution
machinery (33, 65). TNF- and the related FAS ligand (FASL) can trigger apoptosis in susceptible cells by activating their
cell-surface receptors TNFR1 and Fas (Apo-1/CD95), respectively. The
cytoplasmic regions of these receptors contain a death domain that is
essential for the signal-transduction pathway. The death domain
interacts with other proteins that contain this motif and associate
with TNF-R (TRADD) or FAS (FADD/MORT-1) proteins (63). Recently, a new protein, MACH/FLICE/Mch5/caspase-8, was shown to
interact with these receptor complexes (62) and appears to be a direct link between the signals at the membrane level and the
apoptotic execution machinery (56). How caspase-8 transmits the signal to downstream targets, and especially how other caspases become activated, is not yet understood. Recent studies have pointed out the key role of mitochondria in the apoptotic execution in cell-free systems (42-44, 50, 99). Mitochondria appear to
act through the release of soluble factors, in particular cytochrome c and a 50-kDa protease, whose translocation to the
cytoplasm is regulated by Bcl-2 or members of this family
(59).
We investigate here whether apoptosis was induced in U937 cells after
parvovirus H-1 infection by measuring the cleavage of the PARP enzyme
and the formation of apoptotic bodies. Results presented in this study
give evidence for the participation of ICE-like enzymes, in particular
caspase-3 (CPP32/apopain/Yama) as effectors of H-1 virus-induced
killing. An early event during H-1 virus infection of U937 cells is the
rapid decrease of their content in c-myc transcripts and
proteins, a feature also observed in TNF--treated cells (data not
shown) and interpreted in terms of a possible role of this oncoprotein
in apoptosis. Furthermore, the four RU clones, selected for their
resistance to parvovirus H-1 infection, were also found to resist
apoptosis in response to TNF-. Altogether, our data suggest a
possible interconnection between the apoptotic pathways activated by
TNF- and parvovirus H-1.
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MATERIALS AND METHODS |
Cells and virus.
The human promonocytic cell line U937
(83) and four subclones designated RU (52) were
cultured in RPMI 1640 medium (Gibco BRL) supplemented with 10%
heat-inactivated fetal calf serum in a 5% CO2 atmosphere
at 37°C.
H-1 parvovirus was propagated in NB-E cells and purified as described
previously (10). H-1 virus inoculation and titration by
plaque assay were performed according to published methods (10). Wild-type H-1 virus was inactivated by UV irradiation (258 nm, 170 kJ/m2). Recombinant H-1 virus expressing the
green fluorescent protein (GFP) from jellyfish (104) was
produced by cell cotransfection with an H-1 virus infectious plasmid in
which the VP genes had been replaced by the GFP gene and with a helper
plasmid expressing the capsid genes as described by Kestler et al.
(40a). The recombinant virus was purified on cesium gradient
as previously described (10).
Immunoblot analysis.
Cultures (106 cells) were
inoculated with H-1 parvovirus at a multiplicity of infection (MOI) of
10 PFU per cell or treated with TNF- (Sigma) (1 or 50 ng/ml) and
cycloheximide (Sigma) (10 µg/ml). The rabbit polyclonal anti-TNF-
blocking antibody (IC-Chemikalien, Ismaning, Germany) was used at a
concentration of 10 µg/ml of RPMI medium containing 0.1% bovine
serum albumin (BSA). After being treated, cells were collected by
low-speed centrifugation, suspended in 100 µl of RIPA buffer (10 mM
Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.5%
sodium deoxycholate; 0.1% sodium dodecyl sulfate [SDS]) containing 3 M urea, 10 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride, and
incubated for 1 h on ice. The samples were centrifuged at
18,000 × g to eliminate DNA and cellular debris. After
the protein concentration was determined (Bio-Rad assay), 50 to 150 µg of the total protein was diluted in an equal volume of 100 mM
Tris-HCl (pH 6.8)-5% SDS-10% 2-mercaptoethanol-20%
glycerol-0.1% bromophenol blue, subjected to 8 or 0.1% SDS-15%
polyacrylamide gel electrophoresis, and electrotransferred onto a
nitrocellulose membrane (Amersham). Nonspecific binding sites were
blocked by incubating the membrane for at least 2 h in
phosphate-buffered saline (PBS) containing 5% BLOTTO powdered milk and
0.1% Tween-20 (Sigma). Blots were further incubated with the indicated
antibodies and visualized with an enhanced chemiluminescence kit and
horseradish peroxidase-conjugated second antibody according to the
manufacturer's recommendation (Amersham). Antibody directed against
PARP (clone CII10) was a generous gift of Guy Poirier (Quebec City,
Quebec, Canada). Antibody specific for CPP32 p11 (clone C-17) was
purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).
