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INTRODUCTION |
Apoptosis is initiated by a wide variety of stimuli, including
developmental signals, cellular stress, and disruption of the cell
cycle. Although the cellular mechanisms underlying apoptosis vary
according to the type of stimuli involved, it is generally believed
that apoptosis can be induced by the activation of pro-apoptotic signaling or inhibition of survival signaling. A well known survival signal pathway is mediated by the transcription factor, nuclear factor
B (NF-
B)1 (1-3).
NF-
B exists as a dimeric complex, comprising different members of
the Rel family: p50, p52, p65, c-Rel, and RelB; all of which contain a
well conserved amino-terminal 300 amino acid region. This part of the
sequence, known as the "Rel homology region," is responsible for
DNA binding, dimerization, and nuclear localization (1, 2). In
unstimulated cells, NF-
B is located in the cytoplasm as an inactive
complex through interactions with inhibitory proteins, I
B
and
I
B
. Upon cellular stimulation processes, such as cytokine induction or ionizing radiation, I
Bs are rapidly phosphorylated and
degraded. This in turn releases NF-
B, allowing translocation into
the nucleus, binding to specific
B sites, and subsequently inducing
the expression of target genes responsible for cell survival (4-8).
Previous studies have shown that activation of NF-
B is inhibited by
a variety of mechanisms. The prevention of degradation or induction of
I
B synthesis leads to the inhibition of NF-
B activation (9-11).
Inhibition of p65 phosphorylation, which is important for the
activation of p65, has also been known to suppress transcriptional
activity of NF-
B (12). Moreover, the cleavage of NF-
B by caspases
inhibits its activity during apoptosis (13, 14). These reports indicate
that inactivation of NF-
B activity plays a crucial role in the
apoptotic pathway.
Vitamin K3 (menadione) induces growth arrest and apoptosis
in various cancer cell lines (15). Despite a broad-range effect on
growth suppression of cancer cells, its hydrophobicity presents difficulties for use as an anticancer drug. Earlier experiments focusing on a search for new naphthoquinone analogs with polar groups
led to the discovery of 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (NA) by our group (16). NA induced growth inhibition more potently than
other analogs and natural VK3, without producing ROS in
human hepatocarcinoma cells (15, 16). In view of the fact that, as yet,
little is known about the mechanism of action of NA, this study
involves a detailed investigation into the molecular function of NA in apoptosis.
In this report, we demonstrate that NA inhibits tumor
necrosis factor
(TNF-
) induced activation of NF-
B
and induces apoptosis of HeLa cells. NA potently inhibits
transcriptional activity of NF-
B through caspase-3-mediated
proteolytic cleavage of p65 at Asp97. Truncated p65 leads
to the inhibition of transcriptional activity of NF-
B, and therefore
the promotion of NA-induced apoptosis, while in contrast, uncleavable
mutant p65 protein protects cells from apoptosis. These observations
suggest that the activation of NF-
B is inhibited by
caspase-3-mediated cleavage of p65, indicating that the survival
pathway mediated by NF-
B might be abolished during NA-induced apoptosis.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
Human cervical epitheloid
carcinoma (HeLa) cells were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10%
heat-inactivated fetal bovine serum (Life Technologies, Inc.) at
37 °C, in a humidified incubator with an atmosphere of 5%
CO2. Unless specified otherwise, all reagents were
purchased from Sigma.
Cell Proliferation and Apoptosis Assays--
HeLa cells were
seeded in 35-mm plates at an initial density of 2 × 104 cells/plate. Cells were cultured for 16-20 h in
complete medium and treated with 15 µM NA, 100 ng/ml
TNF-
or a combination of NA and TNF-
for 24 h. After
staining cells with trypan blue solution (0.4%), cell survival was
quantified by measuring dye exclusion. Cell viability was determined as
a percentage of control cells. For the apoptosis assay, HeLa cells were
transfected with plasmids encoding p65, p65D97A, or
p65
with pEGFP-C1 plasmid (CLONTECH) that encodes green fluorescence protein. Apoptotic cells were identified by their rounded
morphology, compared with the stead-out morphology of non-apoptotic
cells. The number of apoptotic cells were counted and presented as a
percentage of the total population of fluorescent cells.
