From the Division of Biochemistry, Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom
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ABSTRACT |
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Apoptosis involves the activation of a
cascade of interleukin-1 converting enzyme-like proteases
(caspases), a group of cysteine proteases related to the prototype
interleukin-1
-converting enzyme (caspase-1). These proteases cleave
specific intracellular targets such as poly(ADP-ribose) polymerase,
DNA-dependent protein kinase, and nuclear lamins. We show
here that apoptosis can be induced by double-stranded RNA. The
induction of apoptosis by double-stranded RNA and other agents leads to
the cleavage by a caspase of the signal transducer and activator of
transcription factor, STAT1 which is pivotal in the signal transduction
pathways of the interferons and many other cytokines and growth
factors. The product of this cleavage is no longer able to mediate
interferon-activated signal transduction and the cleavage event may
play a role in regulating the apoptosis response itself.
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INTRODUCTION |
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Apoptosis is the process of programmed cell death, and is a common
occurrence in embryogenesis and in the destruction of infected, damaged, or senescent cells (1, 2). The biochemical processes involved
in apoptosis can be divided into two discrete phases. A variable period
of time elapses following exposure to apoptosis-inducing signals before
cells become committed to undergo apoptosis. This phase is followed by
the execution phase (more correctly a suicide phase, since apoptosis is
cell autonomous) which, unlike the commitment phase, is irreversible.
The execution phase is the best understood, and is characterized by
changes in the cytoplasmic membrane, breakdown of the nuclear envelope,
condensation of chromatin structure, and eventual destruction of the
chromatin (laddering). Execution is associated with the activation of a
family of cysteine-proteases (caspases) which are related to the enzyme
required to process interleukin-1
(IL-1
)1 from its precursor
form (IL-1
-converting enzyme, caspase-1). It is not clear whether
any single caspase can cause cell death, since these enzymes appear to
be arranged in a complex network. Furthermore, to date, although
several substrates have been identified for caspases, no single
substrate cleavage has been shown to be essential for apoptosis, and
the consensus of opinion is that cell death occurs "by a thousand
cuts" (3).
In addition to activation through occupation of surface receptors such as Fas or tumor necrosis factor, apoptosis can also be induced by intracellular signals such as DNA damage, inhibition of protein synthesis, and viral infection. We are interested in the specific means by which viral infection can cause cells to undergo apoptosis (4). Apoptosis of virally infected cells is important since it allows macrophages to destroy a cell before the completion of the viral life cycle and the release of infectious particles (5). The significance of this is reflected by the fact that many viruses encode specific inhibitors of caspases (6-11). One of the best viral inducers of apoptosis is influenza (12-14). It has been proposed that double-stranded RNA (dsRNA) is generated during influenza viral replication, and that this leads to the transcriptional activation of Fas antigen that is a key factor in inducing apoptosis (15, 16).
In this paper we show that dsRNA is capable of inducing apoptosis
without the requirement for de novo protein synthesis,
indicating that susceptible cells contain a pre-existing pathway for
the execution of apoptosis if exposed to dsRNA. This is highly
reminiscent of the transcriptional induction of -interferon
(
-IFN) in response to viral infection or dsRNA treatment (17). While
investigating the effects of this dsRNA-induced apoptosis on the
induction of
-IFN we discovered that the signal transducer and
activator of transcription molecule, STAT1, becomes cleaved after
aspartic acid 694 by a caspase. STAT1 is a transcription factor that
becomes phosphorylated on tyrosine 701 by members of the JAK kinase
family in response to IFNs and many other cytokines and growth factors (18-20); this phosphorylation is required for dimerization and subsequent DNA binding and is thus essential for effective signal transduction. The removal of the C terminus of STAT1 by proteolysis consequently renders STAT1 unable to participate in signal
transduction. This cleavage is also caused by inducers of apoptosis
other than dsRNA, and may play a role in the down-regulation of
cellular responses during cell death.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
HeLa (subclone E (21)) and U3A (22) cells were
grown in Dulbecco's modification of Eagle's medium supplemented with
Glutamax (Life Technologies) + 10% fetal bovine serum (Life
Technologies). For dsRNA treatments, HeLa cells were grown until they
were 80-90% confluent, primed by treatment with 500 units/ml human
-IFN (Wellferon, a mixture of naturally produced
-IFN subtypes;
Glaxo-Wellcome) for 18 h, and then induced using 100 µg/ml dsRNA
(poly(I)-poly(C); Pharmacia) for the lengths of times shown in the
figures. Where indicated, cycloheximide was added to the medium at 50 µg/ml, at this concentration, protein synthesis is inhibited to
>99.9% as determined by incorporation of labeled amino acids into
total protein (23). The caspase inhibitor ZVAD
(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Enzyme System
Products) was dissolved in Me2SO and added to cells at 100 µM at the same time as dsRNA. TPCK (Sigma) was dissolved in methanol and added to cells at 100 µM at the same time
as dsRNA. Etoposide was dissolved in Me2SO and added to
cells for 18 h at 50 µM. IFN treatment of U3A cells
used 500 units of human
-IFN/ml or 500 units of human
-IFN/ml for
3 h where indicated.
