From the Clinical Research Center, Samsung Biomedical
Research Institute and ** Department of Medicine, Samsung Medical
Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea and § Graduate School of East-West Medical Science,
Kyunghee University, Seoul, 130-701, Korea
Received for publication, August 22, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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We investigated the molecular mechanism of the
synergism between interferon The pleiotropic proinflammatory cytokine
TNF In many cases, the anti-tumor effect of TNF In the current work, we utilized ME-180 human cervical cancer cells to
investigate the molecular mechanism of synergistic anti-tumor effects
of IFN Cell Line and Reagents--
ME-180 cervical cancer cell line was
obtained from ATCC (Manassas, VA) and grown in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 2 mM
glutamine, and penicillin-streptomycin (Life Technologies, Inc.).
Recombinant human IFN Assessment of Cytotoxicity by
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Bromide (MTT)
Assay--
Cells (3 × 104/well) were seeded in
96-well plates and treated with various combinations of cytokines for
the indicated time periods. The optimal concentrations of the cytokines
for the cytotoxic action were 100 units/ml for IFN Morphological Analysis of Apoptotic Cells--
Morphological
changes in the nuclear chromatin of cells undergoing apoptosis were
detected by staining with 2.5 µg/ml bisbenzimide Hoechst 33258 fluorochrome (Calbiochem), followed by examination on a fluorescence
microscope. In some experiments, cytokine-treated cells were
double-stained with propidium iodide (PI, 2.5 µg/ml) and Hoechst
33258 (2.5 µg/ml) to distinguish apoptotic cells from necrotic cells.
Intact blue nuclei, condensed/fragmented blue nuclei,
condensed/fragmented pink nuclei, and intact pink nuclei were
considered viable, early apoptotic, late apoptotic, and necrotic cells,
respectively (13). Transmission electron microscopy was carried out
essentially as previously described (13). In brief, cells were fixed in
4% glutaraldehyde, 1% paraformaldehyde, 0.2 M phosphate,
pH 7.2, at 4 °C for 2 h. After two washes in 0.2 M
phosphate, the cell pellet was post-fixed with 2% OsO4 in
the same buffer for 30 min. The pellet was dehydrated in ethanol and then in 100% propylene oxide, followed by embedding overnight at
37 °C for another 3 days at 60 °C. Ultrafine sections were cut
and examined on an electron microscope (Hitachi H7100, 75 kV).
DNA Ploidy Analysis--
Cells were suspended in
phosphate-buffered saline, 5 mM EDTA and fixed by adding
100% ethanol dropwise. RNase A (40 µg/ml) was added to resuspended
cells, and the incubation was carried out at room temperature for 30 min. PI (50 µg/ml) was then added for flow cytometric analyses.
Assessment of Caspase Activity--
Caspase-3- or -8-like
activity was measured using a caspase assay kit (Pharmingen, San Diego,
CA) according to the supplier's instruction. In brief, caspase-3 or -8 fluorogenic substrates (Ac-DEVD-AMC or Ac-IETD-AMC) were incubated with
cytokine-treated cell lysates for 1 h at 37 °C, then AMC
liberated from Ac-DEVD-AMC or Ac-IETD-AMC was measured using a
fluorometric plate reader with an excitation wavelength of 380 nm and
an emission wavelength of 420- 460 nm.
Western Blot Analysis--
Cells were lysed in triple-detergent
lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM
NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 1 mM phenylmethylsulfonyl fluoride). Protein
concentration in cell lysates was determined using the Bio-Rad protein
assay kit. An equal amount of protein for each sample was separated by
10 or 12% SDS-polyacrylamide gel electrophoresis and transferred to
Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech). The
membranes were blocked with 5% skim milk and sequentially incubated
with primary antibodies (rabbit anti-human IRF-1, Santa Cruz; rabbit
anti-human STAT1 and anti-human phospho-STAT1, New England Biolabs) and
horseradish peroxidase-conjugated secondary antibodies (anti-rabbit
IgG, Amersham Pharmacia Biotech), followed by ECL detection (Amersham
Pharmacia Biotech).