DNA Fragmentation--
Cells were lysed in 400 µl of lysis
buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl,
25 mM EDTA (pH 8.0), 0.5% sodium dodecyl sulfate (SDS),
0.1 mg/ml proteinase K), and incubated at 50 °C for 15 h. DNA
was prepared using phenol/chloroform extraction and precipitated with
ethanol. After treatment with RNase A (0.1 mg/ml) for 2 h, DNA
samples were separated by electrophoresis on 1.5% agarose gels, and
visualized with ethidium bromide staining.
Preparation of Nuclear Extracts--
Cells were washed twice
with ice-cold phosphate-buffered saline, harvested by
centrifugation, and lysed in 400 µl of buffer A (10 mM
HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride) for 15 min. Samples were made up to a
volume of 25 µl with 10% (v/v) Nonidet P-40 and vortexed for 10 s. After centrifugation at 2,700 × g for 30 s,
individual supernatant fractions (cytosolic extracts) were transferred
to new tubes. Pellets were treated with ice-cold Buffer C (20 mM HEPES (pH 7.9), 0.4 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and
incubated for 15 min at 4 °C, followed by centrifugation at 20,000 × g for 5 min. Supernatants (nuclear extracts)
were transferred to new tubes and kept frozen at
70 °C until
further use.
Electrophoretic Mobility Shift Assay--
The NF-
B-specific
oligonucleotide (5'-AACGGGACTTTCCGCTGGGGACTTTCCG-3') (Santa Cruz
Biotechnology, Santa Cruz, CA) was end-labeled with
[
-32P]ATP (Amersham Pharmacia Biotech) using T4
polynucleotide kinase (Roche Molecular Biochemicals). Electrophoretic
mobility shift assays were performed by incubating 10 µg of nuclear
extract with 2 µg of poly(dI-dC) (Amersham Pharmacia Biotech) in
binding buffer (5 mM HEPES (pH 7.9), 5 mM
MgCl2, 50 mM KCl, 0.5 M
dithiothreitol, 10% glycerol) for 10 min at 37 °C. After
incubation, end-labeled double-stranded oligonucleotide probe was added
and the reaction mixture incubated for 10 min at 37 °C. Samples were
separated by native polyacrylamide gel electrophoresis in low ionic
strength buffer, following which dried gels were subjected to autoradiography.
Transient Transfection and Luciferase Assay--
HeLa cells were
transiently transfected with NF-
B luciferase reporter plasmid for
12 h using the LipofectAMINE method according to manufacturer's
instructions (Life Technologies). To assess the variation in
transfection efficiencies, a control plasmid pCMV
(CLONTECH) that expressed the LacZ gene
was used. Following 48 h transfection, cells were treated with NA,
TNF-
, or a combination of these compounds for 24 h. With a view
to investigating the transcriptional activity of NF-
B, cells were
transfected with p65, p65D97A, or
p65, together with
NF-
B luciferase reporter plasmid. Luciferase assays were performed
after 48 h transfection. Induction of luciferase expression was
determined by Lumat LB9501 (Berthold), and standardized to the
constitutive level of
-galactosidase activity.
Western Blot Analysis--
Western blot analysis was carried out
on 10 µg of nuclear extract. Following sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were
electrophoretically transferred to polyvinylidene difluoride
(Schleicher and Schuell). Membranes were blocked with TBS containing
0.5% Tween 20 in 5% skimmed milk, and subsequently incubated with the
following primary antibodies: anti-p65 COOH terminus (SC-372, Santa
Cruz), anti-p65 NH2 terminus (SC-109, Santa Cruz),
anti-PARP (N-20, Serotech), anti-Fas (G254-274, Pharmingen), anti-FasL (C-178, Santa Cruz), anti-caspase-3 (C31720, Transduction Laboratory), anti-caspase-9 (SC-7885, Santa Cruz), and cytochrome c (65981A, Pharmingen). Membranes were washed with TBST and
treated with secondary antibody conjugated with peroxidase. The
quantity of protein present was detected using the enhanced
chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech),
followed by exposure to film.
In Vitro Cleavage Assay--
In vitro transcription
and translation were performed with the TNT-coupled reticulocyte system
(Promega) and [35S]methionine (1,000 Ci/mmol, Amersham
Pharmacia Biotech) according to the manufacturer's instructions.