Monoclonal antibodies against c-Myc (clone 9E10) and Max (C-17) were
purchased from Sigma and Santa Cruz Biotechnology, respectively. The
rabbit polyclonal serum SP8 directed against carboxy-terminal peptides
of NS1 has been already described (21). His-tagged VP-2
protein from H-1 virus was expressed in bacteria and used to raise a
rabbit polyclonal antiserum in our laboratory.
RNA extraction and Northern blotting.
Total cellular RNA was
isolated at different times postinfection by the modified guanidium
isothiocyanate method described by Chomczynski and Sacchi
(11). Briefly, 106 infected cells were
resuspended in 0.5 ml of 4 M guanidium isothiocyanate, 25 mM sodium
citrate (pH 7), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. After 0.5 ml of water-saturated phenol and 0.1 ml of chloroform were added, the
sample was mixed and further incubated on ice for 20 min. The RNAs were
precipitated from the aqueous phase with 1 volume of isopropanol at
20°C. After centrifugation, the RNA pellet was solubilized in
formamide at 50°C for 15 min and stored at 70°C.
The RNAs were fractionated by electrophoresis on a 1%
agarose-formaldehyde gel. After transfer under high salt conditions
to
a Hybond-N
+ nylon filter (Amersham), the samples were
prehybridized with
salmon sperm DNA (100 µg/ml) and hybridized
overnight at 42°C
with a
32P-labeled randomly primed
specific c-
myc DNA probe (exon2,
EcoRV-
BglII
fragment) in the presence of 50%
formamide and 5% dextran sulfate.
Membranes were then washed under
highly stringent conditions (15
min at 42°C and 30 min at 55°C in
0.2× SSC [1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate]-0.1%
SDS) and autoradiographed.
Nucleus Hoechst staining.
At different times after infection
(MOI of 5 PFU per cell) or after treatment with TNF- (10 ng/ml),
cultures (106 cells) were collected by low-speed
centrifugation, suspended in 100 µl of PBS, and allowed to settle for
15 min on slides pretreated with 1 mg of poly-L-lysine
(Sigma) per ml before fixation with 4% formalin. After being washed
with PBS, the slides were incubated with Hoechst staining at a
concentration of 75 µg/ml for 30 min at 4°C, washed twice with PBS,
and examined under a fluorescent Leitz microscope at 450 nm after the
samples had been excited at 330 nm.
| |
RESULTS |
Parvovirus H-1 induces apoptosis in the U937 cell line by
activating members of the caspase family.
With very few
exceptions, the replication of autonomous parvoviruses in cultures of
permissive cell lines has been described to be accompanied by typical
cytopathic changes (e.g., cell shrinking and nuclear morphologic
changes) (80) that can now be considered characteristic of
cells undergoing apoptosis. This prompted us to investigate the
molecular mechanisms underlying H-1 virus-induced cell death in the
human monocytic cell line U937. This cell line had been shown to be
very sensitive to the cytopathic effect of H-1 virus, with fewer than
0.1% of cells surviving 4 days after infection at an MOI of 100 PFU/cell (52). Two criteria of apoptosis were considered in
this study: morphologic changes (i.e., the appearance of apoptotic
bodies) and cleavage of the enzyme PARP, a known substrate for some
proteases of the caspase family that play a key role in programmed cell
death (34, 89).
As shown in Fig.
1, typical apoptotic
bodies could be observed in cultures infected with H-1 virus (5 PFU/cell), affecting
30% to more than 60% of the cells at 15 h
(Fig.
1A) and 25 h (Fig.
1B) postinfection, respectively. In
contrast, these morphologic
alterations were hardly detectable in
mock-infected cultures (Fig.
1C). U937 cells undergoing apoptosis after
treatment with TNF-
(10 ng/ml), a known inducer of programmed cell
death in this cell
line (
31,
64,
96), are shown for
comparison in Fig.
1D.
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FIG. 1.
Induction of morphologic changes in U937 cells infected
with H-1 virus. Cultures (106 cells) were infected with H-1
virus (5 PFU/cell) (A and B), mock infected (C), or treated with
TNF- (10 ng/ml) (D) and further incubated for 15 h (A) and
25 h (B, C, and D). Cells were collected by low-speed
centrifugation and suspended in PBS before being fixed in 4% formalin
on poly-L-lysine-coated slides, stained with Hoechst
solution, and examined by fluorescence microscopy.
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|
PARP, a 116-kDa enzyme implicated in DNA single-strand break repair,
has been shown to be cleaved into two specific fragments
(85 and 23 kDa) during the onset of apoptosis (
40,
46). This
cleavage
is induced by members of the ICE-like cysteine protease
family, in
particular the CPP32/YAMA/apopain/caspase-3 (
86).