Transient Transfection--
ME-180 cells in 6-well plates were
co-transfected with 1 µg of human STAT1 cDNA, dominant-negative
mutant STAT1 cDNA (kindly provided by Dr. Hirano, Osaka University,
Japan), human IRF-1 cDNA (kindly provided by Dr. Taniguchi,
University of Tokyo), or phosphorylation-defective dominant-negative
mutant I NF- Immunofluorescence Staining--
ME-180 cells seeded onto
chamber slides (Lab-Tek, Nalge Nunc International, Naperville, IL) were
fixed in 4% paraformaldehyde for 30 min at room temperature and then
in cold methanol for 10 min at Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared from ME-180 cells treated with cytokines as previously
described (18). Synthetic double-strand oligonucleotides of consensus
NF- IFN IFN Inhibition of Cytoprotective NF- Inhibition of Caspases Directed ME-180 Cells to Undergo Necrotic
Cell Death--
We next investigated whether the activation of
caspases is involved in the IFN Here we present evidence that STAT1/IRF-1 pathways initiated by
IFN Although further works are necessary to completely delineate the
downstream signaling pathways after STAT1/IRF-1 in IFN Because IFN Our results indicate that IFN In conclusion, we report a novel signal transduction of IFN (IFN
) and tumor necrosis factor
(TNF
) documented in a variety of biological occasions such as tumor
cell death and inflammatory responses. IFN
/TNF
synergistically
induced apoptosis of ME-180 cervical cancer cells. IFN
induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited
IFN
/TNF
-induced apoptosis, whereas IRF-1 transfection induced
susceptibility to TNF
. Dominant-negative I
B
transfection
sensitized ME-180 cells to TNF
. IFN
pretreatment attenuated
TNF
- or p65-induced NF-
B reporter activity, whereas it did not
inhibit p65 translocation or DNA binding of NF-
B. IRF-1 transfection
alone inhibited TNF
-induced NF-
B activity, which was reversed by
coactivator p300 overexpression. Caspases were activated by
IFN
/TNF
combination; however, caspase inhibition did not abrogate
IFN
/TNF
-induced cell death. Instead, caspase inhibitors directed
IFN
/TNF
-treated ME-180 cells to undergo necrosis, as demonstrated
by Hoechst 33258/propidium iodide staining and electron microscopy.
Taken together, our results indicate that IFN
and TNF
synergistically act to destroy ME-180 tumor cells by either apoptosis
or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways
initiated by IFN
play a critical role in IFN
/TNF
synergism by
inhibiting cytoprotective NF-
B. IFN
/TNF
synergism appears to
activate cell death machinery independently of caspase activation, and
caspase activation seems to merely determine the mode of cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 exerts a wide variety
of biological activities such as induction of septic shock, activation
of local inflammatory responses, and fever generation as an endogenous
pyrogen (1). TNF
also kills various tumor cell lines in
vitro and mediates anti-tumor effect in vivo (2). TNF
exerts its biological effects by binding to two types of cell
surface receptors with molecular masses of 55 kDa (p55) and 75 kDa
(p75). TNF
cytotoxicity is mostly mediated by p55 receptors (3).
After the ligation of p55 receptors, a canonical apoptotic signal
transduction pathway is initiated. The cytoplasmic death domain of p55
receptor interacts with the death domain of intracellular adapter
molecules such as TRADD (TNF receptor-associated death domain protein)
and FADD (Fas-associated death domain protein), which leads to the
activation of initiator caspases (4). This, in turn, triggers the
caspase cascade and ultimately results in apoptotic cell death.
was enhanced by IFN
(5) or metabolic inhibitors such as cycloheximide and actinomycin D
(6). Although these metabolic inhibitors are believed to block the
synthesis of cytoprotective proteins, the effects of IFN
might be
mediated by the induction of new proteins that increase the sensitivity
of target cells to TNF
. IFN
/TNF
synergism also has been
reported in biological responses other than tumor cell killing. For
instance, the two cytokines synergistically up-regulated the expression
of numerous genes, including ICAM-1 (intercellular adhesion molecule
1), IP-10, and major histocompatibility complex class I heavy
chain (7-9). However, the molecular mechanism of the synergism between
the two cytokines is not clearly understood. It has been reported that
IFN
increases the expression of TNF
receptors (10). However,
because the sensitivity of the cells to TNF
is not simply correlated
with the level of TNF
receptor expression (11, 12), up-regulation of
TNF
receptor alone does not adequately explain the cytokine
synergism in the anti-tumor action.
