From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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It is likely that endogenous inhibitors of the
apical caspases such as caspase-9 exist to prevent undesirable
activation of caspase cascades. A naturally occurring variant of
caspase-9 named caspase-9S was cloned from human liver. Caspase-9S is
missing most of the large subunit of caspase-9, including the catalytic site, but has the intact prodomain and small subunit. Caspase-9S did
not show apoptotic activity in transfection analysis. Overexpression of
caspase-9S inhibited apoptosis induced by caspase-9, indicating that
caspase-9S is an endogenous dominant-negative of caspase-9. Moreover,
caspase-9S inhibited apoptosis induced by tumor necrosis factor(TNF)- Apoptosis, an evolutionarily conserved and genetically regulated
biological process, plays an important role in the development and
homeostasis of multicellular organisms (1, 2). Transmembrane receptor
molecules including TNF1-R1,
Fas, and TRAIL-R are known to activate apoptotic signaling pathways
following ligand binding. TNF- Most evidence implicates the activation of caspases as an essential
step in apoptotic signaling (1, 2). All caspases, so far identified,
are initially synthesized as inactive zymogens composed of the
prodomain plus large and small subunits. Generation of the active
caspase requires sequence-specific proteolytic cleavage to convert the
zymogen to a corresponding active enzyme (1, 2). Several studies have
shown that caspase-3 is pivotal in the execution of apoptosis (12, 13).
Recently, caspase-3 was shown to liberate a DNase termed CAD
(caspase-activated DNase) from an
inhibitor of CAD (ICAD) by cleaving the ICAD protein (14-16). This
process leads to DNA degradation, a hallmark event in apoptosis. Although many inducers of apoptosis lead to activation of caspase-3 activation and other terminal caspases, the pathways leading to this
event are not well established. It is likely that numerous mechanisms
exist to limit the activation of caspase-3 and other terminal caspases.
For example, nitric oxide blocks caspase-3 (and potentially other
caspases) activation and activity by modifying cysteine residue of the
catalytic site (17-20). The anti-apoptotic p35 protein is
enzymatically processed and forms a stable caspase-p35 complex,
preventing autoproteolytic activation of caspases (21).
A mechanism for the activation of caspase-3 involving the release of
cytochrome c from the mitochondria has been identified (22).
TNF- Previously, Northern blotting analyses suggested the possible existence
of different forms of caspase-9 (29, 30). To date, only one form of
caspase-9 has been cloned and characterized. Therefore, to better
understand the functional role of caspase-9 in apoptosis, we cloned
another form of caspase-9, named caspase-9S and characterized.
Expression Plasmids--
The full-length caspase-9 and
caspase-9S cDNAs obtained by PCR from human liver cDNA library
(Invitrogen) were cloned into pcDNA3 (Invitrogen). The full-length
caspase-3 cDNA was amplified by PCR from pKV-caspase-3 (from Dr. R. Talanian) and cloned into pcDNA3. The pCMV-Bax was provided by Dr.
E. White (Rutgers University, New Brunswick, NJ). The expression
plasmids for Apaf-1 (22) and FADD (26) described previously were
provided by Dr. X. Wang and Dr. V. Dixit, respectively.
Molecular Cloning of Caspase-9S cDNA--
Human caspase-9S
cDNA was cloned by PCR from a human liver cDNA library
(Invitrogen) using two primers, 5'-ATGGACGAAGCGGATCGGCGGC-3' (5'-primer) and 5'-TTATGATGTTTTAAAGAAAAGTTTTTTC-3' (3'-primer). PCR was
carried out for 60 s at 95 °C, 60 s at 55 °C, and
70 s at 72 °C for 35 cycles. The resulting PCR products (1250 and 800 bp) were subcloned into pBluescript II SK(+) (Stratagene) and sequenced.
RT-PCR and Southern Blotting Analysis--
Total RNA was
isolated from MCF-7, A549, HeLa, SK-OV-3, RL95-2, HepG2, and MRC-5 as
described (31) and subjected to RT-PCR. The same primer set and
conditions used for caspase-9S cDNA cloning were employed for PCR.