Aliquots of 3 µl of in vitro translated
[35S]methionine-labeled p65 were incubated with 2 µl of
recombinant caspases (caspase-3, caspase-1, or caspase-8) in the
presence or absence of 10 µM YVAD-fmk or Ac-DEVD-CHO in
reaction buffer (20 mM HEPES (pH 7.4), 100 mM
NaCl, 0.5% Nonidet P-40, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) for 90 min at 30 °C.
[35S]Methionine-labeled p65 and mutants
(p65D97A or p65D469A) were incubated for 90 min
with 200 µg of cell lysates prepared from cells treated with NA for
24 h at 30 °C. Samples were subsequently subjected to 10%
SDS-PAGE and autoradiography.
Mutagenesis and Plasmid Construction--
Individual mutants of
p65 were generated by site-directed mutagenesis. For the
p65D97A mutant, p65 DNA (100 ng) was incubated with 100 pmol of mutagenic oligomers
(5'-GAAAGGACTGCCGGGCTGGCTTCTATGAGGC-3' and
5'-GCCTCATAGAAGCCAGCCCGGCAGTCCTTTC-3'), and for the
p65D469A construct, the oligomers
(5'-ACCTGGCATCCGTCGCCAACTCCGAGTTTCA-3' and
5'-TGAAACTCGGAGTTGGCGACGGATGCCAGGT-3') were employed. The mutagenesis reaction was performed by polymerase chain reaction using
cloned pfu polymerase (Stratagene), polymerase chain
reaction was performed as follows: 50 s at 95 °C, 1 min at
55 °C, 15 min at 68 °C for 12 cycles. Base substitutions of these
constructs were confirmed by automated DNA sequencing.
Additionally, a NH2 terminus deletion mutant (
p65)
was produced by polymerase chain reaction, using the following primers:
5'-GGGAAGCTTGGCTTCTATGAGGCTGAGC-3' and
5'-GGGGATCCTTAGGAGCTGATCTGACTCAGC-3.
Preparation of Cytosolic Fractions--
Cells were washed twice
with ice-cold phosphate-buffered saline, resuspended in 5 volumes of
Buffer A (50 mM Tris Cl (pH 7.5), 1 mM EDTA, 50 mM 2-mercaptoethanol, 0.2% bovine serum albumin, 10 mM KH2PO4, 0.4 M
sucrose, 0.1 M phenylmethylsulfonyl fluoride, and 1 µg/ml
pepstatin) and incubated on ice for 20 min. Following incubation, cells
were treated with Nonidet P-40 (0.1%) and vortexed for 10 s.
Unbroken cells and nuclei were pelleted by centrifugation at 6,000 × g for 5 min at 4 °C. Supernatant fractions were
additionally centrifuged at 10,000 × g for 10 min at
4 °C to pellet mitochondria. After the second round of centrifuging,
supernatants containing cytosol were transferred to new tubes.
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RESULTS |
NA-induced Apoptosis in HeLa Cells--
NA has been shown
to induce growth inhibition in human hepatocarcinoma cells (16). The
effect of NA on growth in HeLa cells was investigated by treating cells
with NA and examining cell viability. We observed a decrease in
viability at 12 h, and entire loss of viability after 48 h,
indicating that cell death occurs in a time-dependent
manner (Fig. 1A). HeLa cells
treated with TNF-
demonstrated continuous proliferation, thereby
signifying resistance to cell death. However, cells treated with NA for
1 h prior to the addition of TNF-
once again showed significant cell death. In contrast, pretreatment with TNF-
notably reduced cell
death induced by NA. In order to determine whether cells are killed via
apoptotic processes, we examined internucleosomal DNA fragmentation (a
well known biochemical characteristic) after treatment with NA.
Apoptotic DNA laddering was faintly observed at 12 h (data not
shown), and clearly present at 24 h following NA treatment. DNA
fragmentation was not observed in cells exposed to TNF-
, but
appeared during pretreatment with NA (Fig. 1B). These
results imply that NA induces cell death in HeLa cells via an apoptotic
pathway, and attenuates TNF-
-induced resistance against
apoptosis.

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Fig. 1.
Effect of NA on the induction of apoptosis in
HeLa cells. A, HeLa cells were treated with 15 µM NA or 100 ng/ml TNF- for 24 h, TNF- for
1 h before treatment with NA (TNF- + NA), or NA for 1 h
prior to treatment with TNF- (NA + TNF- ). Cell survival was
quantified at the indicated intervals by trypan blue dye exclusion. The
percentage of cell survival was defined as the relative number of
surviving untreated cells. B, apoptotic DNA fragmentation of
the same samples was visualized by ethidium bromide staining, following
1.5% agarose gel electrophoresis.