/TNF
. We also studied the role of caspase activation in
ME-180 cell death by IFN
/TNF
synergism. Our results indicate that
1) IRF-1 induction after STAT1 activation by IFN
plays a central
role in synergistic tumor cell death by IFN
/TNF
, 2)
IFN
-induced IRF-1 inhibits cytoprotective NF-
B transactivation, 3) IFN
/TNF
induces ME-180 cell death regardless of caspase
activation, and caspase activation dictates only the mode of cell death
between apoptosis and necrosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased from R&D Systems (Minneapolis,
MN). Recombinant human TNF
was generously provided by Dr. T. H. Lee (Yonsei University, Seoul, Korea). Caspase inhibitors (z-VAD-fmk,
benzyloxycarbonyl-Val-Ala-Asp(OCH3)-CH2-fluoromethyl ketone; BD-fmk,
t-butoxycarbonyl-Asp(OCH3)-CH2F;
z-DEVD-fmk,
benzyloxycarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2-fluoromethyl ketone; z-IETD-fmk,
benzyloxycarbonyl-Ile-Glu(OCH3)-Thr-Asp(OCH3)-CH2-fluoromethyl ketone) were purchased from Enzyme Systems (Livermore CA), and cathepsin B inhibitor FA
(benzyloxycarbonyl-Phe-Ala-CH2-fluoromethyl ketone)
and MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H, also called
Z-LLL) were from Calbiochem. All other chemicals were obtained from
Sigma, unless stated otherwise.
and 10 ng/ml for
TNF
. In some experiments, cells were pretreated with caspase
inhibitors or MG-132 for 1 h before cytokine treatment. After
cytokine treatment, the medium was removed, and MTT (0.5 mg/ml) was
added, followed by incubation at 37 °C for 2 h in
CO2 incubator. After a brief centrifugation, supernatants
were carefully removed, and Me2SO was added. After
insoluble crystals were completely dissolved, absorbance at 540 nm was
measured using a Thermomax microplate reader (Molecular Devices).
Results were presented as means ± S.E. (n = 3).
B
(14) together with 0.2 µg of lacZ gene
(pCH110, Amersham Pharmacia Biotech) using LipofectAMINE reagent (Life
Technologies, Inc.). 48 h after the transfection, cells were
treated with cytokines. After another 48 h, the cells were fixed
with 0.5% glutaraldehyde for 10 min at room temperature and stained
with X-gal (5-bromo-4-chloro-3-indolyl
-D-galactopyranoside; 1 mg/ml) in 4 mM
potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM magnesium chloride at 37 °C for detection of blue
cells. At least 200 blue cells were counted for each experiment, and
transfection efficiency was 10-35%. Results were presented as
means ± S.E. (n = 3).
B Reporter Assays--
NF-
B reporter activity was
measured using the dual-luciferase reporter assay system (Promega,
Madison, WI). In brief, ME-180 cells in 12-well plates were
co-transfected with 0.5 µg of NF-
B-responsive reporter gene
construct carrying two copies of
B sequences linked to luciferase
gene (IgG
NF-
B-luciferase, generously provided by Dr. G. D. Rosen, Stanford University, Stanford, CA) (15) together with 0.1 µg
of Renilla luciferase gene under hamster sarcoma virus
thymidine kinase promoter (pRL-TK, Promega) using LipofectAMINE reagent
(Life Technologies, Inc.). 24 h after the transfection, cells were
treated with cytokines. After 5 h, activities of firefly
luciferase and Renilla luciferase in transfected cells were
measured sequentially from a single sample using the dual-luciferase reporter assay system (Promega). Results were presented as firefly luciferase activity normalized to Renilla luciferase
activity. In some experiments, cells were co-transfected before
cytokine treatment with NF-
B p65 (16) or coactivator p300 expression plasmid (0.5 µg; kindly provided by Dr. Livingston, Harvard Medical School, Boston, MA) (17) along with NF-
B-responsive reporter plasmid
(0.5 µg) and pRL-TK (0.1 µg). Results were presented as means ± S.E. (n = 3).
20 °C. Fixed cells were
permeabilized in 0.1% Triton X-100, 0.1% sodium citrate for 3 min at
4 °C and then sequentially incubated with mouse anti-p65 antibody
(Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated anti-mouse
IgG, and streptavidin-fluorescein isothiocyanate. Stained cells were
examined on a fluorescent microscope.
B binding sequence, GAT CCC AAC GGC AGG GGA (Promega), were
end-labeled with [
-32P]ATP using T4 polynucleotide
kinase. Nuclear extract was incubated with the labeled probe in the
presence of poly- (dI-dC) in a binding buffer containing 20 mM HEPES at room temperature for 30 min. For supershift
assays, a total of 0.2 µg of antibodies against p65 or p50
subunit of NF-
B were included in the reaction. DNA-protein complexes
were resolved by electrophoresis in a 5% nondenaturing polyacrylamide
gel, dried, and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and TNF
Synergistically Induced the Apoptosis of ME-180
Cells--
First we screened several tumor cell lines to assess their
sensitivity to IFN
/TNF
-induced cytotoxicity (data not shown). Cytotoxic synergism between IFN
and TNF
was most evident in ME-180 cells. Although either cytokine alone exhibited no significant cytotoxicity, the combination of the two cytokines significantly reduced ME-180 cell viability (Fig.