As an internal control for RT-PCR, glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified by PCR at the same experimental condition. PCR products were run in 1% agarose gel, transferred onto
the nitrocellulose membrane, and subjected to Southern blotting as
described (31). The DNA fragment encompassing the 294th amino acid to
the stop codon of caspase-9 (Fig. 1B) was used as a probe for Southern blotting.
Western Blotting--
Preparation of whole cell lysates and
Western blotting was performed as described (20) using anti-human
caspase-9 monoclonal antibody (PharMingen) or polyclonal anti-human
caspase-9 antibody (Dr. X. Wang).
Apoptosis Assays--
MCF-7 cells were transiently cotransfected
with Production of Recombinant TRAIL and Caspase-9S--
A human
TRAIL cDNA fragment (amino acids 114-281) obtained by PCR was
cloned into the pET-23d (Novagen) plasmid, and expressed protein was
purified using the His-bind Resin and Buffer Kit (Novagen).
To purify recombinant caspase-9S protein, the full-length caspase-9S
cDNA was cloned into pQE30 (Qiagen) bacterial expression vector.
Expression and purification followed the procedures described to purify
recombinant TRAIL.
Protein Binding Assay--
The cDNA fragment corresponding
to residues 1-97 of Apaf-1 was obtained by PCR and cloned into the
mammalian expression vector pCMV-GST to generate the GST-Apaf-1(1-97)
fusion protein. C-33A cells grown on 60-mm plates were transiently
transfected with 5 µg of pCMV-GST or pCMV-GST-Apaf-1(1-97) in order
to express the GST or GST-Apaf-1(1-97) proteins. Thirty-six hours
after transfection, whole cell lysates were prepared with RIPA lysis
buffer (1% Nonidet P-40, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl
fluoride, 2 µg/ml leupeptin, and 1 mM sodium
orthovanadate in phosphate-buffered saline) and incubated overnight at
4 °C with in vitro translated 35S-caspase-9
or 35S-caspase-9S in binding buffer (10 mM
Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol, and 0.5% Nonidet P-40) in the
presence or absence of purified caspase-9S. The reaction mixtures were
further incubated with glutathione-agarose beads for 2 h at
4 °C, and the beads were pelleted by centrifugation. Beads were
washed five times with binding buffer and boiled for 3 min in 1 × sample buffer. The resulting supernatants were loaded onto 15% SDS gel
and visualized by autoradiography.
Molecular Cloning of Caspase-9S cDNA--
To clone a caspase-9
variant, we employed PCR. PCR using a set of primers encompassing the
open reading frame of caspase-9 generated two products (1250 and 800 bp) from human liver cDNA library (data not shown). Nucleotide
sequencing revealed that the 1250- and 800-bp PCR products coded for
full-length caspase-9 and a shorter variant of caspase-9, respectively.
The shorter variant of caspase-9 was named caspase-9S (caspase-9 short
form, GenBankTM accession number AF110376). Analyses of
nucleotide and deduced amino acid sequences revealed that caspase-9S
was missing sequences coding for most of the large subunit (p17) of
caspase-9, including the catalytic site (QACGG) (Fig.
1, A and B). The
prodomain and small subunit (p12) of caspase-9 were intact in
caspase-9S (Fig. 1, A and B). The molecular mass
of caspase-9S was estimated to be 30 kDa, and the in vitro
translated product migrated as a 30-kDa protein on SDS gels (Fig.
1C).
Coexpression of Caspase-9 and Caspase-9S in Various Cell
Lines--
We next examined whether caspase-9S transcript is expressed
normally in cells. RT-PCR/Southern blotting detected transcripts representing caspase-9 as well as caspase-9S in most cell lines (caspase-9 transcript in A549 cells was only detectable after overexposure) (Fig. 2A).
RT-PCR/Southern blotting also revealed that the ratio of caspase-9 to
caspase-9S varied in the different cell lines. In MCF-7, RL95-2 (Fig.