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TNF-
-induced NF-
B Activation Is Inhibited by NA
Treatment--
The results illustrated in Fig. 1 suggest that NA might
destroy survival signaling induced by TNF-
, and therefore induce cell apoptosis. NF-
B, a representative anti-apoptotic factor activated by TNF-
, protects cells from TNF-
-induced apoptosis (3). The key question that requires address is whether NA overrides TNF-
-induced resistance by blocking NF-
B activation. NF-
B
activity was examined by electrophoretic mobility shift assay, using
32P-labeled NF-
B-specific oligonucleotide (see Fig.
2A for details). Treatment of
HeLa cells with TNF-
resulted in remarkable activation of NF-
B,
while NA failed to induce NF-
B activation. Moreover, TNF-
-induced
activation of NF-
B was strongly repressed by pretreatment with NA.
We next examined the transcriptional activity of NF-
B, using a
luciferase assay. It was observed that TNF-
-induced luciferase expression is significantly inhibited upon pretreatment with NA (Fig.
2B), indicating that NA inhibits TNF-
-induced NF-
B
activation and therefore obstructs NF-
B-stimulated anti-apoptotic
signaling pathway. Our results suggest that inhibition of NF-
B
activity may be part of the mechanism responsible for the apoptotic
effect of NA.

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Fig. 2.
NA reduces DNA binding activity and
transcriptional activity of NF- B induced by
TNF- . A, HeLa cells were
treated simultaneously with NA and TNF- for 30 min, or NA or TNF-
individually for 10 min before treatment with the other compound.
Nuclear extracts were prepared and subjected to electrophoretic
mobility shift assay with a radiolabeled double-stranded B
oligonucleotide. The arrow on the left-hand side
of the panel indicates the NF- B binding complex. B, HeLa
cells were transfected with a NF- B luciferase reporter plasmid (0.5 µg) for 48 h along with the same samples as in Fig.
1A. After treatment with the drugs for 12 h, luciferase
activity was determined. The diagram is depicted as fold induction at
the basal level. Each column represents an average (± S.E.) of three
individual experiments.
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NA Does Not Prevent Nuclear Translocation of p65--
It is known
that NF-
B sequesters in the cytoplasm due to interactions with
I
B, and translocates to the nucleus in response to extracellular
stimuli (2, 17). With a view to understanding the molecular mechanism
of NA, we examined whether NA-mediated NF-
B suppression is achieved
by perturbance of nuclear translocation of NF-
B. HeLa cells were
exposed to NA or TNF-
for 30 min, and the level of p65, a subunit of
NF-
B in nuclear fractions, was assessed. As shown in Fig.
3A, exposure of cells to
TNF-
results in an increase in the level of p65 protein in cell
nuclei, in contrast to nuclear p65 in NA-treated cells, where no
increase is observed. However, the amount of TNF-
-induced nuclear
p65 is not reduced by pretreatment with NA, indicating that NA does not
interrupt nuclear translocation of p65 induced by TNF-
. From the
above data, we conclude that inhibition of TNF-
-induced NF-
B activation by NA is not due to prevention of nuclear translocation of
p65.

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Fig. 3.
NA does not interrupt nuclear translocation
of p65. A, HeLa cells were treated with either NA or
TNF- , or co-treated with these compounds for 30 min. Nuclear
extracts (10 µg) of the cells were separated by 10% SDS-PAGE and
Western blotting was performed with anti-p65 antibody. B,
cells were treated with the compound as in Fig. 1A, and
protein levels were monitored by Western blot analysis, using
antibodies for p65 or PARP. Note that the asterisk on the
right side of the Western blot of p65 indicates nonspecific
bands.
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p65 Is Cleaved during NA-induced Apoptosis--
The next step was
to investigate whether there are any changes in the amount of nuclear
p65 at 24 h when cells undergo apoptosis induced by NA treatment.
It was found that the protein level of nuclear p65 in NA-treated cells
is not altered, compared with that of control or TNF-
-treated cells
(Fig. 3B). The cleavage of poly(ADP-ribose) polymerase
(PARP), a predominant biochemical hallmark of apoptosis, was further
observed in cells. Interestingly, the smaller band of p65
(Mr ~ 55,000) (
p65) was recognized in NA-treated cells, but not in the control or TNF-
-treated cells (Fig.