1A). The cytokine cytotoxicity was dependent on the dose of IFN
used. However, concentration higher
than 100 units/ml did not further increase the cytotoxicity (Table
I). The reduction of cell viability was
due to apoptosis as demonstrated by Hoechst 33258 staining and DNA
ploidy analysis. IFN
/TNF
treatment induced nuclear condensation
and fragmentation (Fig. 1B) and led to the appearance of
sub-diploid cells (Fig. 1C), which are hallmarks of
apoptotic cells. DNA ploidy assays also indicated that the effect of
IFN
/TNF
was not due to the growth arrest as was shown by the
absence of decrease in the percentage of cells in the S phase.
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Fig. 1.
IFN /TNF
synergistically induces ME-180 cell apoptosis. A combination
of IFN
(100 units/ml) and TNF
(10 ng/ml), but not either cytokine
alone, induced ME-180 cell death. Cell viability was assessed by MTT
assays after treatment with the cytokines for 48 h (A).
Induction of ME-180 cell death was due to apoptosis, as demonstrated by
chromatin condensation in Hoechst 33258 staining (B) or the
appearance of sub-G1 peak in flow cytometric analyses
(C) at 24 h after cytokine treatment.
Dose response of cytokine cytotoxicity
/TNF
Synergism Involved IFN
-induced STAT1 Activation
and IRF-1 Induction--
Based on our results that the combination of
IFN
and TNF
, but not either cytokine alone, induced ME-180 cell
death, we explored the possibility that IFN
sensitizes ME-180 cells
to TNF
-mediated cytotoxicity. This was first tested by sequential
treatment of ME-180 cells with the two cytokines. After IFN
treatment, TNF
alone was sufficient to induce a significant
cytotoxicity in ME-180 cells (Table II).
However, sequential treatment with TNF
and then with IFN
did not
have the same effects, indicating that IFN
confers susceptibility to
TNF
on ME-180 cells through induction or up-regulation of certain
genes in ME-180 cells. Because STAT1 and IRF-1 are known to be
canonical intracellular signal-transducing molecules in IFN
signaling, we investigated the involvement of STAT1/IRF-1-signaling
pathways in IFN
/TNF
synergism on ME-180 cell apoptosis. IFN
,
but not TNF
, induced phosphorylation of STAT1 and up-regulated IRF-1
expression in ME-180 cells (Fig. 2).
Furthermore, the transfection of phosphorylation-defective dominant-negative mutant of STAT1 significantly inhibited
IFN
/TNF
-induced ME-180 cell death, indicating that IFN
-induced
STAT1 activation is critical for the induction of TNF
susceptibility
(Fig. 3A). We next asked
whether IRF-1, a downstream mediator of STAT1, is responsible for the
priming effects of IFN
. Transfection of IRF-1 conferred TNF
susceptibility on ME-180 cells in a dose-dependent manner,
indicating a central role for IRF-1 in the sensitization of ME-180
cells to TNF
-induced apoptosis (Fig. 3, B and
C).
Cytotoxic effects of sequential treatment of cytokines
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Fig. 2.
IFN activates STAT1
(A) and induces IRF-1 expression (B)
in ME-180 cells. Western blot analyses demonstrated that treatment
of ME-180 cells with IFN
induced STAT1 expression (24-h treatment)
as well as its phosphorylation (30-min treatment) (A).
IFN
also induced IRF-1 expression at 24 h after treatment, and
the expression was further increased at 48 h after the treatment
(B). However, TNF
alone did not change the expression of
either STAT1 or IRF-1. C, untreated control; I,
IFN
(100 units/ml); T, TNF
(10 ng/ml); D,
IFN
plus TNF
.
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Fig. 3.
A key role for STAT1/IRF-1 signaling in
IFN /TNF
synergism. A, transient transfection of
phosphorylation-defective STAT1 dominant-negative mutant (DN STAT1)
significantly inhibited IFN
/TNF
cytotoxicity, as demonstrated by
counting blue cells co-expressing lacZ at 48 h after
cytokine treatment (IFN
, 100 units/ml; TNF
, 10 ng/ml).
B, transfection of IRF-1 cDNA (1 µg) induced
susceptibility to TNF
. In contrast to empty vector (pcDNA3)
transfectants, treatment of IRF-1 transfectants with TNF
alone for
48 h significantly decreased the number of blue cells.
C, the effects of IRF-1 were dependent upon the dose of
IRF-1 cDNA (0.1, 0.5, 1, and 2 µg) used in the transient
transfection. The number of blue cells upon transfection with an empty
vector without TNF
treatment was set to 100%.