2A), HepG2, and MRC-5 cell lines (data not shown), the
expression level of caspase-9 mRNA was higher than that of
caspase-9S. In contrast, HeLa and SK-OV-3 cell lines, caspase-9S
mRNA expression was detected to be higher than that of caspase-9
(Fig. 2). We have tested two antibodies (obtained from PharMingen and
Dr. X. Wang) for their capacity to detect caspase-9 and caspase-9S
proteins by Western blot analysis (Fig. 2B). Both antibodies
detected only caspase-9 protein. MCF-7 and HeLa cells expressed more
caspase-9 protein than A549 cells, correlating with the corresponding
message levels.
Dominant-negative Action and Inhibitory Role of Caspase-9S in
Apoptosis--
To examine caspase-9S function by transient
transfection, the MCF-7 cell line was chosen by two reasons: first,
caspase-9S expression was detected to be lower than caspase-9
expression in the cell line; second, the cell line was easily killed by
apoptosis stimuli such as TNF-
Overexpression of caspase-9 or caspase-3-induced apoptosis (Fig.
3A). In contrast, overexpression of caspase-9S did not lead to apoptosis (Fig. 3A), indicating that the deleted region
of caspase-9, including the catalytic site, is crucial for its normal function. The cysteine residue of the catalytic site is well conserved throughout the caspase family members and required for catalytic function. Loss of the catalytic site in caspase-9S most likely accounts
for the null activity. Previously, an artificial mutant caspase-9
(C287A), where the catalytic site cysteine residue (Cys, 287th amino
acid) was mutated to alanine (Ala), demonstrated dominant-negative activity against wild-type caspase-9 (22, 26). Thus, we next examined
whether caspase-9S also exhibits dominant-negative activity against
wild-type caspase-9. Overexpression of caspase-9S protected cells from
apoptosis induced by caspase-9 (Fig. 3B). This indicated that caspase-9S functions as a dominant-negative inhibitor of caspase-9. Overexpression of caspase-9S also inhibited apoptosis induced by Bax and FADD (Fig. 3B) as well as by TNF- Overexpression of Caspase-9S Inhibits Apaf-1/Caspase-9-mediated
Apoptosis--
Previously, Apaf-1 was shown to directly interact with
and activate procaspase-9 during apoptosis (22). To examine the
functional role of caspase-9S in this process, MCF-7 cells were
transiently transfected with plasmid constructs expressing caspase-9,
caspase-9S, or Apaf-1, individually or in combination (Fig.
4A). Transfection of the
vector did not significantly affect cell viability (column 1). Cells overexpressing caspase-9 exhibited moderate cell death (column 2), whereas cells overexpressing caspase-9S were
mostly viable (column 3). Overexpression of Apaf-1 alone
(column 4) or coexpression of Apaf-1 and caspase-9S
(column 5) did not induce apoptosis. However, when
coexpressed with caspase-9, Apaf-1 significantly increased apoptosis
(column 2 versus column 6). This
result is in agreement with the previous findings that showed direct
activation of caspase-9 by Apaf-1 (22). Coexpression of caspase-9S
inhibited apoptotic cell death induced by the combination of Apaf-1 and caspase-9 (column 7).
In order to activate caspase-9, Apaf-1 must interact with the prodomain
of caspase-9 (22). Our data (Fig. 1, B and C, and Fig. 3) suggest that caspase-9S may bind to Apaf-1 through its prodomain as does caspase-9 and compete with caspase-9 for binding to
Apaf-1 preventing caspase-9 activation. Therefore, we performed in vitro binding assays to examine this possibility directly
(Fig. 4B). Since the N-terminal 97 amino acids of Apaf-1 has
been shown to be responsible for interacting with the prodomain of
caspase-9 (22), we used GST-Apaf-1(1-97) for our binding assays. As
shown previously (22), in vitro translated caspase-9 bound
to GST-Apaf-1(1-97) (lane 5). Under the same experimental
conditions, caspase-9S also efficiently bound to GST-Apaf-1(1-97)
(lane 6). However, neither caspase-9 nor caspase-9S bound to
GST alone (lanes 3 and 4), indicating that
specific interactions occur between Apaf-1(1-97) and caspase-9S. Importantly, the binding of caspase-9 to Apaf-1 was abrogated by
caspase-9S (lane 7). These functional and binding assay
results indicate that caspase-9S plays as a dominant-negative
inhibitor, at least in part, by blocking Apaf-1-caspase-9 interaction
through an interaction with Apaf-1.
