
[Cancer Research 60, 7039-7047, December 15, 2000]
© 2000 American Association for Cancer Research
Identification of a Caspase-2 Isoform that Behaves as an Endogenous Inhibitor of the Caspase Cascade1
Nathalie Droin,
Myriam Beauchemin,
Eric Solary2 and
Richard Bertrand2,3
Research Centre of the University of Montreal Hospital Centre, Notre Dame Hospital, Montreal Cancer Institute, Montreal, Quebec, H2L 4M1, Canada [N. D., M. B., R. B.], and INSERM U517, Faculty of Medicine and Pharmacy, 21033 Dijon, France [N. D., E. S.]
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ABSTRACT
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Procaspase-2
is one of the aspartate-specific cysteine proteases that are activated
in response to various apoptotic stimuli. Two isoforms of human
procaspase-2 have been described initially. Overexpression of the long
isoform (caspase-2L) promotes cell death whereas the short isoform
(caspase-2S) antagonizes some apoptotic pathways. In the present study,
we identified two additional CASP-2 mRNAs, designated
CASP-2L-Pro and
CASP-2S-Pro. The proteins encoded by these
isoforms corresponded to the prodomain of procaspase-2L and -2S, in
which the last
-helix of their caspase recruitment domains was
deleted. Caspase-2L-Pro mRNA and protein were detected in a
series of human tissues and cell lines. Yeast 2-hybrid assays and
immunoprecipitation studies indicated that caspase-2L-Pro
can interact with procaspase-2L and the adaptor protein RAIDD/CRADD,
but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of
recombinant caspase-2L-Pro negatively interfered with
cytochrome c/dATP-mediated activation of the caspase cascade in a
cell-free system. In transient expression studies of human B lymphoma
Namalwa cells, overexpression of caspase-2L-Pro weakly
induced apoptosis, which was prevented by a D83A/E87A double mutation.
In stable selected CASP-2L-Pro-transfected
Namalwa cells, overexpression of caspase-2L-Pro delayed
apoptotic DNA fragmentation induced by death receptor agonists
(anti-Fas antibodies, tumor necrosis factor-
) and DNA
topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and
prevented etoposide-induced activation of the caspase cascade.
These inhibitory effects were not observed in stable transfected
cells expressing the D83A/E87A double mutant. Altogether, these data
indicated that the caspase-2L-Pro isoform functions as an
endogenous apoptosis inhibitory protein that antagonizes caspase
activation and cell death.
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INTRODUCTION
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The evolutionary conserved process known as apoptosis plays an
essential role in normal development, tissue homeostasis, and immune
responses, whereas its dysregulation is involved in the pathogenesis of
various diseases (1, 2, 3, 4)
. Apoptosis is also one of the
cell-death mechanisms that can be triggered in cancer cells by
cytotoxic drugs (5, 6, 7)
. This death process usually
requires the activation of a series of cysteine aspartate-specific
proteases, referred to as caspases (8)
. These enzymes are
produced as zymogens, known as procaspases, that require proteolytic
cleavage to constitute a catalytically active endopeptidase
(9, 10, 11)
. Fully mature caspases are heterotetramers that
contain two large and two small subunits. In addition to the four
subunits that constitute the active enzyme, procaspases contain an
amino-terminal prodomain of varying length (12)
. The
caspase gene family contains 14 mammalian enzymes, of which 11 have
been cloned in humans (11)
. Some of these enzymes
(caspase-1, -4, -5, and -11) are involved mainly in cytokine
processing. Other caspases can be subdivided into two groups on the
basis of their prodomain length. A simple view of the proteolytic
cascade activated during apoptosis involves caspases with a long
prodomain (caspase-8, -9, and -10) as initiator enzymes that cleave and
activate effector caspases with a short prodomain (caspase-3, -6, and
-7). In turn, these latter enzymes cleave a series of key cellular
proteins, which results in cell dismantling. Actually, in
vitro studies have shown that effector caspases can cleave and
activate caspases with a long prodomain, suggesting feedback
amplification loops (13)
.
Activation of initiator caspases requires adaptor molecules that
interact with their prodomains. These prodomains contain either a death
effector domain, or
DED,4
or a
caspase recruitment domain, or CARD. Both the DED and the CARD function
as interaction motifs that allow specific protein-protein interaction.
Interaction between CARDs in the adaptor molecule APAF-1 and in the
prodomain of procaspase-9 mediates the recruitment, oligomerization,
and activation of the enzyme that, in turn, activates effector caspases
such as caspase-3 and -7 (14, 15, 16, 17)
. Similarly, interaction
between DEDs in the adaptor FADD/MORT1 and the prodomain of
procaspase-8 mediates the recruitment of the enzyme in a
death-initiating signaling complex. FADD/MORT1 contains another
protein-protein interaction domain known as the DD, also found in the
COOH-terminal intracytoplasmic part of plasma membrane death receptors.
Interaction between DDs comes after ligand-dependent receptor
activation (18, 19, 20, 21, 22, 23)
. All these interaction domains have a
similar three-dimensional structure consisting of six or seven
-helices organized in an antiparallel arrangement
(24, 25, 26)
.
Human procaspase-2L, initially described as Ich-1 L (27)
,
is a long prodomain-containing enzyme that is activated in many cell
types in response to various apoptotic stimuli, including growth factor
withdrawal, DNA damaging agents, TNF-
, and Fas ligation
(27, 28, 29, 30, 31)
. The role of caspase-2L in the proteolytic
cascade that leads to cell death remains poorly understood. The
caspase-2L prodomain contains a CARD that interacts with a similar
domain of the adaptor molecule RAIDD/CRADD (29
, 32) . This latter molecule also contains a DD that can associate
with the DD of the serine/threonine kinase RIP (32)
, an
enzyme that interacts with TNFR-1 via TRADD (33)
.