B Activity by
IFN
--
TNF
is known to initiate both death and survival
signals, and recent studies on TNF
-induced survival signal suggested
an important role of NF-
B activation (19-22). Thus, we investigated how IFN
induces susceptibility to TNF
-induced cytotoxicity by examining the role of NF-
B in ME-180 cell death and its possible regulation by IFN
. Treatment of ME-180 cells with a proteasome inhibitor (MG-132), which is known to inhibit NF-
B activation (23),
rendered the cells sensitive to TNF
-induced apoptosis (Fig.
4A), suggesting the
cytoprotective role of NF-
B. Also, upon the transfection of
phosphorylation-defective dominant-negative mutant I
B
, TNF
alone induced a significant cytotoxicity, further supporting the
cytoprotective role of NF-
B (Fig. 4B). NF-
B reporter assays indicated that IFN
pretreatment attenuated TNF
-induced NF-
B activity, suggesting that IFN
synergizes with TNF
for ME-180 cell apoptosis by inhibiting TNF
-induced cytoprotective NF-
B activity (Fig. 4C). IFN
pretreatment, however,
did not inhibit nuclear translocation of p65 (Fig.
5) or DNA binding of NF-
B induced by
TNF
treatment (Fig. 6). Also, IFN
did not inhibit TNF
-induced degradation of I
B
(data not
shown). However, IFN
treatment did inhibit the NF-
B reporter
activity induced by transfection of p65 subunit of NF-
B (Fig.
4D), indicating that IFN
directly inhibited
NF-
B-mediated transactivation within the nuclei without affecting
the nuclear translocation or DNA binding of NF-
B. We next studied if
IRF-1 mediates this inhibitory action of IFN
on NF-
B.
Transfection of IRF-1 alone was sufficient to inhibit TNF
-induced
NF-
B activity, indicating a central role of IRF-1 in the inhibition
of NK-
B transactivation by IFN
(Fig.
7A). We also investigated the
possible mechanism of interference between IRF-1 and NF-
B.
Transfection of p300 coactivator abrogated the inhibitory effect of
IFN
treatment (Fig. 7B) or IRF-1 transfection (Fig.
7C) on TNF
-induced NF-
B activity, suggesting the
possibility of coactivator competition between IFN
-induced IRF-1 and
TNF
-induced NF-
B.
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Fig. 4.
Inhibition of cytoprotective
NF- B by IFN
.
A, inhibition of NF-
B by proteasome inhibitor MG-132
sensitized ME-180 cells to TNF
. ME-180 cells were treated with
either MG-132 (0.5 µM) alone or in combination with
TNF
(10 ng/ml) for 48 h, and then cell viability was assessed
by MTT assays. B, inhibition of NF-
B by transfection of
dominant-negative mutant I
B
(DN I
B
)
also rendered ME-180 cells sensitive to TNF
treatment. Viability of
ME-180 cells co-transfected with dominant-negative I
B
and
lacZ was significantly decreased by TNF
treatment (24 h),
in contrast to the cells co-transfected with an empty vector
(pcDNA3) and lacZ. C and D,
NF-
B reporter assays revealed that pretreatment (24 h, 100 units/ml)
of ME-180 cells with IFN
inhibited TNF
-induced NF-
B activity
(C). IFN
treatment (48 h) also inhibited NF-
B reporter
activity induced by p65 transfection (NF-
B
p65) (D). Transiently transfected cells were
treated with cytokines for the indicated time period before NF-
B
reporter assays (C and D).
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Fig. 5.
No effects of IFN on
nuclear translocation of p65 subunit of
NF-
B. As compared with untreated control
(A), TNF
treatment (45 min, 10 ng/ml) induced nuclear
translocation of p65 (B), which was not affected by IFN
pretreatment (24 h, 100 units/ml) (C).
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Fig. 6.
No significant effects of
IFN on DNA binding of
NF-
B protein. A, IFN
pretreatment (100 units/ml, 24 h) did not significantly affect
TNF
-induced
B sequence binding of NF-
B proteins (lanes
4 and 5). The identity of DNA-complexed proteins was
confirmed by supershift assays using antibodies (Ab) against
p65 (lane 6), p50 (lane 7), or both (lane
8). B, ME-180 cells were similarly treated with
increasing doses of IFN
and TNF
as indicated, and then NF-
B
was detected by electrophoretic mobility shift assay. IFN
at all
concentrations tested did not significantly influence TNF
-induced
DNA binding of NF-
B, indicating that the inability of IFN
to
inhibit TNF
-induced DNA binding of NF-
B was not due to the low
dose of IFN
used.