Western Blotting Analysis-- Cells were lysed in sample buffer (24) (500 µl/9 cm plate) and extracts (15 µl) were fractionated on 7.5% polyacrylamide/SDS gels. Gels were transferred to 0.45-µm polyvinylidene difluoride membranes (Pierce), blocked, incubated with specific antisera, and washed as recommended by the manufacturers. Experiments shown in this work used a polyclonal STAT1 antibody raised against the N-terminal 194 amino acids (Transduction Laboratories catalogue number G16930); we have also used monoclonal antibodies raised against the C terminus (Santa Cruz catalogue number sc-417) and an internal fragment (Transduction Laboratories catalogue number S21120) to characterize the STAT1* product (data not shown). STAT2, STAT3, and STAT6 antibodies were all monoclonal products raised against the respective N-terminal regions (Transduction Laboratories; catalogue numbers STAT2 = S21220; STAT3 = S21320; STAT6 = S25420) while the human PARP antibody was a rabbit polyclonal raised against a synthetic internal peptide (GVDEVAKKK; Upstate Biotechnology; catalogue number 06-557). The monoclonal antibody (MAbSV5-P-k) (25) recognizing amino acids 95-108 of the P and V proteins of simian virus 5 was obtained from Dr. R. Randall (University of St. Andrews). All protein-antibody interactions were detected with enhanced chemiluminescence (Amersham) using horseradish peroxidase-conjugated sheep anti-mouse (Amersham) and donkey anti-rabbit (Amersham) IgG as secondary antibodies for monoclonal and polyclonals, respectively. For re-probing, filters were stripped as described by the manufacturers.
Plasmid Construction--
A cDNA sequence for human
STAT1 was obtained by polymerase chain reaction and verified by
partial sequencing and functional complementation in U3A cells (see
Fig. 4). This cDNA was inserted between the NcoI and
XbaI sites of the EF1
promoter vector pEFplink2 (a kind
gift of Dr. R. H. Treisman, Imperial Cancer Research Fund) to
generate pEFSTAT1
. The STAT1
cDNA was then epitope-tagged at
the N terminus using oligonucleotides encoding the amino acids 95-108
(GKPIPNPLLGLDST) of the P and V proteins of simian virus 5 to
generate pEFSTAT1
SV5. Single amino acid changes to
alanine or glutamic acid at aspartic acid 694 or to a stop codon at
glycine 695 were introduced into pEFSTAT1
SV5 using recombinant
polymerase chain reaction to generate pEFSTAT1
SV5(D694A),
pEFSTAT1
SV5(D694E), and pEFSTAT1
SV5(G695/STOP),
respectively. The amino acid changes were confirmed by sequencing. The
wild type STAT1
sequence and STAT1
genes containing the D694A and
G695/STOP changes were transferred from these plasmids into the vector
pT7
plink (26) as NcoI to XbaI fragments to
generate pT7
STAT1
, pT7
STAT1
(D694A), and pT7
STAT1
(G695/STOP), respectively. The reporter plasmid for
-IFN contained 4 copies of the 9-27 ISRE core (27)
(AGGAAATAGAAACTG) arranged in tandem upstream of the BamHI
site of ptk
(
39)lucter (21), while the reporter plasmid for
-IFN contained 2 copies of the IRF-1 GAS site (28) (TTTCCCCGAAA)
arranged in tandem upstream of the BamHI site of
ptk
(
39)lucter.