Since
U937 cells express high levels of PARP, time course experiments
were
carried out to investigate whether infection with parvovirus
H-1 led to
the cleavage of this enzyme. At different times postinfection
(MOI of
10 PFU/cell), proteins were extracted and analyzed by
Western blotting
with the appropriate antibodies. As shown in
Fig.
2A, the cleavage of PARP could be
observed at 10 h postinfection
and reached its maximum after
18 h. No cleavage of PARP was detected
in mock-treated cells. The
production of the viral nonstructural
(NS; 86 kDa) protein NS-1 in
phosphorylated and unphosphorylated
forms and of the capsid proteins
VP-1 (88 kDa) and VP-2/3 (68
and 65 kDa, respectively) was determined
at the same time intervals.
As seen in Fig.
2B and C, respectively, the
NS and VP proteins
could be detected by Western blotting as early as
6 h postinfection,
thus preceding the cleavage of PARP.
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FIG. 2.
Cleavage of PARP and activation of CPP32/caspase-3 in
parvovirus H-1-infected cells. Total protein extracts were prepared at
different times after U937 cell infection with H-1 virus (10 PFU/cell).
Aliquots (70 or 150 µg) of proteins were analyzed by Western blotting
with various antibodies. (A) Monoclonal PARP antibody. The bands
corresponding to full-size PARP (116 kDa) or its cleavage product (85 kDa) are indicated. (B) Polyclonal anti-NS-1 serum. The phosphorylated
(upper band) and nonphosphorylated (lower band) forms of NS-1 are
marked. (C) Polyclonal antiserum directed against VP-1 and VP-2 capsid
proteins. (D) CPP32/caspase-3 antibody. The molecular sizes are as
indicated.
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We further determined whether the cleavage of PARP correlated with the
activation of CPP32/caspase-3, a cysteine protease
known to use PARP as
a substrate in vitro (for a review, see reference
94). CPP32 is produced as a proenzyme that has an
apparent molecular
size of 32 kDa and is activated as a result of its
cleavage into
two subunits, p11 and p20, of 11 and 20 kDa,
respectively. These
subunits are associated in a heterotetrameric
structure (
22,
86,
87). As shown in Fig.
2D, the level of
the proenzyme caspase-3
decreased about 8 h after infection, while
the p11 fragment became
visible as a weak band on the Western blot due
to the relatively
poor affinity of the antibody for the cleaved
molecule. The p20
subunit is not recognized by the commercial antibody
and could
therefore not be detected in these experiments. Altogether,
these
results strongly suggest that H-1 virus-induced apoptosis in the
U937 cell line involves the activation of at least some members
of the
caspase family. Apoptosis induced by H-1 infection does
not seem to be
restricted to the monocytic cell line U937. Indeed,
although PARP
cleavage could not be detected in two reference
fibroblast lines, we
were able to reproducibly observe other signs
of apoptosis, such as a
DNA ladder in the mouse A9 cells and the
EJ-
ras
oncogene-transformed Fisher rat cells (FR-EJ4), after infection
with
the minute virus of mice (MVMp) and the H-1 virus, respectively
(data
not shown).
In order to determine whether H-1 virus adsorption and/or uptake is
sufficient to trigger apoptosis or whether de novo synthesis
of viral
proteins is required, U937 cultures (10
6 cells) were
inoculated with UV-inactivated H-1 virus (100 PFU/cell)
(Fig.
3, lane 9). For comparison, parallel
cultures were infected
with nonirradiated H-1 virus (MOI of 1.7 PFU/cell; Fig.
3, lanes
2 to 4) or with nonirradiated recombinant H-1
virus in which the
genes encoding the capsids were replaced by the GFP
gene from
jellyfish (
104) (MOI of 1.7 PFU/cell; Fig.
3,
lanes 5 to 7).
A batch of cells was also infected with intact virus at
a low
MOI (0.03 PFU/cell; Fig.
3, lane 8), corresponding to the
contamination
of the recombinant virus stock with wild-type particles.
After
protein extraction at different times posttreatment, the cleavage
of PARP and the production of viral NS-1 and VP-1/VP-2 proteins
were
analyzed by Western blotting. As shown in Fig.
3A, the recombinant
H-1/GFP virus induced PARP cleavage (lanes 5 to 7) as efficiently
as
did the wild-type virus (lanes 2 to 4), whereas the UV-irradiated
H-1
virus failed to do so even at the very high dose of particles
equivalent to the 100 PFU/cell that was used (lane 9). The result
is in
keeping with the known cytotoxic activity of the parvoviral
NS
proteins, in particular NS-1 (
8), suggesting that de novo
synthesis and accumulation of NS-1 proteins, for which the wild-type
and recombinant viruses are both competent, were necessary to
commit
cells into the apoptotic pathway, as indicated by the PARP
cleavage. As
illustrated in Fig.