3B). The truncated p65 (
p65) is assumed to be a cleavage product generated during the apoptotic process. Experiments were conducted to analyze whether the appearance of
p65 is correlated with NA-induced apoptosis. As shown in Fig.
4A, the
p65 band firstly
appears in cells treated with 10 µM NA for 24 h. In
a time course experiment, this band was observed at 12 h after the addition of 15 µM NA (Fig. 4B). In contrast to
the results obtained with p65, we could not observe any cleavage of
p50, which is another component of NF-
B. Since PARP cleavage was
observed at 24 h after treatment with NA (Fig. 4B, last
panel), cleavage of p65 should be an earlier event. Our
observations suggest that
p65 is the cleavage product of p65
produced during NA-induced apoptosis.

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Fig. 4.
Cleavage of p65 correlates with NA-induced
apoptosis. HeLa cells were treated with 0-20 µM NA
for 24 h (A), or with 15 µM for an
indicated time period (B). Nuclear extracts (10 µg) were
fractionated by 10% SDS-PAGE, and subjected to immunoblotting with
p65, p50, and PARP antibodies, respectively. The asterisk in
the middle panel indicates a nonspecific band.
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p65 Is Cleaved at Asp97 by Caspase-3--
It is known
that various proteins are cleaved by caspase-3 during apoptosis
(18-21). To evaluate whether caspases are responsible for p65
cleavage, cells were incubated with a caspase-3-specific inhibitor
(Ac-DEVD-CHO), and an inhibitor for a broad spectrum of caspases
(zVAD-fmk) for 1 h prior to treatment with NA. Cleavage of p65 was
monitored by Western blot analysis. Upon treatment with Ac-DEVD-CHO, we
failed to observe the appearance of both the cleavage product (Fig.
5A) and DNA fragmentation
(Fig. 5B), which indicates that the cleavage of p65 is
catalyzed by caspase-3. To provide direct evidence of this theory,
in vitro-translated [35S]p65 was incubated
with recombinant caspase-1, caspase-3, and caspase-8. It was noted that
while p65 was cleaved by caspase-3 and inhibited by Ac-DEVD-CHO (Fig.
6), caspase-1 and caspase-8 had no effect
on the cleavage of p65. In view of the data obtained, we conclude that
p65 is a substrate of caspase-3 in vitro.

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Fig. 5.
Caspase inhibitors block cleavage of p65 and
DNA fragmentation. HeLa cells were incubated in the absence or
presence of 10 µM Ac-DEVD-CHO or 10 µM
zVAD-fmk for 1 h prior to treatment with 15 µM NA.
Cells were harvested at 24 h after NA treatment, and divided into
two samples for DNA fragmentation and Western blot analysis.
A, equal amounts of nuclear cell extracts were separated by
SDS-PAGE (10%) and subjected to Western blot analysis with anti-p65
antibody. B, DNA fragmentation was visualized by ethidium
bromide staining, following agarose gel electrophoresis (1.5%).
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Fig. 6.
p65 is cleaved by caspase-3 in
vitro. 35S-Labeled wild-type p65 was
incubated with recombinant caspase-3 in the absence or presence of
caspase inhibitors (Ac-DEVD-CHO or zVAD-fmk), caspase-1, or
caspase-8 for 90 min at 30 °C. The reaction mixtures were
subjected to 10% SDS-PAGE and the p65 fragment was visualized
by autoradiography.
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Caspase-3 has been shown to recognize and cleave specific substrates
containing a conserved DXXD consensus tetrapeptide motif (22). Putative recognition sites for caspase-3 exist in the amino acid
sequence of p65
(Asp-Cys-Arg-Asn97-Gly98,
Asp-Thr-Asp-Asp294-Arg295,
Ala-Ser-Val-Asp469-Asn470). In order to
identify the cleavage site of p65 recognized by caspase-3, in
vitro labeled [35S]p65 was incubated with caspase-3,
and Western blot analysis was performed using antibodies against the
COOH and NH2 termini of p65. The
p65 band was recognized
by an antibody against the COOH terminus, but not by
anti-NH2 terminus antibody (Fig.