Transfections-- HeLa cells were transfected with 2 µg of DNA and 8 µl of LipofectAMINE (Life Technologies Inc.) according to the manufacturers instructions. U3A cells were transfected using calcium phosphate precipitation with 10 µg of effector, 10 µg of reporter, and 2 µg of transfection control plasmid (pSV2CAT (29)). For reporter gene assays, lysates were prepared and assayed for luciferase and chloramphenicol acetyltransferase activity as described previously (21). Luciferase activity was corrected to the chloramphenicol acetyltransferase activity to normalize for variations in the transfection efficiency. Transfection experiments were repeated at least three times.
Apoptosis Assay--
Laddered cytoplasmic DNA was prepared from
Triton X-100 lysates and labeled with [-32P]dATP using
the terminal transferase activity of Taq DNA polymerase (30). Reaction products were fractionated by electrophoresis on 1.8%
agarose gels in 0.089 M Trisborate, 0.002 M
EDTA, and after drying the gel, visualized by autoradiography.
In Vitro Cleavage Assay--
Bacterial lysates were prepared
from Escherichia coli BL21.DE3pLysS carrying a pET plasmid
encoding recombinant caspase-3 (kindly provided by Dr. N. McCarthy,
ICRF) or the control plasmid pET15b, as described previously (31). One
microgram of lysate was incubated with 1 µl of
[35S]methionine-labeled STAT1 or mutant forms of
STAT1
(prepared by in vitro translation of linearized
pT7
STAT1
, pT7
STAT1
(D694A), or pT7
STAT1
(G695/STOP) in
a standard TnT T7 quick coupled transcription/translation kit reaction;
Promega Corp.), in a 10-µl reaction containing 25 mM
Hepes, pH 7.5, 5 mM dithiothreitol, 5 mM EDTA,
0.1% CHAPS at 37 °C for 1 h. The reactions were stopped by the
addition of sample buffer (24) then analyzed by fractionation on a
7.5% polyacrylamide/SDS gel followed by autoradiography.
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RESULTS |
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We have previously shown that -IFN expression can be induced in
HeLa cells in response to treatment with dsRNA (21, 23). Induction
occurs at the level of transcriptional initiation and does not require
new protein synthesis, although the extent of induction can be much
enhanced by a pretreatment with type I IFN (priming). We noted that
during the induction process, cells exhibited an altered morphology
consistent with the activation of apoptosis. To determine whether
apoptosis could be induced by dsRNA, we treated HeLa cells with
poly(I)-poly(C), which was added directly to the growth medium, and
prepared DNA from induced cells. Fig. 1
shows that a nucleosomal ladder characteristic of cells undergoing
apoptosis is produced in response to dsRNA. When cycloheximide was
included in the induction medium the degree of laddering was
significantly enhanced, although consistent with results in other
systems cycloheximide alone could cause some laddering (32-36).
Although it is not possible to determine from these results whether
dsRNA and cycloheximide induce apoptosis through a common mechanism,
the fact that the extent of apoptosis seen with the combination of
dsRNA and cycloheximide is greater than that seen with either inducer
alone demonstrates that cycloheximide cannot be blocking the dsRNA
effect. Thus de novo protein synthesis cannot be required
for the induction of apoptosis by dsRNA, a result that is consistent
with the recent observations of others (37). Inclusion of the caspase
inhibitor ZVAD (38) in the induction mixture prevented DNA laddering
(Fig. 1) and completely blocked the observed cell death (data not
shown).