RAIDD/CRADD was proposed to recruit procaspase-2L to TNFR-1 in a manner
similar to the recruitment of procaspase-8 to the Fas death receptor
via FADD/MORT1 (29
, 32)
. However, whereas disruption of
the CASP-8 gene in mice completely abolishes TNF-
-induced
cell death, CASP-2 null mice do not exhibit any significant
defect in TNF-
mediated apoptosis, which suggests that caspase-2L
does not play a key role as an initiator enzyme in this cell death
pathway (34
, 35) . In addition to its recruitment at
TNFR-1, procaspase-2L has been shown to be released from mitochondria
upon apoptotic stimulation, accumulating in the nucleus in which the
protein colocalizes with RAIDD to subnuclear dot-like and filamentous
structures, and migrating to the Golgi apparatus where it specifically
cleaves golgin-160 (36, 37, 38, 39)
. In a cell-free system
established to reconstitute the proteolytic cascade activated by
cytochrome c released from the mitochondria upon apoptotic
stimuli, procaspase-2L was found to be cleaved by effector caspases
such as caspase-3, which suggests its possible downstream involvement
in the cascade (40)
.
Whereas overexpression of procaspase-2L induces apoptosis in various
cell types, a truncated isoform has been initially described whose
overexpression prevents the death of cells deprived of growth factors
(27)
. Alternative splicing of CASP gene mRNA
has been identified for several other caspases, including caspase-1,
-6, -7, -8, -9, and -10 (22
, 23
, 41, 42, 43, 44, 45, 46, 47)
. Ectopic
transient overexpression of some of these isoforms demonstrated
pro-apoptotic activity, because of either intrinsic proteolytic
activity or the presence of a DED or a CARD involved in either the
recruitment and autoprocessing of procaspases (10)
or the
formation of insoluble filamentous structures termed death effector
filaments (36
, 48)
. Conversely, overexpression of
alternative spliced isoforms of the CASP-9 gene, which
encode procaspase-9b, inhibits the activation of procaspase-9 by
competitively interacting with the CARD of APAF-1 (46
, 47)
, whereas caspase-8
3 impedes Fas- and TNFR-1-induced cell
death (23)
.
Here, we report the identification of two additional isoforms of the
CASP-2 gene. These isoforms encode proteins designated
caspase-2L-Pro and caspase-2S-Pro
because they correspond to caspase-2L and to caspase-2S prodomain,
lacking the last
-helix of their CARD domains. We focused on
caspase-2L-Pro, which is shown to function as an
endogenous apoptosis inhibitory molecule by interfering with
procaspase-2L activation and the proteolytic cascade.
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MATERIALS AND METHODS
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Cell Lines and Culture.
All cell lines were obtained from the American Type Culture Collection
(Manassas, VA) and grown in suspension culture in RPMI 1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2
mM L-glutamine, 100 units/ml penicillin and 100
µg/ml streptomycin in an atmosphere of 95% air and 5%
CO2 at 37°C. Cell culture products were
purchased from Life Technologies, Inc. (Grand Island, NY). To ensure
exponential growth, the cells were resuspended in fresh medium 24 h before each treatment. For DNA labeling, they were grown with
[14C]-thymidine (0.03 µCi/ml) for 24 h,
then chased overnight in isotope-free medium before to drug treatment.
Drugs and Chemicals.
Radioactive precursors [
-32P]-dCTP (>3000
Ci/mmol) and [2-14C]-thymidine (59 mCi/mmol)
were procured from ICN BioMedicals (Costa Mesa, CA). Etoposide (VP16),
CPT, cycloheximide, and recombinant TNF-
were purchased from the
Sigma Chemical Co. (St. Louis, MO), and stock solutions made in
Me2SO were stored at -80°C. The agonist
anti-human Fas mAb (IgM, clone CH-11) was obtained from Upstate
Biotechnologies (Lake Placid, NY). The specific fluorogenic derivative
peptides Ac-DEVD-AMC, Ac-LEHD-AFC, Ac-VEID-AFC, z-VDVAD-AFC, and
MCA-VDVADGWK(DNP)-NH2 were acquired from Bachem Bioscience Inc. (King
of Prussia, PA) and Calbiochem-Novobiochem Corporation (San Diego, CA).
All other chemicals were of reagent grade and purchased from Sigma
Chemical Co., ICN BioMedicals, Roche Molecular Biochemicals (Montreal,
Quebec, Canada), or other local sources.
cDNA Cloning, Site-directed Mutagenesis and Transfection.
Human CASP-2L-pro and
CASP-2S-Pro cDNAs were first cloned by RT-PCR
from mRNA using CASP-2L- and
CASP-2S-specific adaptor primers containing
NotI sequences at their respective ATG start and TGA stop
codons. All amplified fragments were directly cloned into pTarget
vector (Promega, Charbonnières, France) and sequenced according
to the manufacturers instructions (Pharmacia Biotech, Orsay, France).
The open reading frame of CASP-2L-pro
cDNA was subcloned into pTarget vector by PCR with
CASP-2L-pro-specific primers, PRO
sense primer encoding HA epitope tag sequences
(5'-ATGTATCCTTATGATGTTCCTGATTATGCTGCCGCTGACAGGGGACGCAGG-3') and PRO
antisense primer sequences (5'-TCAGGAGTGCAAGGCTTCACAGAAGGCATCAAAAG
CTTGGGG-3'). CASP-2L-pro
site-directed mutagenesis was performed on the
pTarget-HA-CASP-2L-pro plasmid, using
primer PRO sense and primer MUT-2 L antisense
(5'-TCAGGAGTGCAAGGCTGCACAGAAGGCAGCAAAAGCTTGGGG-3')
containing the mutations. The pTarget-His-CASP-2L vector was
generated by PCR using the CASP-2L-specific primers, with
the sense primer encoding His repeated sequences. The
pCDNA3-FLAG-RAIDD vector was obtained from Dr. V. M. Dixit
(University of Michigan Medical School, Ann Arbor, MI).
In transient expression experiments, all transfections were performed
by electroporation at 270 V (Gene Pulser; Bio-Rad, Hercules, CA) with
10 µg of each vector, and the cells were incubated for 48 h at
37°C before analysis. To generate stable transfected cell
populations, 10 x 106 Namalwa
cells were electroporated at 270 V with 10 µg of each plasmid,
including empty pTarget vector or pTarget encoding
HA-CASP-2L-Pro or
HA-CASP-2L-ProMut. The electroporated
Namalwa cells were selected during two months in RPMI 1640 media
supplemented with 10% FCS and 2 mM
L-glutamine in the presence of 1.5 mg/ml G418.