Our data (Figs. 3 and 4) indicate that caspase-9S is an endogenous
inhibitor of apoptosis. TNF-
It has been shown that the MCF-7 cell line does not express caspase-3
(35). We have observed this as well (data not shown). However, the cell
line is easily killed by various apoptosis-inducing molecules that
promote the cytochrome c release. This suggests that
Apaf-1/caspase-9 system also functions to activate a downstream executioner caspase(s) other than caspase-3 in this cell line. Caspase-3-like caspases including caspase-6 and caspase-7 have been
identified, and it has been shown that caspase-9 also activates caspase-7 (36). The capacity of caspase-9 to cleave/activate multiple
terminal caspases may explain why the defects observed in caspase-9
knockout mice (27, 28) are more serious than those observed in
caspase-3 knockout mice (12). Inappropriate activation of apical
caspases such as caspase-9 may cause undesirable activation of
downstream caspases, which will be detrimental to cells. Thus,
caspase-9S may guard against unwanted caspase-9 activation. FLIP
(FLICE-inhibitory protein) acts through a similar mechanism to block
procaspase-8, another apical caspase, which interacts with the
transmembrane death receptor molecules including TNF-R1 and Fas
(37).
, TNF factor-related apoptosis-inducing ligand (TRAIL), Bax, or Fas-associated death domain-containing protein (FADD) as
well as the combination of Apaf-1 and caspase-9. In vitro
binding assays demonstrated that caspase-9S binds to Apaf-1 and blocks the binding of caspase-9 to Apaf-1. Coexpression of caspase-9 and
caspase-9S mRNA was identified in various cell lines. Thus, caspase-9S acts as a dominant-negative inhibitor of caspase-9 activation, at least in part, by blocking Apaf-1-caspase-9 interaction.
INTRODUCTION
Top
Abstract
Introduction
References
and Fas ligand (or agonistic Fas
antibody) induce trimerization of the receptor molecules, eliciting
interaction of the receptors with cellular adaptor proteins such as
TRADD (3, 4) and FADD (5-7). FADD has been shown to directly interact
with and activate procaspase-8 (8-10). This signaling event is
followed by activation of caspase cascades leading to cleavage of
cytosolic, cytoskeletal, as well as nuclear proteins and DNA. Although
TRAIL-R is also known to induce cellular events similar to those
activated by TNF-
and Fas, the molecular signaling events for this
receptor are only partially characterized (11).
, Fas ligand (or agonistic Fas antibody), and TRAIL are known to
induce the release of cytochrome c from the mitochondria during apoptosis (23). Recent studies have demonstrated that Bcl-2
family member Bid links receptor-mediated apoptotic signals to
cytochrome c release from mitochondria (24, 25). The
C-terminal portion of Bid generated by caspase-8 activated through Fas
signaling pathway targets mitochondria and induces cytochrome
c release (24, 25). However, the detailed biochemical
mechanism by which cleaved Bid induces cytochrome c release
from mitochondria remains to be resolved. Once released, cytochrome
c is now known to bind to Apaf-1, a mammalian homologue of
C. elegans Ced-4, inducing a conformational change of Apaf-1
(22). Apaf-1 associated with cytochrome c and dATP directly
activates procaspase-9 (22) by interacting with the prodomain, termed
CARD (caspase recruitment domain)
of procaspase-9 (22, 26). Thus, caspase-9 is positioned at the apex in
the apoptotic signaling cascades activated by released cytochrome
c. The activated caspase-9 then is able to activate procaspase-3 (22). These findings suggest that caspase-9 may be a
central regulator of caspase-3 in a cytochrome
c-dependent signaling pathway involving Apaf-1.
Thus, caspase-9 activation is a potential key site for the regulation
of apoptosis. This hypothesis is supported not only by results obtained
with caspase-9 knockout mice where caspase-3 activation is
blocked (27, 28), but also by recent findings that a mutant caspase-9
generated by changing 287th cysteine to an alanine exhibits
dominant-negative activity and inhibits apoptosis (26).