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HeLa cells contain two splice variant forms of STAT1, referred to as
STAT1 (91 kDa) and STAT1
(84 kDa), respectively (Fig. 2A). While investigating the
role of STAT1 in the induction of
-IFN expression we discovered that
exposure of HeLa cells to dsRNA for 2 h generated a novel form of
STAT1 (STAT1*), migrating with slightly faster mobility
than the 84-kDa form (STAT1
; Fig. 2A). Since the
production of STAT1* occurs with similar kinetics to the
activation of apoptosis, we investigated whether STAT1*
generation was associated with the activation of caspases. To determine
whether the production of STAT1* required de
novo protein synthesis, we repeated the induction of HeLa cells
with dsRNA in the presence of cycloheximide. Although cycloheximide
alone could also generate STAT1* to some degree, the
combined treatment of HeLa cells with dsRNA and cycloheximide led to
enhanced production of STAT1* (Fig. 2B) and was
accompanied by the loss of both STAT1
and -
. These results
suggested that STAT1* is derived from STAT1
and -
by
post-translational modification. To determine whether other members of
the STAT family are similarly affected we examined the state of STAT2
and STAT3 (Fig. 2C) and STAT6 (data not shown) in extracts
from dsRNA- and cycloheximide-treated HeLa cells without seeing
equivalent changes in mobility. By contrast, the well defined caspase
substrate, PARP, is cleaved under the same conditions (Fig.
2C).
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Fig. 2D shows that STAT1* generation in cells
treated with dsRNA and cycloheximide could be completely blocked by
treating cells with the caspase inhibitor ZVAD, whereas the serine
protease inhibitor, TPCK, was unable to affect cleavage. By contrast,
TPCK completely inhibited dsRNA-dependent activation of
NF-B under the same conditions (39), while ZVAD had no effect (data
not shown). The cleavage of PARP was similarly blocked by ZVAD but not
by TPCK (data not shown). To test whether STAT1* could be
generated by other inducers of apoptosis, we treated HeLa cells
with the DNA topoisomerase II inhibitor etoposide (40). This treatment
also resulted in the production of STAT1*, although we note
that this treatment reproducibly leads to preferential cleavage of
STAT1
over STAT1
(Fig. 2D). Taken together, these results indicate that the dsRNA/cycloheximide induction mixture triggers apoptosis in HeLa cells and that during this process STAT1
and -
are converted to a shorter form, presumably by proteolytic cleavage.
Because STAT1 and -
differ at their C termini, but are converted
to a single product, it seemed likely that STAT1* is
generated by a single cleavage event occurring near the C terminus of
the protein. By using a panel of antibodies we were able to show that
STAT1* lacks the C-terminal end of STAT1
and -
(data
not shown). Since a caspase-mediated cleavage was strongly implicated
by the above data, we inspected the C-terminal end of the human STAT1
protein sequence for an appropriate motif. We noticed a potential
cleavage site for caspases at the aspartic acid residue at position 694 (Fig. 3A). Cleavage at this
site in STAT1 would produce a species with a predicted molecular mass
of 81 kDa, in line with the observed size of STAT1*. To
verify that this sequence was indeed a target for cleavage, we created
an epitope-tagged wild type STAT1
expression vector, and ones in
which the aspartic acid residue at position 694 was changed to alanine
(D694A) or glutamic acid (D694E). When the epitope-tagged wild type
form of STAT1
was expressed in HeLa cells, a protein with mobility
similar to that of the endogenous form of STAT1
(91 kDa) could be
observed (Fig. 3B). A small amount of a protein with similar
mobility to STAT1* (81 kDa, Fig. 3B) could also
be observed in untreated cells; this product is generated as a result
of a low level of apoptosis induced by the transfection conditions, and
can be blocked with ZVAD (data not shown). When cells were treated with
dsRNA and cycloheximide the conversion of the 91- to the 81-kDa
truncation form was much enhanced (Fig. 3B). The 81-kDa form
migrated with the same mobility as a protein produced by expression of
a form of STAT1
truncated after amino acid 694 (G695/STOP) (Fig.