In Vitro Transcription/Translation Experiments and
Production of Recombinant Caspase-2L-Pro Protein.
In in vitro transcription/translation studies,
procaspase-2L, procaspase-2S and caspase-2L-Pro
proteins were generated by TNT®-coupled
transcription/translation assay, according to the manufacturers
instructions (Promega), with purified pTarget-CASP-2L,
pTarget-CASP-2S, and
pTarget-HA-CASP-2L-pro plasmids. To
generate recombinant protein,
CASP-2L-pro cDNA was first amplified
by PCR with specific adaptor primers containing EcoRI
sequences at the ATG start codon and XhoI sequences at the
TGA stop codon. The PCR product was inserted in the pCR-TOPO vector (TA
cloning system; InVitrogen, San Diego, CA) and then subcloned in the
bacterial expression vector pET-30a (+)-His-TAG (Novagen, Madison, WI)
at the EcoRI and XhoI sites. Escherichia
coli BL21 (DE3) was transformed with purified plasmids and
recombinant protein expression induced for up to 15 h by adding
100 µM isopropylthio-ß-galactoside to
exponentially growing bacteria at room temperature. The bacteria were
collected by centrifugation, resuspended in 5 mM
imidazole, 0.5 M NaCl, 20
mM Tris-HCl (pH 7.9), and samples were sonicated
on ice. After centrifugation at 12,000 x g
for 20 min, bacterial lysates were applied to a charged and
equilibrated Chelating-Sepharose (Amersham Pharmacia Biotech,
Piscataway, NJ) chromatography column. The column was then washed with
10 v of 5 mM imidazole, 0.5 M NaCl, 20
mM Tris-HCl (pH 7.9), 6 v of 50
mM imidazole, 0.5 M NaCl, 20
mM Tris-HCl (pH 7.9), and the bound protein was
eluted with 6 v of 1.0 M imidazole, 0.5
M NaCl, and 20 mM Tris-HCl
(pH 7.9). Proteins were concentrated and dialyzed several times in a
Centricon Plus-20 centrifugal device (Millipore, Bedford, MA). The
purity of recombinant caspase-2L-Pro was measured
by SDS-PAGE and Coomassie Blue R-250 staining (not shown).
Expression Studies and Southern Blotting.
cDNAs from normal human tissues were obtained from InVitrogen, and
genomic DNA from transfected lines was extracted by a salting-out
procedure (49)
. CASP-2L and
CASP-2L-pro cDNAs were amplified with
sense primer (5'-ATGGCCGCTGACAGGGGACGC-3') and antisense primer
(5'-TCAGGAGTGCAAGGCTTCAC-3'). The presence of
pTarget-HA-CASP-2L-pro and
pTarget-HA-CASP-2L-ProMut in the
transfected lines was monitored by PCR using sense and antisense
primers directed to internal vector sequences. PCR was performed in a
reaction mixture containing 200 µM 2'-deoxynucleotide
5'-triphosphate mix, 50 mM KCl, 1.5 mM
MgCl2, 10 mM Tris-HCl (pH
8.3), 50 ng of each primer, and 0.5 units of Taq DNA polymerase
(Perkin-Elmer Biosystem, Markham, Ontario, Canada). The reaction
mixtures were heated for 5 min at 94°C and amplified for 35 cycles
with denaturation at 94°C for 50 s, annealing at 45°C for
50 s, and extension at 72°C for 30 s. The amplified
products were electrophoresed on 1.2% agarose gel in Tris-acetate
buffer (pH 8.0) and gel-stained with ethidium bromide. For Southern
blot analysis, DNA was transferred by capillarity to GeneScreen nylon
membranes (Dupont-NEM Research Products, Boston, MA). After DNA
denaturation and fixation, blots were hybridized with a
[
-32P]-dCTP labeled
EcoRI-EcoRI Casp-2L-Pro DNA
restriction fragment. Hybridizations were undertaken overnight at
42°C in a solution containing 2 x SSC, 2 x Denhart, 2% (w/v) SDS, 50% (v/v) formamide, and 100 µg/ml
salmon sperm DNA. The blots were washed twice for 20 min at 42°C in
0.2 x SSC and 0.1% (w/v) SDS, then for 45 min at
56°C in 0.02 x SSC and 0.05% (w/v) SDS. DNA was
visualized by autoradiography on Kodak X-AR film.
Protein Extraction, Coimmunoprecipitation and Western Blot
Analysis.
The antibodies used in this study included anti-human procaspase-2 pAb
that recognize the NH-2 terminal domain of procaspase-2 L
[Ich-1(Ab-1); Calbiochem-Novabiochem Corporation], anti-HA peptide
epitope tag mAb (clone 12CA5; Roche Molecular Biochemicals) and
anti-FLAG peptide epitope tag mAb (M2; Upstate Biotechnologies). For
protein extraction, the cells were washed twice in PBS, lysed in buffer
containing 150 mM NaCl, 50 mM Tris-HCl, (pH
8.0), 0.1% Na-SDS, and 0.5% Na-desoxycholate in the presence of
protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride,
2.5 µg/ml pepstatin, 10 µg/ml aprotinin, 2.5 µg/ml trypsin
inhibitor, and 5 µg/ml leupeptin) for 30 min, then centrifuged (20
min; 15,000 x g), and supernatants
collected. For immunoprecipitations, control and transfected Namalwa
cells were collected by centrifugation, washed twice with ice-cold PBS,
and then homogenized in lysis buffer [50 mM Tris
(pH 7.4); 150 mM NaCl; 1 mM
NaVO4; 1% BSA; 1% NP40; 2
mM phenylmethylsulfonyl fluoride; 1 mg/ml
aprotinin] at 4°C for 30 min. Extracts were centrifuged and
supernatants collected. Specific anti-HA or anti-FLAG monoclonal
antibodies were added for 1 h, and immunocomplexes were captured
and precipitated with protein A/G-Sepharose (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA). For Western blots, after SDS-PAGE and
electrotransfer to nitrocellulose membranes (Amersham Pharmacia
Biotech), the membranes were incubated for at least 2 h with 5%
nonfat milk in PBST (PBS, 0.1% Tween 20) and for 23 h at room
temperature with primary antibodies. Horseradish peroxidase-conjugated
goat antirabbit or antimouse secondary antibodies were then added for
3060 min, and proteins revealed by enhanced chemiluminescence reagent
(Amersham Pharmacia Biotech) and autoradiography.