3B, the accumulation of NS-1
proteins after
infection with either wild-type H-1 virus (lanes
2 to 4) or recombinant
H-1/GFP virus (lanes 5 to 7) was comparable,
while the accumulation of
VP polypeptides, as seen with the wild-type
virus, was hardly
detectable with the recombinant virus (Fig.
3C, lanes 5 to 7). This
finding is in agreement with the GFP substitution
for VP in the
recombinant virus, since the residual level of VP
production can be
attributed to the emergence of a small proportion
of wild-type viruses
during the production of recombinant H-1/GFP
virus stocks
(
40a). It should be stated that this wild-type contamination
of the recombinant stock did not account for the ability of the
latter
to trigger apoptosis, since an equivalent inoculum of pure
wild-type
H-1 virus (MOI of 0.03 PFU/cell), which yielded the
same production of
VP proteins as had the recombinant stock but
without any detectable
NS-1 accumulation (Fig.
3B and C, lane
8), failed to induce PARP
cleavage as measured by Western blotting
(Fig.
3A, lane 8). Altogether,
these data point to NS proteins
as the major effector of the induction
of PARP cleavage (Fig.
3A) and the formation of apoptotic bodies (data
not shown), although
an additional contribution of the capsid proteins
cannot be ruled
out.
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FIG. 3.
Induction of the cleavage of PARP in the presence of
parvoviral NS proteins. Western blot analyses were carried out with
protein extracts prepared from U937 cells at different times after
infection, with intact (lanes 2 to 4, lane 8) or UV-irradiated (lane 9)
wild-type H-1 virus or with a recombinant H-1 virus in which the VP
genes were replaced by the jellyfish GFP (lanes 5 to 7). MOIs are given
as intact wild-type virus PFU equivalents per cell. (A) PARP cleavage.
(B and C) Accumulation of the H-1 virus proteins NS-1 (B) and VP-1 and
VP-2 (C).
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It is now well established that members of the Bcl-2 protein family are
key regulators of apoptosis (
73). Some of these
factors,
such as the human Bcl-2 protein or Bcl-xl, are suppressors
of cell
death, whereas other members of the family, e.g., Bax
and Bak, promote
programmed cell death. Bcl-2 and related proteins
can form channels in
vitro and may participate in the regulation
of mitochondrial
permeability transition and in the release of
apoptogenic protease
activators, in particular cytochrome
c and
apoptosis-inducing factor from the mitochondria (
44). This
prompted
us to investigate whether H-1 virus-induced apoptosis involved
modifications in the accumulation of three members of this family:
Bcl-2, Bax, and Bcl-xl. To this end, Western blotting analyses
were
performed with protein extracts from infected U937 cells
with specific
antibodies. No significant change in the steady-state
levels of these
proteins could be observed up to 28 h postinfection
(MOI of 10 PFU/cell), suggesting that H-1 virus-induced apoptosis
is not mediated
by a gross alteration of their intracellular accumulation
(data not
shown). However, it cannot be ruled out that the parvovirus
affects the
posttranslational modification of these proteins or
the expression of
other members of the Bcl-2 family.
c-Myc is rapidly downregulated in parvovirus-induced
apoptosis.
c-Myc, which is known to be involved in the regulation
of cell proliferation and differentiation, was consistently found to sensitize cells to the induction of apoptosis by several treatments (3, 35). Knowing that c-Myc is overexpressed in U937 cells (18), these data prompted us to investigate the possible
role of this oncoprotein in H-1 virus-induced apoptosis. It should be
stated that, although the deregulation of c-Myc in the induction of
apoptosis is well documented for some cell lines, there is no evidence
for the requirement of the downregulation of overexpressed c-Myc during
the execution of apoptosis. The rapid decrease of c-Myc in U937 cells
committed to differentiation led us hypothesize that this might be also
the case in U937 cells committed to apoptosis. The accumulation of
c-Myc in protein extracts prepared from H-1 virus-infected U937 cells
(MOI of 10 PFU/cell) at various times postinfection was analyzed by
Western blotting with a specific monoclonal antibody. As shown in Fig.