3B), as expected if STAT1* is generated by
cleavage at this site. In contrast to the wild type form, the D694A and
D694E mutant forms of STAT1
were not cleaved upon induction (Fig.
3B). Reprobing the Western blot with the STAT1 antibody
showed that the endogenous STAT1
and -
were cleaved in each case
(Fig. 3B). These results confirmed the presence of a
cleavage site at aspartic acid 694 in STAT1, and demonstrated that this
site is cleaved in both STAT1
and -
during apoptosis. To confirm
that STAT1 can be cleaved by a caspase, we tested whether recombinant
STAT1 can be cleaved by recombinant caspase-3 since the cleavage site
in STAT1 is similar to that seen in several caspase-3 substrates (Fig.
3A). Fig. 4 demonstrates that
the 91-kDa STAT1 protein is cleaved by caspase-3 to give an 81-kDa
product that has the same mobility as STAT1
truncated after amino
acid 694 (G695/STOP). This cleavage is not seen with control extracts, nor if the aspartic acid residue in the caspase cleavage consensus site
was changed to alanine (D694A).
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Activation of STAT1 by - and
-IFN, by cytokines such as IL-6 and
growth factors such as platelet-derived growth factor, involves a
single phosphorylation on tyrosine 701 (19), and for maximal activity,
serine 727 (53). Once phosphorylated, STAT1 can homo- or heterodimerize
to produce complexes capable of binding to DNA and activating
transcription (19). Clearly, cleavage of STAT1 after amino acid 694 would produce a truncated form of the protein that cannot be
phosphorylated on tyrosine 701, and thus should not be activated by
- or
-IFN. To test this, we examined the ability of
STAT1* to rescue responses to
- and
-IFN in U3A cells
which lack functional STAT1 (54). Responses to
-IFN were assessed
using a reporter promoter containing a 15-mer "core" sequence that
is a strong binding site for the type I-IFN-activated transcription
factor ISGF3 (IFN stimulatable response element (55, 56)), while responses to
-IFN were assessed using a reporter promoter containing a GAS site from the IRF-1 promoter that is a strong binding site for
the
-IFN-activated factor (GAF). In contrast to wild-type STAT1, the
truncated form (G695/STOP) was completely unable to rescue either
-IFN (Fig. 5A) or
-IFN
(Fig. 5B) responses. Altering amino acid 694 from an
aspartic acid to an alanine (D694A) did not interfere with either
signal transduction pathway (Fig. 5, A and B).
These results demonstrate that the effect of the proteolytic cleavage
of STAT1 during apoptosis would be to impair the ability of cells
undergoing apoptosis to respond to a variety of cytokine- and growth
factor-mediated signals.
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DISCUSSION |
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The data we have presented indicate that cells contain a
dsRNA-sensitive switch which triggers a pre-established program of both
apoptosis and -IFN production. In neither case is concurrent protein
synthesis required, and as a result there must be a means by which
dsRNA can activate the caspases. Studies on the Fas and tumor necrosis
factor receptors indicate that upon activation the receptors recruit
adaptor molecules through cytoplasmic "death domains" (57-60);
these adaptors in turn recruit additional molecules which contain
caspase motifs (61-63). Once recruited these molecules activate the
downstream caspase cascade. One possible candidate for the mediation of
the dsRNA signal is the cellular enzyme protein kinase R (PKR). This
kinase is known to function to down-regulate protein synthesis in
virally infected cells by phosphorylating the translational initiation
factor eukaryotic initiation factor-2
(64), but has also been shown
to play a role in the activation of NF-
B by dsRNA (65-68). It is
tempting to speculate that PKR may also signal to the apoptotic
execution machinery. Consistent with this it has been shown that
overexpression of PKR can cause apoptosis in cell culture (69), while
mouse embryonic fibroblasts lacking PKR are unable to undergo
apoptosis in response to dsRNA (37). Interestingly, PKR deficiency also
impairs responses to other inducers of apoptosis (37, 70). The
identification of a link between PKR and the caspases represents a
significant future goal.