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Fig. 7.
IRF-1 mediates
NF- B-inhibiting effects of
IFN
probably through coactivator
competition. A, transfection of IRF-1 inhibited
TNF
-induced NF-
B reporter activity in a manner similar to IFN
pretreatment. B, transfection of coactivator p300 abrogated
IFN
-mediated inhibition of NF-
B reporter activity. Transiently
transfected cells were treated with cytokines for an indicated time
period before NF-
B reporter assays (IFN
, 100 units/ml; TNF
, 10 ng/ml). C, ME-180 cells were co-transfected with NF-
B
reporter construct and the indicated plasmids, and then the luciferase
activity was measured after 24 h. Co-transfection of coactivator
p300 also abolished the IRF-1 transfection-mediated inhibition of
NF-
B reporter activity. CBP, cAMP-response element-binding protein
(CREB)-binding protein.
/TNF
-induced apoptosis of ME-180
cells. Cytokine-induced apoptosis of ME-180 cells was accompanied by
the activation of caspase-3-like activity, as demonstrated by the
cleavage of Ac-DEVD-AMC in IFN
/TNF
-treated cells (Fig.
8). Cytokine treatment also induced the
cleavage of Ac-IETD-AMC, indicating concurrent activation of
caspase-8-like activity (data not shown). However, pretreatment with
broad-spectrum caspase inhibitors such as z-VAD-fmk or BD-fmk failed to
inhibit ME-180 cell death by IFN
/TNF
synergism despite the
activation of multiple caspases (Fig.
9A). Instead, IFN
/TNF
in
the presence of caspase inhibitors unexpectedly induced the necrosis of
ME-180 cells, as judged by the swelling of dying cells on a light
microscope (data not shown). Hoechst 33258/PI staining and electron
microscopy confirmed the necrosis of the cells (Fig. 9, B
and C). To study the involvement of individual caspases in the switching process from apoptosis to necrosis, cells were pretreated with inhibitors specific for individual caspases instead of z-VAD-fmk. Because we observed the activation of caspase-3 and -8 in the cytokine-treated ME-180 cells, we tested the effects of z-DEVD-fmk and
z-IETD-fmk alone or in combination. The z-DEVD-fmk and z-IETD-fmk acted
additively in conversion from apoptosis to necrosis, suggesting the
involvement of multiple caspases in determining the mode of ME-180 cell
death (Table III).
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Fig. 8.
Activation of caspase-3-like activity by
IFN /TNF
in ME-180
cells. IFN
/TNF
treatment (IFN
, 100 units/ml; TNF
, 10 ng/ml) induced cleavage of DEVD-AMC, indicating activation of
caspase-3-like activity. Pretreatment of ME-180 cells with z-VAD-fmk
before cytokine treatment completely inhibited the caspase activity
(IFN
, 100 units/ml; TNF
, 10 ng/ml).
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Fig. 9.
Induction of necrotic death by
IFN /TNF
in the
presence of caspase inhibitors. A, pretreatment of
ME-180 cells with broad spectrum caspase inhibitors such as z-VAD-fmk
or BD-fmk did not block the cytokine-induced cytotoxicity as measured
by MTT assays at 48 h after the treatment (IFN
, 100 units/ml;
TNF
, 10 ng/ml). B and C, pretreatment with
z-VAD-fmk switched the mode of cell death from apoptosis to necrosis as
judged by Hoechst 33258/PI double-staining (B) and electron
microscopy (C). In Hoechst 33258/PI double-staining, cells
with blue intact nuclei were viable cells, whereas those with blue
fragmented nuclei were early apoptotic cells. Cells with pink intact
nuclei were necrotic cells, whereas cells with pink fragmented nuclei
were late apoptotic cells. The values in the parentheses below the
photographs represent the percentage of apoptotic (early or late) or
necrotic cells out of the total 500 cells counted (B).