Caspase Activity Assays.
For control and drug-treated cell extracts, cells were homogenized at
4°C for 30 min in lysis buffer containing 100 mM Hepes
(pH 7.5), 5 mM EDTA, 5 mM DTT, 20% glycerol
(or 10% sucrose for caspase-6 extracts), and 0.3% NP40, centrifuged
(10,000 x g for 15 min at 4°C), and
supernatants collected and frozen at -80°C. Typically, 200 µg of
extracted proteins per assay were used to monitor caspase activities.
For the cell-free system, cell-free extracts were generated from U937
cells based on methods described previously (40
, 50 , 51)
.
Briefly, 24 x 108 cells were
pelleted and washed twice with cold PBS before a single wash with 5 ml
of ice-cold cell extract buffer [20 mM
Hepes-KOH (pH 7.5), 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 100 µM phenylmethylsulfonyl
fluoride, 10 mg/ml leupeptin, and 2 mg/ml aprotinin]. The cells were
pelleted, resuspended in 2 v of ice-cold cell extract buffer and
transferred to a 2-ml Dounce-type homogenizer. They were allowed to
swell under hypotonic conditions for 20 min on ice. They were then
disrupted with 2050 strokes of a B-type pestle. Lysis was confirmed
by examination of a small aliquot of the suspension under a light
microscope. The lysates were centrifugated at 15,000 x g for 15 min at 4°C, and supernatants were frozen in
aliquots at -80°C until required. Caspase activation was initiated
by adding 50 µg/ml cytochrome c and 1
mM dATP in 100 µg cell-free extract per assay
in the absence or presence of various amounts of purified recombinant
caspase-2L-Pro protein.
Caspase activities were measured by monitoring fluorescence
continuously in a dual luminescence fluorometer (LS 50B; Perkin-Elmer)
using specific emission and excitation wavelengths for the fluorogenic
derivative peptides z-VDVAD-AFC, Ac-DEVD-AMC, Ac-LEHD-AFC, Ac-VEID-AFC,
and MCA-VDVADGWK(DNP)-NH2. Reactions were performed in cuvettes and
temperature maintained at 37°C in a water-jacketed sample
compartment. The assay mixture contained 100 mM Hepes (pH
7.5), 20% (v/v) glycerol [or 10% (v/v) sucrose for caspase-6
substrate], 5 mM DTT, 5 mM EDTA, and 100
µM fluorogenic peptide substrates. Enzyme activities were
determined as initial velocities expressed as relative
intensity/min/mg.
Quantitation and Analysis of DNA Fragmentation.
The kinetics of DNA fragmentation were measured by a filter DNA elution
assay reported previously (52)
. DNA was extracted by a
salting-out procedure (49)
. After electrophoresis in 1.8%
agarose gel in Tris-borate-EDTA buffer (pH 8.0), DNA was visualized by
ethidium bromide staining.
Yeast Two-Hybrid System.
The MatchMaker LexA 2-hybrid system (Clontech Laboratories, Inc., Palo
Alto, CA) was used as described elsewhere (53)
.
CASP-2L-pro cDNA was subcloned in
2-hybrid plasmid pLexA vector, and Casp-2L, FADD,
RAIDD and APAF-1 cDNAs were subcloned in the
2-hybrid plasmid pB42AD. Sequential transformations were performed by
the lithium acetate method in yeast strain EGY48, and the cells were
plated on histidine-deficient (for pLexA marker), tryptophan-deficient
(for pB42AD marker), and histidine/tryptophan/leucine-deficient (for
LEU2 reporter gene assay) minimal SD
induction/selection media. APAF-1 cDNA was kindly provided
by Dr. X. Wang (University of Texas Southwestern Center, Dallas, TX),
FADD and RAIDD cDNAs by Dr. V. M. Dixit
(University of Michigan Medical School). pLexA-p53, pLexA-Lam, and
pB42AD-SV40Large T from Clontech Laboratories served as positive and
negative controls.
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RESULTS
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Molecular Cloning of CASP-2L-Pro cDNA.
A set of primers encompassing the open reading frame of
Casp-2L consistently generated two amplified fragments of
1308 and 1090 bp by RT-PCR. Both fragments were amplified in several
human cell lines (HL60, U937, K562, KCL22, Jurkat, BV173, and HT29) and
hybridized with a specific CASP-2L probe (data not shown).
Nucleotide sequence analysis revealed that the 1308-bp fragment encoded
CASP-2L ORF, whereas the 1090-bp fragment represented a
shorter variant form that was designated
CASP-2L-pro (Fig. 1A)
.
CASP-2L-pro contains the first 256
nucleotides of CASP-2L and then differs by an alternative
splicing that deletes the next 218 nucleotides. The deletion generates
a translational frameshift with a stop codon 18 nucleotides downstream
(Fig. 1, A and B)
.
CASP-2L-pro encodes a protein of 91
amino acids that contains the prodomain of procaspase-2L in which the
CARD is deleted of the last
-helix and lacks the subsequent large
and small subunits of procaspase-2L (Fig. 1C)
. The
calculated molecular weight of caspase-2L-Pro was
estimated at Mr 11,000, and the
in vitro translated product migrated at
Mr 15,000 on SDS gels (Fig. 1D)
. A similar variant isoform of CASP-2S was
identified by RT-PCR using a set of primers encompassing the open
reading frame of CASP-2S and was designated
CASP-2S-Pro (data not shown).

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Fig. 1. Nucleotide and deduced amino acid sequences of
caspase-2L-Pro. A, nucleotide sequence
alignment of CASP-2L and
CASP-2L-pro. GT- and AG-conserved
splice sites are underlined in CASP-2L sequence.
B, organization of
CASP-2L-pro. GT- and AG-conserved
splice sites are surrounded. CASP-2L and
CASP-2L-pro stop codons are indicated.