7A). Moreover, the
p65
mutant generated during NA-induced apoptosis was not recognized by the
NH2 terminus-specific antibody (Fig. 7B). Taken
together, the results clearly demonstrate that p65 is cleaved by
caspase-3 at the NH2 terminus. To determine the exact
cleavage site, we introduced point mutations at Asp97
(p65D97A) and Asp469 (p65D469A).
Wild-type (wt) p65 and the two p65 mutants were translated in
vitro and incubated with cell lysates prepared from NA-treated cells. As shown in Fig. 7C, p65D97A was
resistant to caspase-3 in cell lysates, while wt p65 and p65D469A mutant proteins were cleaved. Our results confirm
that caspase-3 cleaves the
Asp-Cys-Arg-Asp97-Gly98 motif of p65 during
NA-induced apoptosis. The findings so far implicate that NF-
B might
be inactivated through caspase-3-mediated cleavage of p65 after the
Asp97 residue during NA-induced apoptosis.

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Fig. 7.
Caspase 3-mediated cleavage of p65 occurs at
the NH2 terminus. A, in vitro
translated 35S-labeled p65 was incubated with recombinant
caspase-3 alone, or in the presence of 10 µM caspase
inhibitors, Ac-DEVD-CHO or zVAD-fmk. The reaction mixture was separated
by 10% SDS-PAGE and subjected to Western blot analysis with specific
antibodies against the COOH and NH2 termini of p65,
respectively. B, HeLa cells were treated with NA for the
indicated time period. Cell lysates were subjected to SDS-PAGE (10%),
followed by Western blot analysis using polyclonal antibody against the
NH2 terminus of p65. C, HeLa cells were treated
with 15 µM NA for 24 h and cell lysates were
prepared. Aliquots of 5 µl of in vitro translated
[35S]Met-p65 and mutants
([35S]Met-p65D97A or
[35S]Met-p65D469A) were incubated with 200 µg of cell lysate in the absence or presence of Ac-DEVD-CHO for 90 min at 30 °C. Reaction mixtures were further subjected to 10%
SDS-PAGE, and autoradiography was performed on the dried gel.
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Effect of Caspase-3-mediated Cleavage of p65 on Transcriptional
Activity of NF-
B and NA-induced Apoptosis--
The role of
caspase-3-mediated p65 cleavage on NF-
B activity and
apoptosis was further investigated. The NH2
terminus of p65 contains a 300-amino acid Rel homology domain, which
includes a DNA-binding domain, dimerization domain, and the nuclear
translocation signal. In order to examine whether the transcriptional
activity of NF-
B is disrupted by cleavage of p65, HeLa cells were
co-transfected with NF-
B-luciferase reporter plasmid together with
wild-type (wt) p65, caspase-3 resistant mutant (p65D97A),
and caspase-3-cleaved COOH-terminal fragment of p65 (
p65), respectively, following which luciferase activity was monitored. NF-
B activity was reduced to half that of control in the
p65-transfected cells, while a 2-fold increase was observed in cells
transfected with p65 or p65D97A (Fig.
8A). This suggests that
cleavage of p65 at Asp97 impairs its DNA binding ability,
and therefore leads to a reduction in the transcriptional activity of
NF-
B.

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Fig. 8.
Effect of p65 cleavage on
NF- B activity and NA-induced apoptosis.
A, HeLa cells were transfected with expression plasmids
encoding p65, p65D97A, or p65 (0.5 µg each), together
with NF- B-luciferase plasmid (0.5 µg). Cells were harvested
48 h after transfection, and a luciferase assay was performed.
Experiments were performed in triplicate and averaged. B,
cells were co-transfected with a pEGFP-C1 plasmid together with
increasing concentration of p65, p65D97A, or p65 (0.5, 1, and 2 µg of DNA). After treatment of cells with NA for 12 h,
cells that underwent morphological changes were counted in
GFP-expressing cells as a measure of apoptosis. The results depicted
are representative of three separate experiments.
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Experiments were subsequently conducted to analyze the effect of p65
cleavage on NA-induced apoptosis. Cells were expressed with different
concentrations of p65, p65D97A, and
p65, together with
GFP. NA-induced apoptosis was monitored by counting the number of dead
GFP-positive cells after treatment with NA for 12 h (see Fig.