EXPERIMENTAL PROCEDURES
-galactosidase (pCMV
-gal) and test expression plasmids using
Lipofectin (Life Technologies, Inc.) according to the manufacturer's
instructions. At the indicated time points after transfection, the
number of round blue apoptotic cells out of the total number of blue
cells was determined for each condition.
RESULTS AND DISCUSSION
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Fig. 1.
Molecular cloning and deduced amino acid
sequence analysis of caspase-9S. A, the deduced amino
acid sequence of caspase-9S was aligned with that of caspase-9
(GenBankTM accession number U56390). The box
indicates the catalytic site of caspase-9. The dotted line
describes missing amino acids. The putative sequence-specific cleavage
sites (29, 30) are denoted by asterisks. The mismatched
amino acid residue indicated by the arrowhead may result
from sequencing error. Extensive nucleotide sequencing of other
cDNA clones confirmed alanine (A) residue as the right
amino acid. The nucleotide sequence of caspase-9S has been submitted to
GenBankTM with accession number AF110376. B,
molecular structures of caspase-9 and caspase-9S are shown
schematically. The putative cleavage sites (130, 315, and 330) and
catalytic site (QACGG) of caspase-9 are indicated. C, the
full-length cDNA fragments encoding caspase-9 and caspase-9S
identified in panel A were subcloned into pcDNA3 and
in vitro translated using the TNT-coupled
transcription/translation kit (Promega) in the presence of
[35S]methionine. The translated products were
fractionated on a 15% SDS gel and visualized by autoradiography.
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Fig. 2.
Coexpression of caspase-9 and caspase-9S in
various cell lines. A, RT-PCR was carried out with RNA
(lanes 5-9) isolated from various cell lines as described
under "Experimental Procedures." As positive controls, the
expression constructs containing caspase-9 and caspase-9S cDNA
(Fig. 1C) were used in the PCR step (lanes 3 and
4). The resulting RT-PCR or PCR products were resolved on
1% agarose gel and visualized by ethidium bromide staining. After
transferred onto nitrocellulose membrane, RT-PCR/PCR products were also
subjected to Southern blotting as described under "Experimental
Procedures." As an internal control for RT-PCR,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
was amplified by PCR at the same experimental condition and used for
normalization. The lanes 1 and 2 are shorter
exposed results of lanes 3 and 4. B,
80 µg of each whole cell lysate (lanes 3-5) was
fractionated on 15% SDS-gel, and caspase-9 protein was detected by
Western blotting using anti-human caspase-9 monoclonal antibody
(PharMingen). Equal loading of the samples was monitored by Coomassie
staining (data not shown). As positive controls, in vitro
translated caspase-9 and caspase-9S proteins (Fig. 1C) were
loaded onto lane 1 and 2, respectively. Tested
caspase-9 antibody failed to detect caspase-9S protein as shown in
lane 2. The arrowhead indicates a nonspecific band.
(without transcription or translation
blocker) and TRAIL (see Fig.
3C).
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Fig. 3.
Dominant-negative action and inhibitory role
of caspase-9S in apoptosis induced by various molecules.
A, MCF-7 cells plated at 1.0 × 105/12-well
were transiently transfected with 0.2 µg of pCMV -gal plus 0.5 µg
of vector, caspase-9, caspase-9S, or caspase-3 expression plasmid using
Lipofectin (Life Technologies, Inc.) according to the manufacturer's
instructions. Fifteen hours after transfection, cells were stained for
-galactosidase expression and examined for morphological evidence of
apoptosis as described previously (Kumar et al. (38)). The
experiments were carried out at triplicate. The bar
indicates standard error. B, 0.2 µg of pCMV
-gal plus
1.5 µg of vector or caspase-9S expression plasmid were transiently
transfected with 0.5 µg of caspase-9, Bax, or FADD expression
plasmids or vector into MCF-7 cells plated at 3.0 ×105/6-well. Twenty hours after transfection, apoptosis
assays were performed as described in the legend to A.