C, deduced amino acid sequence of
caspase-2L-Pro compared with procaspase-2L.
Boxes indicate -helices of the caspase recruitment
domain. D, Western blots of procaspase-2L,
procaspase-2S, and HA-caspase-2L-Pro generated in
vitro by TNT®-coupled transcription/translation assay. The
translated products were fractionated on 12% SDS-PAGE, detected by
anti-caspase-2 or anti-HA antibodies, and revealed by enhanced
chemiluminescence.
|
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Expression of CASP-2L-Pro.
On the basis of PCR and Southern blot analyses (Fig. 2, A and B)
,
CASP-2L-pro was found to be
constitutively expressed in a series of normal human tissues, including
skin, breast, and colon, whereas its expression was virtually
undetectable in lung, spleen, brain, testis, and ovary. A protein of
Mr 15,000, corresponding to the
Mr of
caspase-2L-Pro, was also detected in cellular
extracts prepared from human B lymphoma Namalwa, monocytic-like
leukemic U937 and promyelocytic HL60 cells using polyclonal antibodies
raised against the NH-2 terminal domain of procaspase-2L (Fig. 2C)
. These antibodies also recognized procaspase-2L as a
Mr-48,000 protein (Fig. 2C)
. A protein of Mr 15,000
was also detected in HeLa and Jurkat cells, suggesting that
caspase-2L-Pro is expressed in many cell types
(data not shown).

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Fig. 2. Expression of caspase-2L-Pro in
human tissues and cancer cell lines. A, PCR analysis of
CASP-2L and CASP-2L-pro
expression in normal human tissues. The PCR products were analyzed on
agarose gel stained by ethidium bromide after electrophoresis.
CASP-2L and CASP-2L-pro
fragments are indicated by arrows. The 205-bp fragment
observed in the ovary is a nonspecific PCR product. B,
Southern blot analysis of PCR products using a
[ -32P]-dCTP
EcoRI-EcoRI Casp-2L-Pro DNA
restriction fragment as probe. C, Western blot analysis of
procaspase-2 L and caspase-2L-Pro in Namalwa,
U937, and HL60 cell lines. Two hundred-µg proteins were loaded per
lane. After electrophoresis and transfer, procaspase-2L and
caspase-2L-Pro were detected by caspase-2
polyclonal antibodies raised against the prodomain of caspase-2L and
revealed by enhanced chemiluminescence.
|
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Caspase-2L-Pro Specifically Interacted with
Procaspase-2L and RAIDD.
Previous studies revealed that procaspase-2 L interacted with the
adaptor protein RAIDD/CRADD, an interaction that was suppressed by
deletion of the prodomain of procaspase-2L (29
, 32)
. In a
yeast 2-hybrid assay, caspase-2L-Pro interacted with both
procaspase-2L and RAIDD/CRADD, whereas it did not interact with
FADD/MORT1 or APAF-1 (Fig. 3A)
. These interactions were
also observed in cotransfected Namalwa cells where
pTarget-HA-CASP-2L-pro was
transiently transfected with either pCDNA3-FLAG-RAIDD or
pTarget-His-CASP-2L. Immunoprecipitations with anti-FLAG or
anti-HA monoclonal antibodies were followed by immunoblottings with
anti-FLAG (Fig. 3B)
and anti-procaspase-2 antibodies (Fig. 3C)
. The caspase-2L-Pro/procaspase-2L
interaction appeared weaker than the
caspase-2L-Pro/RAIDD interaction, both in the
yeast 2-hybrid assay and in cotransfected cells.

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Fig. 3. Caspase-2L-Pro interacts with
procaspase-2L and RAIDD. A, in a yeast 2-hybrid assay, EGY
48 yeast cells containing caspase-2L-Pro fused to
the LexA DNA-binding domain were grown in selective medium in the
presence of various proteins fused to the B42 DNA activation domain, as
indicated. The positive control in the two-hydrid assay represents
pLexA p53 and pB42 SV40 T, and the negative control represents pLexA
lamin C and pB42 SV40 T. B, Namalwa cells were transiently
transfected by electroporation with empty pTarget,
pCDNA3-FLAG-RAIDD, or cotransfected with
pCDNA3-FLAG-RAIDD and
pTarget-HA-CASP-2L-pro, as indicated.
Coimmunoprecipitation experiments (IP) on transfected
Namalwa cells were performed with anti-Flag or anti-HA antibodies, as
indicated, and blotting was revealed by anti-Flag antibodies before
enhanced chemiluminescence. In the control (C),
coimmunoprecipitation experiments were undertaken by anti-HA antibodies
and A/G-Sepharose beads. C, Namalwa cells were transiently
transfected by electroporation with empty pTarget or cotransfected with
pTarget-HIS-CASP-2 L and
pTarget-HA-CASP-2L-pro.
Immunoprecipitation experiments (IP) were performed by
anti-HA antibodies (HA), and blotting was revealed by
anti-caspase-2 antibodies before enhanced chemiluminescence. In the
control (C), coimmunoprecipitation experiments were
conducted with anti-HA antibodies (diluted) and A/G-Sepharose beads.
|
|
Caspase-2L-Pro Interfered with Caspase Activities
in Vitro.
To determine whether caspase-2L-Pro could modulate the
activation of other caspases, we used a cell-free system, described
previously, in which the caspase cascade is activated by the addition
of cytochrome c and dATP (40
, 50
, 51)
.
Cell-free extracts were prepared from untreated U937 cells, and caspase
activities were monitored with a series of fluorogenic peptide
substrates, suggesting activation of these caspases, in the absence or
presence of recombinant caspase-2L-Pro protein.
The addition of recombinant caspase-2L-Pro
induced dose-dependent inhibition of z-VDVAD-AFC (caspase-2 L
substrate), Ac-DEVD-AMC (caspase-3, -7, and -2L substrate),
Ac-VEID-AFC (caspase-6 substrate) and Ac-LEHD-AFC (caspase-9 substrate)
hydrolysis (Fig. 4)
. Recombinant Bcl-xL
protein deleted of its COOH-terminal anchor transmembrane domain was
used as a negative control in cellular extracts obtained from apoptotic
U937 cells. The addition of recombinant Bcl-xL protein (up to 20 µg)
had no inhibitory effect on caspase activities (data not shown; Ref.