Effects of various caspase inhibitors on the conversion of ME-180 cell
death from apoptosis to necrosis
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
play a central role in IFN
/TNF
synergism in the induction of ME-180 cell apoptosis. Transfection of dominant-negative STAT1 abolished IFN
/TNF
synergism, whereas transfection of IRF-1
sensitized ME-180 cells to TNF
-induced apoptosis. Thus, STAT1
activation and IRF-1 induction by IFN
appear to be important in
IFN
/TNF
synergism in ME-180 cell apoptosis. However,
dominant-negative STAT1 did not completely abolish cytotoxicity by
IFN
/TNF
, and IRF-1 transfection could not be completely
substituted for IFN
. IFN
induces STAT1 as well as IRF-1, and some
cellular responses to IFN
are reported to be mediated by both STAT1
and IRF-1 (24-25). Neither STAT1 or IRF-1 alone may not explain all of
the priming effect of IFN
in TNF
-induced death. The role of IRF-1
in the induction of apoptosis by DNA damage or IFN
has been
previously suggested (26-28), which supports the proapoptotic action
of IRF-1. Previous work in our laboratory also showed that IRF-1 plays
a central role in IFN
/TNF
-induced apoptosis of pancreatic islet
-cells in autoimmune
diabetes.2 Caspase induction
has been suggested as a possible downstream event after IRF-1 induction
in IFN
-induced apoptosis (28). Although RNase protection assays
revealed that the expression of caspase-1 and -4 was up-regulated by
IFN
treatment in ME-180 cells (data not shown), there remains yet to
be determined how the increases in the expression of these caspases
mediate IRF-1 action. In IFN
/TNF
-induced death of ME-180 cells,
caspases seem to be involved in determining the mode of cell death
rather than decision between death and survival (see below).
/TNF
cytotoxic synergism, our current work indicates that NF-
B is one of
the targets of STAT1/IRF-1 action. We demonstrated that IFN
attenuated TNF
-induced NF-
B reporter activity in ME-180 cells.
Also, the inhibition of NF-
B either by transfection of dominant-negative I
B "super repressor" or by treatment with a proteasome inhibitor (MG-132) rendered ME-180 cells sensitive to
TNF
-induced apoptosis. These results indicate that IFN
sensitizes ME-180 cells to TNF
-induced apoptosis by inhibiting NF-
B-mediated activation of survival signals. Furthermore, this action of IFN
was
mediated by IRF-1. It has been previously reported that IRF-1 and
NF-
B interact in vitro as well as in vivo for
the cooperative induction of inflammatory genes (29-31). In ME-180
cells, however, IRF-1 negatively influenced NF-
B activity. IRF-1
does not seem to directly interact with NF-
B because NF-
B
transcriptional activity was assessed using a reporter construct
containing a
B element but not an IRF-1 response element. Thus, in
ME-180 cells, it is likely that IRF-1 indirectly affects NF-
B
transcriptional activity through the regulation of other factors
modulating the transcriptional activity. We also demonstrated that
IFN
did not block the TNF
-induced translocation of p65 from
cytosol to nucleus or DNA binding of NF-
B but yet inhibited NF-
B
reporter activity. These results suggest that IFN
-induced IRF-1
inhibits the nuclear events of NF-
B transactivation but not
cytosolic events. Our work also showed that transfection of
transcriptional coactivator p300 abolished the inhibition of NF-
B
reporter activity by IFN
. Transcriptional activation by NF-
B
requires multiple coactivators (32). It has been recently reported that
the intracellular amount of the coactivator p300 is limited compared
with other transcriptional factors and that competition for p300 may
regulate transcriptional activity (33). Thus, it is possible that
IFN
-induced IRF-1 competes with TNF
-induced NF-
B for the
common coactivator(s) such as p300, and this competition may be
responsible for the inhibition of NF-
B transactivation. Then what
are the target genes that are induced by NF-
B and are subject to the
inhibitory action of IRF-1? Recently, a role of TNF receptor-associated
factor 1 (TRAF2), TRAF2, c-IAP1 (inhibitor of apoptosis (IAP)) and
cIAP2 was reported in anti-apoptosis mediated by NF-
B (19). These are possible candidates for such target genes. Another puzzling point
is what determines how IFN
acts on NF-
B. Previously, IFN
has
been shown to increase TNF
-induced NF-
B activation in enhancing the expression of multiple genes involved in the inflammatory responses
(34). In sharp contrast, however, our work disclosed that IFN
inhibited TNF
-induced NF-
B in ME-180 cells. This novel signaling
pathway of synergism between IFN
/TNF
involving competition between IRF-1 and NF-
B for p300 coactivator may not be generalized to other cell types, considering previous reports showing different signaling patterns in response to IFN
/TNF
(34). The same stimulus seems to activate distinct signaling pathways depending on the cell
types. Because of this discrepancy in signal transduction pathways, the
final outcome would be different among different cell types. Some cells
would undergo death by IFN
/TNF
, whereas other cells may be
activated by IFN
/TNF
to participate in inflammatory responses.
is also known to activate the STAT1-signaling pathway,
we investigated whether IFN
also synergizes with TNF
to destroy
ME-180 cells. Our results indicated that IFN
and TNF
synergistically induced ME-180 cell death, and this was accompanied by
the activation of STAT1 and inhibition of NF-
B reporter activity by
IFN
in a manner similar to
IFN
.3 Thus, the cytotoxic
priming role of IFN
in IFN
/TNF
synergism presented in the
current study does not seem to be restricted to IFN
. Rather, the
STAT1/IRF-1-signaling pathway that can be initiated by either type I or
type II interferon appears to be critical for the cytotoxic synergism
with TNF
.