8B). Following exposure of cells to NA, apoptosis was
significantly enhanced in
p65-transfected cells. In contrast, p65
and p65D97A inhibited apoptosis in a
dose-dependent manner. However, p65D97A was
more effective at apoptotic inhibition than wild-type p65, suggesting
the requirement of intact p65 for the inhibition of apoptosis. We
conclude from the data that NA induces caspase-3-mediated cleavage of
p65, leading to the disruption of anti-apoptotic function of NF-
B,
and consequently to cell death.
Caspase-3 Is Activated through Caspase-9 Signaling, Rather than the
Caspase-8 Pathway during NA-induced Apoptosis--
The molecular
mechanism linked to NA-induced activation of caspase-3 was further
investigated in this study. Caspase-3 is activated by two main
pathways. Ligation of Fas ligand (FasL) to Fas activates caspase-8,
which in turn cleaves and activates other caspases including caspase-3
(23, 24). The second pathway involves the activation of caspase-3 by
caspase-9. Caspase-9 is activated by cytochrome c released
from mitochondria in the presence of Apaf-1 and dATP during apoptosis
(25, 26). Previous reports demonstrated that ligation of Fas induces
caspase-3-mediated cleavage of p65 at the COOH terminus during
apoptosis in Jurkat T cells (13). This leads to the query as to whether
caspase-3 is activated by an increase in Fas or FasL expression during
NA-induced apoptosis (Fig.
9A). No change in the protein
levels of Fas and FasL were observed following treatment with NA,
indicating that Fas-stimulated caspase-8 signaling pathway is possibly
not related to the activation of caspase-3 in our system.

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Fig. 9.
Caspase-3 is activated through the cytochrome
c-caspase-9 pathway during NA-induced apoptosis.
A, HeLa cells were treated with NA for the indicated time
periods. Expression levels of Fas and FasL were analyzed by Western
blot analysis with either monoclonal or polyclonal antibody against
FasL. B, HeLa cells were treated with TNF- , NA, or a
combination of these compounds for 24 h, and cytosolic extracts
were prepared as described under "Experimental Procedures." Equal
amounts of lysates were resolved by 10% SDS-PAGE (caspase-3 and
caspase-9) or 14% SDS-PAGE (cytochome c), following which
immunoblot analysis was performed using monoclonal antibody for
caspase-3, and polyclonal antibodies for caspase-9 and cytochome
c.
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This leaves the possibility of the second pathway being preferentially
in operation, that caspase-3 is activated through caspase-9. In order
to examine this theory, HeLa cells were treated with TNF-
, NA, or a
combination of these compounds. Cytosolic fractions of the cells were
subjected to Western blot analysis for detection of cytochrome
c and caspase-9 (Fig. 9B). Significant amounts of cytochrome c were released into the cytoplasm in NA-treated
cells, but not in TNF-
-treated cells. Moreover, caspase-9 and
caspase-3 were observed in concurrence with cytochrome c
release. Taken together, these results indicate that NA activates
caspase-3 and leads to p65 cleavage via cytochrome
c/caspase-9 signaling, rather than the Fas/caspase-8 pathway.
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DISCUSSION |
The transcription factor, NF-
B, is activated by various
stimuli, and protects against apoptosis (3, 27, 28). TNF-
can
trigger two different pathways with opposite effects simultaneously, the apoptotic pathway mediated by caspase-8, and survival signaling mediated by NF-
B. It is known that TNF-
does not induce apoptosis unless NF-
B pathway is blocked (29). Previous studies have led to
the discovery of the activation mechanism of NF-
B upon various
apoptotic stimuli (1, 2, 4, 5). However, at present, it is currently
unclear how apoptotic pathways overcome survival signaling mediated by
NF-
B, and consequently induce cell death. In this article, we
investigate whether the anti-apoptotic signaling mediated by NF-
B is
inhibited during apoptosis induced by NA. We show that NA triggers
apoptotic cell death in HeLa cells and destroys resistance to apoptosis
caused by TNF-
. Furthermore, NA inhibits NF-
B activation through
caspase-3-mediated cleavage of p65 at the NH2 terminus
during apoptosis in HeLa cells.