54
).

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|
Fig. 4. Caspase-2L-Pro interferes with
caspase activities in a cell-free system assay. U937 cell-free extracts
were incubated with cytochrome c/dATP in the absence or
presence of different amounts of purified recombinant
caspase-2L-Pro protein. Cleavage of Ac-LEHD-AFC
(caspase-9 substrate), Ac-DEVD-AMC (caspase-3, -7, and -2L substrate),
z-VDVAD-AFC (caspase-2L substrate) and Ac-VEID-AFC (caspase-6
substrate) was monitored. Enzyme activities were determined as initial
velocities expressed as relative intensity/min/mg and the results
expressed as the percentage of relative enzyme activity
according to the formula
[(Vi-Vo/Va-Vo) x 100], where Vi is the initial velocity
measured in activated extract in the presence of various fixed amounts
of inhibitors, Vo is the basal initial velocity measured in
unactivated control extract, and Va is the initial velocity
measured in activated extracts in the absence of inhibitors.
Data points are representative of two independent experiments.
|
|
Transient Transfection of CASP-2L-Pro Induced Low Level
of Apoptosis in Namalwa Cells.
Several point mutations in the prodomain sequence of procaspase-2L,
including L41F, G78R, L89A, F85A, F82A, or D83A/E87A, were found to
prevent procaspase-2L interaction with RAIDD, whereas D83A or E87A
substitutions had no effect on this interaction (29)
. By
directed mutagenesis, we generated the
CASP-2L-pro double mutant carrying
D83A/E87A substitutions
(HA-CASP-2L-ProMut, Fig. 5A
). In transient cell death
assays, transfection of wild-type
CASP-2L-pro increased the level of
DNA fragmentation and apoptosis in Namalwa cells, compared with cells
transfected with the empty vector (Fig. 5B)
. In contrast,
CASP-2L-ProMut transfection did not
increase the level of DNA fragmentation and apoptosis in these cells
(Fig. 5B)
, revealing the importance of a functional CARD
domain for this effect.

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Fig. 5. Transient expression of
caspase-2L-Pro induces apoptosis. A,
schematic representation of HA-caspase-2L-Pro and
HA-caspase-2L-ProMut carrying D83A and E87A.
B, Namalwa cells were transfected with pTarget,
pTarget-HA-CASP-2L-pro or
pTarget-HA-CASP-2L-ProMut, and DNA
fragmentation was quantitated by filter DNA elution assays 48 h
after transfection. The results are expressed as the percentage
of DNA fragmentation. Data points are the means of three
independent experiments (n = 9);
bars, SD.
|
|
Stable Expression of Caspase-2L-Pro Inhibited Apoptosis
Induced by Various Stimuli.
Mixed populations of Namalwa cells transfected with empty pTarget,
pTarget-HA-CASP-2L-pro and
pTarget-HA-CASP-2L-ProMut vectors,
were selected under 1.5 mg/ml Geniticin. PCR analysis of genomic DNA,
using specific primers directed to vector sequences, demonstrated the
presence of the insert in the selected cell population (Fig. 6A)
. Western blot analysis
revealed the presence of endogenous procaspase-2L and exogenous
HA-caspase-2L-Pro wild-type and double mutant in
the transfected lines (Fig. 6B)
. To evaluate the effect of
their expression, cells were exposed to various apoptotic stimuli,
including cell death receptor agonists (anti-Fas mAb, TNF-
) and DNA
topoisomerase I- (camptothecin, CPT) and II- (etoposide, VP16)
inhibitors. The kinetics of DNA fragmentation were monitored by filter
DNA elution assay and visualized by agarose gel electrophoresis.
Overexpression of wild-type caspase-2L-Pro
delayed the occurrence of DNA fragmentation induced by agonist Fas mAb,
VP16, TNF-
, and CPT compared with empty vector-transfected cells, an
effect that was partially or totally prevented by the presence of
caspase-2L-ProMut (Fig. 6, C and D)
.

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Fig. 6. Caspase-2L-Pro overexpression
inhibits apoptosis induced by different stimuli. Stable Namalwa variant
cells transfected with empty,
HA-CASP-2L-pro, and
HA-CASP-2L-ProMut vectors were
selected as bulk culture under 1500 µg/ml Geneticin, as indicated.
A, PCR analysis of genomic DNA showing
HA-CASP-2L-pro, and
HA-CASP-2L-ProMut insertion in these
mixed cell populations. B, Western blot analysis of
procaspase-2L, HA-caspase-2L-Pro, and
HA-caspase-2L-ProMut expression in these mixed
cell populations. Proteins were detected by caspase-2 polyclonal
antibodies and revealed by enhanced chemiluminescence. C,
kinetics of DNA fragmentation were monitored by filter DNA elution
assay in cells treated continuously with 50 ng/ml Fas mAb and 0.8 µg
cycloheximide (upper left panel) or 50 ng/ml TNF- and 0.8
µg cycloheximide (lower left panel), or after 30-min
treatment with 20 µM VP16 (upper right
panel) or 1.0 µM CPT (lower right
panel). The results are expressed as the percentage of DNA
fragmentation. Data points are the means of two independent experiments
(n = 6); bars, SD.
Symbols: pTarget ( ),
pTarget-HA-CASP-2L-pro ( ), and
pTarget-HA-CASP-2L-ProMut ( )
Namalwa variant cells selected under 1.5 mg/ml Geneticin. D,
agarose gel electrophoresis of DNA extracted at the indicated times
after VP16 treatment (20 µM; 30 min), from
pTarget, pTarget-HA-CASP-2L-pro, and
pTarget-HA-CASP-2L-ProMut Namalwa
variant cells selected under 1.5 mg/ml Geneticin.
|
|
Caspase-2L-Pro Interfered with Caspase Activation in
Etoposide-treated Cells.