4A, a significant decrease of the level
of the 65-kDa and the N-terminally extended 68-kDa forms of c-Myc
(81) was apparent as early as 6 h postinfection and
became more pronounced with time, leading to the disappearance of a
detectable c-Myc protein signal by around 18 h postinfection, when
PARP cleavage was maximal. Similarly, the level of
c-myc transcripts decreased with time and became
undetectable by 14 h postinfection, as determined by Northern
blotting analysis (Fig. 4B). This effect was specific since -actin
mRNA remained stable during the same time interval, making it unlikely
that the downregulation of c-myc mRNAs reflected an overall
inhibition of transcription or an enhancement of mRNA degradation in
H-1 virus-infected cells. The downregulation of c-myc
expression occurred concomitantly with the production of viral proteins
(Fig. 2B and C) and preceded the cleavage of PARP (Fig. 2A), thereby
representing an early event in parvovirus-induced apoptosis.
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FIG. 4.
Downregulation of c-Myc during H-1 virus-induced
apoptosis. (A) Western blot analysis of total proteins (70 µg/lane)
extracted from H-1 virus-infected U937 cells (10 PFU/cell) at different
times postinfection with anti-PARP (upper panel) or anti-c-Myc (lower
panel) antibodies. (B) Northern blot analysis of total RNAs extracted
from H-1 virus-infected U937 cells with c-myc (upper part)-
or -actin (lower part)-specific DNA probes. (C) Northern blot
analysis of total RNAs extracted from H-1 virus-infected RU and U937
cells at the indicated times postinfection with c-myc (upper
part)- or -actin (lower part)-specific DNA probes.
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We recently selected from the U937 line rare cell variants that
resisted H-1 virus infection, of which four (RU1, RU2, RU3,
and RU4)
were further characterized (
52). The RU derivatives
were
tested to determine the correlation between c-
myc
downregulation
and H-1 virus-induced apoptosis in U937 cells. Indeed,
all H-1
virus-resistant RU clones were distinguishable from the
sensitive
parental U937 cells because, in the former group, the level
of
c-
myc transcripts remained unaffected up to 18 h
after H-1 virus
infection, as determined by Northern blotting analysis
(Fig.
4C).
Resistance to the killing effect of H-1 virus cosegregates with
resistance to TNF--induced apoptosis in U937 cell derivatives.
Although uninfected RU cells expressed levels of c-myc mRNAs
comparable to the parental U937 cells, three of the clones (RU1, RU2,
and RU4) showed a significant decrease, compared to the parental U937
cells, in the accumulation of c-Myc oncoprotein and also in their
capacity to form tumors in immunodeficient mice. The transforming and
pro-apoptotic properties of c-Myc are believed to require its
association with the partner protein Max (2, 30, 49, 70). In
contrast to c-Myc, Max expression was not significantly altered in RU
cells compared with the parental U937 line (Fig.
5, lower panel). The levels of both
proteins in the RU cells are given as percentages of the U937 values
shown in the upper panel of Fig. 5. While being close to 100% for Max, the relative level of c-Myc was strongly reduced in the RU1 (14%), RU2
(31%), and RU4 (16%) clones. Thus, these three clones showed an
imbalance between Myc and Max favoring the association of Max with
itself or with other partners known to inhibit c-Myc activity (49,
57). It should also be stated that all RU clones resisted the
suppressing effect of the differentiating agent TPA
(12-O-tetradecanoylphorbol-13-acetate) on c-myc
oncogene expression and cell proliferation (52). In addition, a subclone of U937 cells that resisted TPA-induced
differentiation or programmed cell death was previously described by
Hass et al. (28), in which c-myc was continuously
expressed in the presence of this agent.
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|
FIG. 5.
Steady-state levels of c-Myc and Max proteins in
parental U937 cells and H-1 virus-resistant (RU) derivatives. (Upper
panels) Densitometric quantification of c-Myc and Max accumulation,
expressed as percentages of the U937 level. Average values and
standard-deviation bars from three experiments are shown. (Lower
panels) Western blot analysis of c-Myc and Max levels in uninfected
U937 and RU cells, with 50 µg of total protein extracts.
|
|
TNF-
is known to induce differentiation (
78) or
programmed cell death (
31,
64,
96) in parental U937 cells.
c-Myc
has been found to sensitize some human tumor cell lines to
TNF-
-induced
apoptosis (
35), and TNF-
-resistant
fibroblasts could even be
made TNF-
sensitive by the enforced
expression of c-Myc (
41).
Therefore, the altered regulation
of c-Myc expression in RU cells,
in particular its resistance to
downmodulating treatments, prompted
us to investigate whether H-1
virus-resistant RU clones also became
refractory to TNF-
-induced
apoptosis. To this end, RU clones
were incubated in the presence of
TNF-
(50 ng/ml) either alone
or with cycloheximide (10 µg/ml).
Protein extracts were prepared,
and the presence of cleaved PARP was
measured by Western blotting
as an indicator of apoptotic cell death.
H-1 virus-infected cells
(MOI of 10) were analyzed in parallel for
comparison. As illustrated
in Fig.