There is ample evidence in earlier literature to illustrate that the
activity of NF-
B is inhibited by various drugs via a number of
mechanisms. Glucocorticoids inhibit NF-
B activity by increasing
levels of I
B
, and consequently sequestering NF-
B in the
cytoplasm (9). Moreover, NF-
B activation is prevented by
anti-inflammatory drugs like sodium salicylate and aspirin (10), which
prevent degradation of I
B by inhibiting the activity of I
B kinase
(IKK
). Proteosome inhibitors additionally block degradation of
I
B (11). It was reported that 3-deazaadenosine, which is known to
induce apoptosis in human and mouse leukemia cells, inhibits the
transcriptional activity of NF-
B through obstruction of p65
phosphorylation without reducing DNA binding activity (12). Our present
analyses demonstrate that activation of NF-
B is inhibited by
proteolytic cleavage of p65 during NA-induced apoptosis. The cleavage,
induced by caspase-3, occurs at a site in the NH2 terminus
of the protein. Another recent study showed that ligation of Fas
repressed NF-
B activity in Jurkat T cells by inducing proteolytic
cleavage of p65 and p50 during apoptosis (13). Data presented in this
study indicate that p65 cleavage occurs at the COOH terminus,
although also mediated by caspase-3. The cleavage at the COOH terminus
of p65 was further examined during apoptosis induced by deprivation of
growth factor (14), where caspase-6 and -7 are available for the
COOH-terminal cleavage of p65, as well as caspase-3. Note that our
results on the cleavage site of p65 at the NH2 terminus are
significantly different from previous reports, although the fact that
caspase-3 is responsible for the cleavage is universally established.
With a view to explaining the dissimilar findings, we cannot exclude
the possibility that another caspase is involved in the cleavage at
this site, because the cell lysate had a higher activity of caspase
than recombinant caspase-3 on the cleavage of p65 (Fig. 7C).
The identity of these caspases is still somewhat ambiguous. It is
difficult to explain why activated caspase-3 targets only the
NH2 terminus of p65 during NA-induced apoptosis. One
possible explanation for this discrepancy is that the activation
mechanism of caspase-3 or the environment in which caspase-3 targets
p65 in our system might be different from that in the other studies
conducted so far.
It has been demonstrated that caspase-3 activation is regulated by at
least two mechanisms. One involves a direct pathway from caspase-8 (23,
24), and the other is mediated by caspase-9, following the release of
cytochrome c from mitochondria (25, 26). NA induces
apoptosis via cytochrome c release into the cytoplasm and
activation of caspase-9 (Fig. 9). However, the expression profiles of
Fas and FasL do not change during NA-induced apoptosis, suggesting that
caspase-3 is activated by caspase-9 rather than caspase-8. It must
therefore be concluded that two caspase-3 activation pathways exist
differently that both ultimately lead to the cleavage of p65. One might
mediate the NH2-terminal cleavage of p65 in NA-induced
apoptosis, while the other mediates the COOH-terminal cleavage of p65
in Fas-induced apoptosis.
During analyses on NF-
B transcriptional activity using the p65
mutants, it was noted that the p65 mutant truncated at the NH2 terminus (
p65) inhibits
NF-
B-dependent transcription by half that of the control
protein following overexpression (Fig. 8A). These data can
be explained by the functional structure of p65. The NH2
terminus of p65 contains a Rel homology domain within which lies a
DNA-binding domain, dimerization domain, and nuclear localization
signal. Proteolytic cleavage at this site might impair the DNA binding
ability of p65, therefore leading to a decrease in the transcriptional
activity of NF-
B.
Overexpression of the deletion mutant
p65 significantly increases
NA-induced apoptosis, while uncleavable p65 mutant
(p65D97A) blocks apoptosis (Fig. 8B). Several
genes induced by NF-
B are known to down-regulate apoptosis,
including manganese superoxide dismutase (30), zinc finger protein A20
(31), and cellular inhibitor for apoptosis (CIAP) (32). Thus, the
abrogation of transcriptional activity of NF-
B by caspase-3-mediated
cleavage of p65 might diminish the anti-apoptotic effect of NF-
B
during apoptosis, which in turn explains why cells are more sensitive to apoptosis by NA.
In conclusion, we suggest that NA represses NF-
B activity via
caspase-3-mediated cleavage of p65, therefore sensitizing cells to
apoptosis. Although the apoptotic mechanism of NA is currently unclear,
down-regulation of NF-
B activity is evidently one of the active
processes in NA-induced apoptosis. Several anticancer agents are less
effective in the induction of apoptosis because of their concomitant
activation of NF-
B. In this regard, NA might be a promising future
candidate for cancer therapy.