In these studies we have also demonstrated that the transcription
factor STAT1, which is required for the ability of cells to respond to
a wide variety of signals, is cleaved during apoptosis by a
caspase. The protease inhibitor ZVAD used in our studies is a general
caspase inhibitor (38), and we have yet to identify which specific
member(s) of the caspase family is involved in generating STAT1*
in vivo, although we have demonstrated that caspase-3
can cleave STAT1 efficiently in vitro. Although the cleavage
site in STAT1 has a number of similarities to the sites known to be
substrates for caspase-3 (Fig. 3A), we note that it also has
some similarity with substrates for caspase-1, especially the minor
caspase-1 cleavage product of pro-IL-1 (49, 50). While we have yet to test it directly, we speculate that STAT1 may also be a caspase-1 substrate, since caspase-1 appears to be rather promiscuous in its
preferences (71-73). It is also interesting to note that STAT1 is not
cleaved at amino acid 143 despite containing an ELDS motif in this
region, and that STAT3 and STAT6 are also refractory to cleavage
despite containing possible caspase cleavage sites (Fig. 3A). We speculate that cleavage by caspases could be
prevented by the tertiary structure of some proteins.
The C-terminal cleavage product of STAT1 has lost the tyrosine residue
at position 701 and thus cannot be activated by - and
-IFN as
shown here, or presumably by the other ligands that activate by
promoting this phosphorylation. It is pertinent to ask what role STAT1
inactivation could play in the apoptotic response. It has previously
been observed that many cell types, including fibroblast (74) and
hematopoeitic cells (75) can undergo apoptosis as a result of becoming
depleted of growth factors or cytokines and can be rescued by the
addition of defined survival factors, which include STAT1 activators
such as platelet-derived growth factor (74), colony-stimulating factor
(76), and
-IFN (77). It is interesting to speculate that one
consequence of apoptosis is to down-regulate the ability of a cell to
respond to such factors. Since multiple growth factors can protect
cells against apoptosis, we think it unlikely that STAT1 cleavage alone
would be sufficient to render cells insensitive to protection by
survival factors. Nevertheless, inactivation of STAT1 function may be
an example of a class of modifications that ensures that the commitment
phase of apoptosis is irreversible. An alternative role for STAT1
cleavage is suggested by the recent demonstration that STAT1 can play a role in apoptosis by up-regulating the level of caspase-1 in response to cytokines (78). Consistent with this we have determined that the
degree of apoptotic death we observe in response to dsRNA and
cycloheximide is much enhanced if cells are primed by a prior exposure
to either type I or type II IFN (data not shown). When cells are
challenged with an apoptosis-inducing signal, the inactivation of STAT1
would lower the levels of caspase, and thus might play a role in
limiting the propensity of a cell to undergo apoptosis. The resolution
of this issue awaits further study.
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ACKNOWLEDGEMENTS |
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We thank George Stark and Ian Kerr for U3A
cells, Di Watling for Wellferon and -IFN, Richard Treisman for the
pEFplink2 plasmid, Rick Randall for MAbSV5-P-k, Victoria Heaton for
etoposide, Nichola McCarthy for pETCPP32 and helpful discussions, and
Mike Westby, Jo Brown, and David Slack for comments on the
manuscript.
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FOOTNOTES |
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* This work was supported by a Wellcome Trust University Award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Biochemistry,
Dept. of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom.
Tel: 44-181-725-5942; Fax: 44-181-725-2992; E-mail:
s.goodbourn{at}sghms.ac.uk.
1
The abbreviations used are: IL, interleukin;
STAT, signal transducer and activator of transcription; PARP,
poly(ADP-ribose) polymerase; IFN, interferon; TPCK,
N-p-tosyl-L-phenylalanine
chloromethyl ketone; dsRNA, double-stranded RNA; PKR, protein kinase R;
ZVAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; CHAPS,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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