6A (left panel), the cells proved to
be sensitive to both
TNF-
(with or without cycloheximide) and H-1
virus regarding
the induction of PARP cleavage, as expected from the
literature
(
96,
97) and the above-mentioned data,
respectively. In contrast,
all four RU clones resisted not only H-1
virus but also TNF-
alone (or cycloheximide alone [data not
shown]), though to various
extents (Fig.
6A). Interestingly, TNF-
was still
able to trigger
the cleavage of PARP in RU cells in the presence of
cycloheximide,
suggesting that the death machinery is fully functional
in all
RU clones but that it is suppressed by a protein(s) whose
synthesis
is cycloheximide sensitive. It should also be stated that at
early
times (4 h) after treatment with TNF-
alone, a small
proportion
of PARP was cleaved in all RU clones (Fig.
6B), but this
progressively
disappeared at later times. This transience may be
assigned to
the instability of PARP fragments produced in low amounts
in treated
RU cells. Alternatively, a small fraction of the
TNF-
-treated
RU cell population may die through apoptosis and be
eliminated
over time, resulting in the overgrowth of the major
resistant
cells.
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|
FIG. 6.
Comparison of the sensitivity of U937 and RU cells to
TNF-- or H-1 virus-induced apoptosis. PARP cleavage was revealed by
Western blot analysis of the total protein extracts (50 µg). (Upper
panel) U937 and RU cells were treated or mock treated for 24 h
with TNF- (50 ng/ml) alone, with TNF- (50 ng/ml) plus
cycloheximide (CHX) (10 µg/ml), or with H-1 virus (10 PFU/cell) as
indicated at the top of each lane. (Lower panel) Cells were treated
with TNF- (1 ng/ml) for the indicated times.
|
|
A striking parallelism was observed between H-1 virus and TNF-
regarding their abilities to induce apoptosis in the U937
cells and
their failure to do so in the RU variants. This prompted
us to test
whether parvovirus H-1 may induce the production of
TNF-
in infected
cultures, leading to the death of TNF-sensitive
U937 cells but sparing
their TNF-resistant derivatives. Indeed,
infection with several other
viruses has been reported to induce
the production of TNF-
(
82). We were, however, unable to detect
TNF-
by
enzyme-linked immunosorbent assay in the medium of U937
and RU cultures
at different times postinfection (data not shown).
Additional
experiments were carried out in which U937 cells were
infected with H-1
virus (MOI of 5 PFU/cell) and further incubated
in the presence of
polyclonal blocking antibodies directed against
TNF-
and preventing
the binding of this factor to its receptor.
These cultures were then
tested for the cleavage of PARP by Western
blotting at 24 h
postinfection. As shown in Fig.
7, the
anti-TNF-
serum fully inhibited the TNF-
-induced cleavage of PARP
(lanes
1 and 3) but did not prevent H-1 virus from triggering this
cleavage
(lanes 6 and 7). Incubation with BSA did not affect the
cleavage
of PARP induced by either TNF-
or H-1 virus (lanes 2 and
5).
These observations argue against the possibility of H-1 virus
inducing apoptosis indirectly through the stimulation of TNF-
release in the medium.
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|
FIG. 7.
Resistance of H-1 virus-induced apoptosis to blocking
anti-TNF- antibodies. PARP cleavage was analyzed by Western blotting
with total protein extracts (50 µg) of U937 cells that were infected
with H-1 virus (5 PFU/cell), mock treated, or treated with TNF- (10 ng/ml) and further incubated for 24 h in the presence or absence
of blocking anti-TNF- serum. BSA (0.1%) was used as a negative
control.
|
|
| |
DISCUSSION |
Many viruses have been reported to induce programmed cell death or
apoptosis in infected cells (20, 38, 58, 85, 102). The
autonomous parvoviruses were assumed to induce cell death by lysis of
infected permissive cell lines. One study showed ultrastructural evidence of apoptosis in cultures of hematopoietic precursors infected
with parvovirus B19 (60). In the present study we report sequential molecular events associated with morphologic changes characteristic of apoptosis upon parvovirus H-1 infection of the monocytic cell line U937. Hoechst staining of H-1 virus-infected U937
cells indeed reveals the typical apoptotic bodies that also appear in
response to TNF- (Fig. 1). The enzyme PARP has been demonstrated to
be cleaved rapidly and specifically during apoptosis (46) by
ICE-related proteases, in particular caspase-3/CPP32, which is
activated in cells after treatment with various apoptotic agents.