The kinetics of caspase activities were monitored in transfected cells
after VP16 treatment. Overexpression of caspase-2L-Pro
interfered with activation of various caspases involved in the
hydrolysis of Ac-LEHD-AFC (caspase-9 substrate), Ac-DEVD-AMC
(caspase-3, -7 and -2L substrate), MCA-VDVADGWK(DNP)-NH2
(caspase-2L substrate) and Ac-VEID-AFC (caspase-6 substrate). In
accordance with the kinetics of DNA fragmentation, inhibition of
caspase activities observed in Namalwa cells overexpressing wild-type
caspase-2L-Pro was either less significant
[MCA-VDVADGWK(DNP)-NH2 hydrolysis] or absent (Ac-DEVD-AMC,
Ac-VEID-AFC and Ac-LEHD-AFC hydrolysis) in cells overexpressing the
D83A/E87A mutant (Fig. 7)
.
 |
DISCUSSION
|
|---|
Several endogenous proteins that either directly or indirectly
interfere with caspase activation have been identified in human cells.
These proteins include six IAPs, known as NIAP, cIAP-1/HIAP-2/hMIHB,
cIAP-2/HIAP-1/hMIHC, X-IAP, Survivin, and Apollon, that are
structurally characterized by at least one BIR domain
(55)
. The majority of IAPs inhibit some members of the
caspase family, either directly or indirectly, although they also
demonstrated caspase-independent inhibitory mechanisms such as those
mediated by activation of the transcriptional factor NF-
B
(56, 57, 58)
and those that involved a mitotic spindle
checkpoint (59)
. Another protein that interferes
negatively with caspase activation is cellular FLICE-inhibitory protein
(c-FLIP, also called Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT/Usurpin),
an inactive homologue of caspase-8 and caspase-10 that may act as a
competitive inhibitor by impeding the binding of these caspases to the
cytosolic domain of death receptors (60)
. In addition, the
alternative spliced isoform of the CASP-9 gene, which
encodes a short variant of procaspase-9, interferes negatively with
procaspase-9 activation, whereas one isoform of CASP-8
(caspase-8
3) impedes Fas- and TNFR-1-mediated cell death (23
, 46
, 47)
. In the present study, we noted that an alternative
spliced isoform of the CASP-2 gene, which encodes a
prodomain-only form of the caspase in which the CARD is deleted of the
last
-helix, also behaves as an endogenous inhibitor of caspases.
Other CASP genes that have been shown to encode several
isoforms include CASP-1, -6, -7, -8, -9, and -10
(22
, 23
, 41, 42, 43, 44, 45, 46, 47)
. These isoforms are most likely splice
variants (61)
. Their expression depends on the tissue or
the cell line examined. For example, caspase-1
isoform is highly
expressed in peripheral blood neutrophils and placenta
(41)
and the short variant isoform of the
CASP-9 gene is detected mainly in skeletal muscle (46
, 47)
; whereas the various isoforms of the CASP-10 gene
are expressed in several fetal tissues, mainly skeletal muscle, lung,
and kidney, but are virtually undetectable in adult tissues
(45)
. We have described previously the heterogeneous
expression of CASP-2L and CASP-2S isoforms in
various acute myelogenous leukemia samples (62)
. Here, we
show that endogenous expression of
CASP-2L-pro varies widely in normal
human adult tissues, from very low levels in the lung, spleen, brain,
testis, and ovary to higher expression levels in the skin and breast.
The expression of caspase-2L-Pro also varies
among diverse human leukemic cell lines. These results suggest that the
physiological role of the various caspase isoforms in modulating
apoptotic pathways might be highly tissue-specific.
Among the various caspase variants, caspase-2S was demonstrated to
prevent apoptosis of Rat-1 cells when deprived of growth factors
(27)
, caspase-8
3 impeded Fas- and TNFR-1-mediated cell
death (23)
, and the recently described short variant of
caspase-9, which lacks the catalytic site, was reported to negatively
interfere with various apoptotic stimuli by competitively interacting
with the CARD of APAF-1, thus preventing procaspase-9/APAF-1
interaction (46
, 47)
. Conversely, the short isoform of
caspase-10 designated as caspase-10/c, which is essentially a
prodomain-only form of the caspase and also lacks proteolytic activity,
can amplify the TNF-
-mediated death signal by inducing the formation
of perinuclear filamentous structures (45)
. The
CASP-1 gene was reported to encode five isoforms, including
three whose overexpression induces apoptosis, whereas the two others
increase the survival of Baculovirus-infected cells
(41)
. In addition, stable overexpression of the
Mr-11,500 prodomain of caspase-1 in
HeLa cells was recently found to enhance Fas-mediated apoptosis
upstream of caspase-8 activation (63)
. However, the
biological significance of caspase variants remains mostly unclear.
Like several caspase isoforms, the caspase-2L-Pro variant
of caspase-2 lacks any catalytic site. With a yeast 2-hybrid system, we
demonstrated that caspase-2L-Pro can interact with the
adaptor protein RAIDD and, to a lesser extent, with procaspase-2L. The
prodomain of procaspase-2 contains a single copy of CARD that was shown
to interact with the CARD present in RAIDD, probably through
electrostatic bond between the two domains (26)
.
RAIDD also contains a death domain in its COOH-terminal region that
interacts with a death domain present in RIP, a serine/threonine kinase
that is recruited to TNFR-1 and required for TNF-mediated activation of
NF-
B (64)
. It remains unclear whether
interaction between RAIDD and procaspase-2 L is necessary for
activation of caspase-2L and the downstream caspase cascade in response
to TNF or other cytokines (12)
. If this is the case, the
interaction of caspase-2L-Pro with RAIDD could prevent
interaction of procaspase-2L with the adaptor molecule, which could
account for the decreased sensitivity of cells overexpressing
caspase-2L-Pro to TNF-mediated cell death.
Caspase-2L-Pro could also interfere with the formation of
RAIDD-procaspase-2 L complexes that have been observed in the nucleus
after treatment with various apoptosis inducers (38)
.
Regardless of the mechanism, mutations of caspase-2L-Pro
that prevent RAIDD/procaspase-2L interaction suppress the protective
effect of caspase-2L-Pro toward apoptosis induced by
TNF-
and other death stimuli.