C, MCF-7 cells plated at 1.0 × 105/12-well
were transiently transfected with 0.2 µg of pCMV
-gal plus 0.5 µg
of vector or caspase-9S expression plasmid as described in the legend
to A. Fifteen hours after transfection, cells were treated
without or with TNF-
(200 ng/ml) or TRAIL (200 ng/ml) for 5 h
followed by apoptosis assays as described in the legend to
A.
and
TRAIL (Fig. 3C), suggesting that caspase-9S blocks a common
signaling pathway activated by Bax, FADD, TNF-
, and TRAIL.
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Fig. 4.
Overexpression of caspase-9S inhibits
Apaf-1/caspase-9-mediated apoptosis. A, MCF-7 cells
plated at 3.0 × 105/6-well were transiently
transfected with 0.2 µg of pCMV -gal plus 0.5 µg of vector,
caspase-9, Apaf-1, or 1.5 µg of caspase-9S expression plasmid,
individually or in combination. The total amount of DNA for
transfection was adjusted to 2.2 µg with vector. Twelve hours after
transfection, apoptotic cells were counted as described in the
legend to Fig. 2. The experiments were carried out at triplicate. The
bar indicates S.E. B, blocking of caspase-9
binding to Apaf-1 by caspase-9S. The cDNA fragment corresponding to
residues 1-97 of Apaf-1 was prepared by PCR and cloned into the
pCMV-GST vector to generate GST-Apaf-1(1-97) fusion protein. In
vitro translated 35S-caspase-9 (lanes 1,
3, 5, and 7) or in vitro
translated 35S-caspase-9S (lanes 2,
4, and 6) or His-tagged caspase-9S purified from
bacterial cells (lane 7) were analyzed in in
vitro binding assays. For the blocking experiment (lane
7), 20 µg of purified caspase-9S was used. The bound products
and aliquots of
of the input for binding assays were
resolved on a 15% SDS gel and visualized by autoradiography.
, Fas ligand, TRAIL, and Bax are known
to induce the cytochrome c release from mitochondria (23).
This suggests that apoptotic signals initiated by such molecules
involve Apaf-1 and converge on a biochemical event eliciting caspase-9
activation. This may explain why apoptosis induced by such molecules is
blocked by caspase-9S (Figs. 3 and 4) as well as dominant-negative
caspase-9 (C287A) (22, 26). Furthermore, recent studies demonstrated
that apoptosis is significantly blocked in caspase-9 knock-out mice,
leading to embryonic lethality (27, 28). Caspase-3 activation was also
blocked in caspase-9 knockout mice. These in vitro and
in vivo results indicate that caspase-9 plays a critical
role in apoptosis. Our functional and binding assays demonstrated that
caspase-9S acts as an anti-apoptotic molecule by specifically blocking
caspase-9 activation (Fig. 4). However, our data do not rule out the
possibility that caspase-9S may directly inhibit enzymatic activity of
caspase-9. Caspase-1 and 3 are homodimeric proteins (2Xp17/p12)
containing two active sites (32-34). Therefore, caspase-9S could form
an inactive heterodimeric complex with caspase-9.
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ACKNOWLEDGEMENTS |
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We thank Dr. V. Dixit for MCF-7 cell line and FADD expression plasmid, Dr. X. Wang for Apaf-1 expression plasmid and caspase-9 antibody, Dr. E. White for Bax expression plasmid, and Dr. R. Talanian for caspase-3 expression plasmid. We also thank Dr. E. Alnemri for valuable discussion and Dr. D. Johnson for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM44100.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF110376.
To whom correspondence should be addressed: BST W1503, Dept. of
Surgery, University of Pittsburgh School of Medicine, 200 Lothrop St.,
Pittsburgh, PA 15261. Tel.: 412-624-6740; Fax: 412-624-1172; E-mail:
seold+{at}pitt.edu.
The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TRADD, TNF receptor-1-associated death domain-containing protein; FADD, Fas-associated death domain-containing protein; PCR, polymerase chain reaction; RT, reverse transcription; GST, glutathione S-transferase; PARP, poly(ADP-ribose) polymerase.
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REFERENCES |
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