Although PARP cleavage per se does not seem to be essential for the
apoptotic process (91), this event is now generally considered to be a consequence of cells committing apoptosis. We show
here that H-1 virus infection triggers the cleavage of PARP and the
activation of caspase-3/CPP32, which is known to use PARP as a
substrate and to be involved in apoptosis.
Our data strongly suggest that the accumulation of the viral
replicative NS proteins is required to trigger the cleavage of PARP and
the formation of apoptotic bodies. Indeed, UV-inactivated H-1 virus is
unable to induce these apoptotic markers, although it is taken up by
target cells. Furthermore, recombinant H-1 virus expressing the NS
proteins but lacking the capsid genes is fully competent to induce the
cleavage of PARP and cell death (Fig. 3). Although NS proteins could be
cytotoxic in an indirect way, through their functions in viral DNA
replication and transcription (14), a direct effect is
supported by recent studies with transformed cell clones which have
integrated the MVMp NS protein-encoding transcription unit under the
control of an inducible promoter (8, 61). While NS protein
accumulation appears to be necessary for H-1-induced apoptosis, a
possible synergistic effect of the capsids cannot be totally ruled out,
since the recombinant H-1 virus stocks are contaminated with a low
fraction of wild-type virus generated during the production. However,
these contaminating wild-type viruses are present in amounts that are
too small to cause a detectable cleavage of PARP in the absence of the
NS-expressing recombinants (Fig. 3, lane 8). The major cytotoxic
nonstructural product of parvoviruses is the protein NS-1
(8), but the mechanism of NS-1 cytotoxic activity is still
elusive. In this respect, one possible target of NS-1 could be the
c-myc gene. Indeed, c-Myc expression is inhibited at early
times after H-1 virus infection of U937 cells (see Fig. 4), and the
disappearance of both the oncoprotein and its mRNA is concomitant with
the accumulation of the viral NS proteins (Fig. 4 and 1, respectively).
It should be stated, however, that c-myc expression is known
to be controlled at both transcriptional (100) and
posttranscriptional (95) levels. Therefore, it cannot be
ruled out that the observed reduction of c-myc mRNA
steady-state levels in H-1 virus-infected U937 cells (Fig. 4B) results
from alteration in RNA processing besides transcription initiation. The
cytotoxic function of NS-1 was previously found to cosegregate with its
capacity for promoter transregulation (47), leading to the
suggestion that NS-1 may act at least in part by disturbing the
expression of essential cellular genes.
Altogether, our observations raise the questions as to whether c-Myc is
involved in H-1 virus-induced apoptosis. Although present data do not
provide definite clues regarding this question, this possibility should
be considered. Indeed, the c-Myc content of U937 cells decreased very
rapidly after H-1 virus infection and before the onset of PARP
cleavage. Furthermore, the regulation of c-Myc expression proved to be
altered in four RU clones that were derived from the U937 cell line
through selection for H-1 virus resistance and also failed to undergo
PARP cleavage and apoptosis after treatment with TNF- (Fig. 6A).
These clones were previously shown to be impaired with regard to the
downregulation of c-Myc expression and the arrest of cell proliferation
in response to the differentiating agent TPA (52). Although
the four RU clones displayed various steady-state levels of c-Myc
oncoproteins, their respective c-Myc levels all remained unchanged
after H-1 virus infection (Fig. 4C). These data support a role for
c-Myc in H-1 virus-induced apoptosis in U937 cells, although it cannot be excluded that the downregulation of c-Myc is a consequence of
apoptosis in this system. c-Myc, in association with its partner protein Max, is a transcription factor that is essential for the cell
cycle entry and progression and is oncogenic when overexpressed (105). It has been shown that the mitotic cycle gets blocked in cells expressing NS-1, pointing to a possible link between NS
toxicity and cell cycle perturbations (68, 69). It was also
reported that in cells infected with MVMp or Aleutian disease virus of
mink, a massive accumulation of NS-1 takes place at the G1-S transition, leading to cell cycle disturbances
(66). Op De Beeck and Caillet-Fauquet (69)
proposed that NS-1 might exert a direct cytostatic action by inhibiting
host cell DNA replication, possibly because of NS-1-induced nicks in
the chromatin. Another, nonexclusive possibility would be that upstream
regulators of the cell cycle, including relevant transcription factors,
are targets for NS-1. Thus, the downregulation of c-Myc upon H-1 virus infection of U937 cells might lead to their arrest in the cell G0-G1 phase, as is the case when these cells
are treated with agents that induce differentiation (18).
The suppression of c-Myc expression in H-1 virus-infected cells might
be a signal of apoptosis resulting from a conflict between
growth-arresting (c-Myc disappearance) and growth-promoting (serum)
factors. Conversely, c-Myc overexpression has been consistently found
to promote apoptosis by growth factor deprivation or genotoxic agents
(105). Interestingly, c-Myc-associated apoptotic events
share with H-1 virus-induced apoptosis the activation of a
caspase-3-like protease (39) and recruit along a similar
signaling pathway (32).