/TNF
induces death signaling in
ME-180 cells regardless of caspase activation and activation of
caspases determines the final mode of cell death (apoptosis versus necrosis). These results suggest that
IFN
/TNF
-induced apoptotic and necrotic death signaling pathways
have common signaling components, and the mode of cell death depends on
distinct signaling events such as caspase activation. A similar dual
pathway in cell death has been reported in L929 cells transfected with
Fas cDNA (35). Ligation of Fas with anti-Fas antibody induced
apoptosis of these cells. However, pretreatment with z-VAD, which
inhibits activation of caspases, resulted in necrotic death. Moreover, necrosis of Fas-expressing L929 cells was inhibited by reactive oxygen
intermediate (ROI) scavengers such as butylated hydroxyanisol, indicating the involvement of ROI generation in necrotic cell death
pathway. Butylated hydroxyanisol, however, did not block IFN
/TNF
-induced ME-180 cell death in the presence of caspase inhibitors (data not shown), suggesting distinct signal transduction between the two cell types. Nevertheless, dual pathways of death signaling appear to be present in the two cells, and it will be of
great interest to see if this type of response could be found in other
cell types exposed to similar or different death signals. Whether a
cell undergoes apoptosis or necrosis by a given stimulus may be
determined by intracellular milieu (36-38). Intracellular levels of
ATP were reported to be a determinant of manifestation of cell death
(apoptosis versus necrosis) (36). Also, the fact that Bcl-2
blocks both apoptotic and necrotic cell death supports the presence of
common signaling components between the two death-signaling pathways
(37, 38). Although our work cannot provide detailed biochemical
mechanisms of cell death machinery in ME-180 cells, our studies point
out the existence of common components between apoptotic and necrotic
death signaling and the role of caspases in determining the type of
cell death, which may help understand the general cell death mechanism.
/TNF
synergism in the induction of ME-180 cell apoptosis: IFN
synergized with TNF
for apoptosis induction by activating
STAT1/IRF-1 pathway. We also present evidence that NF-
B activation
is a survival signal in TNF
-treated ME-180 cells, and IFN
inhibits this survival mechanism, resulting in synergistic cytotoxicity
with TNF
. Moreover, the mode of ME-180 cell death by IFN
/TNF
synergism was dictated by caspase activation. The novel mechanism of
IFN
/TNF
synergism presented here may also be applicable to other
circumstances, where a similar cytokine synergism could be found such
as autoimmune destruction of self tissues by cytokines.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Kye Young Lee, Tae H. Lee, Jae W. Lee, Minho Shong, Soo Young Lee, Il-Seon Park, and Young S. Ahn for insightful discussions and technical help.
![]() |
FOOTNOTES |
---|
* This work was supported by National Research Laboratory Grants 2000-N-NL-01-C-232 from the Korea Institute of Science and Technology Evaluation and Planning and by Science Research Center Grants from Korea Science and Engineering Foundation.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.
¶ Supported by Brain Korea 21 project from the Ministry of Education, Korea.
These authors contributed equally to this work.
Recipient of Juvenile Diabetes Foundation International
Research Grant 1-1999-760). To whom correspondence should be addressed. Tel.: 82-2-3410-3436; Fax: 82-2-3410-3849; E-mail:
mslee@smc.samsung.co.kr.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M007646200
3 K. Suk, I. Chang, Y.-H. Kim, J. Y. Kim, and M.-S. Lee, unpublished data.
2 K. Suk, I. Chang, Y.-H. Kim, S. Kim, J. Y. Kim, and M.-S. Lee, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TNF, tumor necrosis factor; IFN, interferon; IRF, interferon regulatory factor; STAT, signal transducer and activator of transcription; PI, propidium iodide; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OCH3)-CH2-fluoromethyl ketone; BD-fmk, t-butoxycarbonyl-Asp(OCH3)-CH2-fluoromethyl ketone; z-DEVD-fmk, benzyloxycarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2-fluoromethyl ketone; z-IETD-fmk, benzyloxycarbonyl-Ile-Glu(OCH3)- Thr-Asp(Ome)-CH2-fluoromethyl ketone; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Ac, acetyl; AMC, amidome-thylcoumarin.
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