Although weaker than its interaction with RAIDD, interaction of
caspase-2L-Pro with procaspase-2L could interfere with the
procaspase-2L oligomerization that mediates its activation. Several
recent studies have established that procaspase oligomerization can
induce caspase activation (10
, 65
, 66)
. Homodimerization
of procaspase-1 or procaspase-2, which occurs before caspase
processing, has been shown to require their respective caspase
prodomain (67, 68, 69)
. However, the prodomain-prodomain
interaction alone is not sufficient to stabilize the dimers of Nedd2
precursors, the mouse homologue of human procaspase-2L. In addition to
the prodomain, dimerization of Nedd2 precursor requires the
COOH-terminus of the protein (69)
. Thus, interaction of
caspase-2L-Pro with the prodomain of procaspase-2L may not
be sufficient to induce autoprocessing of the proenzyme but could
prevent its homodimerization. This homodimerization is required for
processing of the precursor caspase and activation, because
prodomain-less caspase-2 does not undergo significant processing into
active subunits (69)
.
In transient cell killing assays, ectopic expression of
caspase-2L-Pro induced very low levels of cell death by
apoptosis. Transient expression of the prodomain of procaspase-8 has
also been shown to induce low levels of apoptosis in both 293T and
MCF-7 cell lines (23)
. A similar observation was made by
transiently expressing CARD- (29)
and DED- (70
, 71)
containing adaptor proteins in various cell lines.
Overexpression of caspase-10/c (45)
as well as that of the
prodomain of caspase-1 (72)
induced high levels of
apoptosis in MCF-7 and 293T cell lines, respectively. Expression of
caspase-10/c above an undefined concentration threshold results in the
formation of insoluble filamentous cytosolic or subnuclear structures
(45)
. These structures apparently act as a scaffold to
recruit and facilitate the autoactivation of cytoplasmic or nuclear
procaspases (38
, 48)
. A truncated RAIDD molecule
consisting of the residues 180, which lacked helix 6 of the CARD, was
unable to form filaments in transfected cells, suggesting that an
intact CARD structure was required for filament formation
(38)
. Because caspase-2L-Pro does not contain
the last
-helix, the formation of filamentous structures might not
account for the limited toxicity of caspase-2L-Pro observed
in transient transfection experiments.
Selected cells that stably overexpress caspase-2L-Pro
showed significant inhibition of agonist Fas Ab-, TNF
-, etoposide-
and camptothecin-induced apoptosis, indicating that
caspase-2L-Pro acted as an inhibitory protein. Our
observation that mutation of amino acids involved in
caspase-2L-Pro interaction with RAIDD suppressed its
anti-apoptotic activity is not sufficient to formally implicate
RAIDD-procaspase-2L interactions in cell death pathways induced by
these stimuli. Wild-type caspase-2L-Pro interfered with
various caspase activities in both etoposide-treated cells and a
cell-free system. Because no interaction was detected between
caspase-2L-Pro and APAF-1 in the yeast 2-hybrid assay,
caspase-2L-Pro may not impede the formation of
APAF-1/procaspase-9 complex. We cannot rule out the possibility that
caspase-2L-Pro interacts with other proteins in these
pathways, e.g., caspase-3 or the recently described APAF-1
homologue designated CARD-4/NOD-1, a protein that interacts with and
activates procaspase-9 (73
, 74) .
In conclusion, caspase-2L-Pro is a caspase variant that is
expressed in several normal human tissues and cancer cell lines and
negatively interferes with death receptor- and drug-mediated apoptosis
by preventing caspase activation. In some cell types,
caspase-2L-Pro could contribute to the prevention of
procaspase-2L autoactivation by interfering either with its prodomain,
thereby preventing dimerization of the proenzyme, or with an adaptor
molecule such as RAIDD to prevent its recruitment. This caspase isoform
could also prevent premature caspase activation by participating in the
complex process that generates active subunits. Regardless of the
mechanism of action of caspase-2L-Pro, these results
indicate that regulation of CASP gene transcription and
alternative splicing could play an important role in the modulation of
apoptotic pathways, including those triggered by anticancer drugs and
cytokines.
 |
ACKNOWLEDGMENTS
|
|---|
We acknowledge Ovid DaSilva (Research Center of the University
of Montreal Hospital Center) for the editorial work on this manuscript.
 |
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the
Medical Research Council of Canada (MT-15019) to R. B. The
E. S. group is a labelized group from the Ligue Nationale
Contre le Cancer. INSERM U517 receives the support of the Conseil
Régional de Bourgogne and ARERS. R. B. is a
chercheur-boursier du Fond de la recherche en santé du
Québec. N. D. received support from the Ministère de
lEducation Nationale, de lEnseignement et de la Recherche, the
Société Française dHématologie, and the
French Ministère des Affaires Etrangères. 
2 The two senior authors contributed equally to
this work. 
3 To whom requests for reprints should be
addressed, at Research Centre of the University of Montreal Hospital
Centre, Notre Dame Hospital, Montreal Cancer Institute, 1560 Sherbrooke
Street East, Room Y-5634, Montreal, Quebec, H2L 4M1, Canada. Phone:
(514) 281-6000, extension 6615; Fax: (514) 896-4689; E-mail: richard.bertrand{at}umontreal.ca 
4 The abbreviations used are: DED, death effector
domain; CARD, caspase recruitment domain; CPT, 20S-camptothecin
lactone; DD, death domain; Ac-DEVD-AMC,
acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin; Ac-LEHD-AFC,
acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin; Ac-VEID-AFC,
acetyl-Val-Glu-Ile-Asp-7-amino-4-trifluoromethylcoumarin;
MCA-VDVADGWK(DNP)-NH2,
7-methoxycoumarin-4yl-acetyl-Val-Asp-Val-Ala-Asp-Gly-Trp-Lys-dinitrophenol-NH2;
RT-PCR, reverse transcription-polymerase chain reaction; TNF-
, tumor
necrosis factor-
; TNFR-1, tumor necrosis factor receptor-1; VP16,
etoposide; z-VDVAD-AFC,
benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin;
mAb, monoclonal antibody, IAP, inhibitor of apoptosis; pAb,
polyclonal antibody; HA, hemagglutinin. 
Received 6/ 5/00.
Accepted 10/25/00.
 |
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