An alternative, but nonexclusive reason for the high sensitivity of
U937 cells to the induction of apoptosis in response to H-1 virus or
TNF- may lie in their relative failure to express antiapoptotic
genes upon treatment with these agents. It is noteworthy in this
respect that the RU cell variants which were selected for their
resistance to H-1 virus infection but proved also to survive treatment
with TNF- become sensitive to TNF- in the presence of
cycloheximide. This makes it unlikely that the failure of RU cells to
respond to TNF--induced apoptosis is due to a deficiency in the
apoptotic machinery, and it raises the possibility that the RU clones
have developed an antiapoptotic defense system which requires de novo
protein synthesis. Members of the Rel/NF-kB family of proteins are
candidate mediators of this defense. Indeed, these transcription
factors are known to counteract the apoptotic pathway triggered by
TNF- (4, 51, 88, 90). In addition, Klefstrom et al.
(41) showed that c-Myc increases the cytotoxic effects of
TNF- in fibroblasts by impairing cell survival signaling via
NF-
B. It is therefore conceivable that the resistance of RU cells to
TNF- is mediated, at least in part, by NF-B or related factors.
It should be stated that these transcription factors control the
expression of specific genes in macrophages, including the inducible NO
synthase (iNOS) gene (25, 98). We have previously demonstrated a constitutive production of NO and oxygen species in the
RU clones (52), in keeping with the probable activation of
NF-B in these cells. It may be speculated that NO metabolites inhibit apoptosis in RU cells, which was proposed to occur in other
systems (54) and may result from S-nitrosylation of cysteine proteases (17). Another protein candidate for the modulation of H-1 virus or TNF--induced apoptosis in the U937 cell system is
the retinoblastoma gene product (pRB), an important regulator of the
cell cycle that is also known to protect cells from apoptosis (1,
27). pRB was recently shown to be postranslationally modified by
a transglutaminase-driven reaction in U937 cells undergoing apoptosis
(67). This resulting pRB polymerization is accompanied by
the release and subsequent degradation of the transcription factor
E2F-1. It has also been demonstrated that pRB is a substrate for
caspases in the context of apoptosis induced by TNF-
(37). These studies point to the importance of pRB
inactivation during apoptosis. It remains to be determined whether this
pRB processing occurs in H-1 virus-infected U937 cells and whether it
is impaired in the H-1 virus and TNF--resistant RU derivatives.
In summary, the present study provides evidence for the involvement of
apoptosis in the death of monocytic U937 cells infected with parvovirus
H-1. The viral NS proteins are likely to be responsible for this
effect. The apoptotic pathway triggered by parvovirus H-1 appears to
share at least some steps with the one activated by TNF-, as is
apparent in particular from the TNF resistance of cell variants
selected for their survival to virus infection. Although the precise
mechanism by which H-1 virus induces apoptosis remains to be
determined, it was observed that treatment with TNF- or H-1 virus
infection of U937 cells is accompanied by a rapid and drastic
downregulation of c-Myc expression prior to the appearance of apoptotic
signs (activation of caspase-3, PARP cleavage, and apoptotic bodies).
This feature, together with the failure of TNF- and H-1
virus-resistant cell variants to downregulate c-Myc expression, suggest
that c-Myc, or factors regulated in a similar way, may play a key role
in the signaling of apoptosis. In addition, cell variants resisting
death inducers may emerge through the constitutive activation of
antiapoptotic effector molecules.
| |
ACKNOWLEDGMENTS |
We are grateful to G. Poirier for the PARP antibodies and to A. Buerkle for his help in PARP immunoblotting. We thank J. Cornelis and
J. C. Jauniaux for helpful and critical discussions, G. Balboni and A. Dege for expert technical assistance, and J. M. Vanacker for his support.
B.R. received a fellowship from the Belgian Fonds National de la
Recherche Scientifique. J.-A.L.-G. was supported by a fellowship from
the Commission of the European Communities (Human Capital and Mobility)
and by the German Cancer Research Center.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: DKFZ, Abt. F0100
and INSERM U375, Postfach 101949, D-69009 Heidelberg, Germany. Phone: 49 6221 424960. Fax: 49 6221 424962. E-mail:
j.rommelaere{at}DKFZ-heidelberg.de.
Present address: Center for Advanced Biotechnology and Medicine,
Piscataway, N.J. 08854-5638.
Present address: Centro de Biologica Molecular Severo Ochoa,
Universidad Autonóma de Madrid, 28049 Madrid, Spain.
| |
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Abstract
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