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INTRODUCTION |
Tumor necrosis factors
(TNFs)1 are produced by
neutrophils, activated lymphocytes, macrophages, natural killer cells,
endothelial cells, and smooth muscle cells, and were reported initially
to induce tumor necrosis (1, 2). Subsequent studies, however, indicated that TNFs also promote the proliferation and survival of some
tumor cell lines (3-5). In patients with leukemias or lymphomas,
elevated levels of serum TNF-
, which are associated with a poor
prognosis, have been reported (6-13). The reasons for the
TNF-
-related poor prognosis of leukemia patients are not well
understood. We and others reported that TNF-
significantly stimulates the growth of several human leukemic cell lines, including Mo7e (14, 15), CMK (3, 5), HU-3, and M-MOK (4). TNF-
also stimulates
the proliferation of primary leukemia cells isolated from patients
(16-18). In an ultrastructural study of primary leukemia cells from
patients, TNF-
did not induce direct cytotoxicity or apoptosis, but
instead stimulated leukemia cells (19). Furthermore, we and others have
found that low doses of TNF-
act synergistically with other
cytokines, such as IL-4, to stimulate Mo7e leukemic cell growth (14,
15). However, the molecular mechanisms of the signaling pathway(s) in
TNF-
-induced leukemic cell proliferation remain unclear.
Extensive studies show that TNF-
is an extremely pleiotropic factor,
which can induce activation of phagocytic and endothelial cells,
induction of prostaglandins, alterations in lipid metabolism, and
regulated expression of major histocompatibility complex antigens, oncogenes, and transcription factors (1, 2). The capacity for TNF-
to induce such a wide variety of effects is attributable, in part, to
its ability to activate multiple signal transduction pathways including
mitogen-activated protein kinases (MAPKs), Jun NH2-terminal
kinase (JNK), nuclear factor
B (NF-
B), and/or STATs in numerous
cell lines (20-24). Although primarily involved in cell proliferation
(25, 26), the activation of MAPK has also been implicated in
apoptosis in some circumstances (27-29). TNF-
-induced
recruitment of the signal transducer FADD to type I TNF receptors may
mediate TNF-
-induced apoptosis, but TNF-
-induced activation of
NF-
B protects against TNF-
-induced apoptosis in some cell lines
(30). Conventional NF-
B is a heterodimer that consists of p65 and
p50 subunits. Both subunits of NF-
B are members of the NF-
B/Rel
family of transcription factors, which also includes c-Rel, RelB, and
p52 (31). The activity of NF-
B is strictly regulated by an
inhibitor, I
B
, that forms a complex with NF-
B and keeps
NF-
B in the cytoplasm (23). When cells receive signals that activate
NF-
B, I
B
is phosphorylated and degraded through a
ubiquitin/proteasome pathway. The degradation of I
B
triggers the
translocation of NF-
B from the cytoplasm to the nucleus. Although
increasing evidence shows that the activation of NF-
B is involved in
cell activation and proliferation, several lines of evidence also
indicate that activation of NF-
B may result in apoptosis (32-34).
The fact that TNF-
stimulates the proliferation of human leukemic
cell lines as well as primary leukemia cells led us to investigate the
TNF-
-induced activation of signaling pathways in Mo7e and other cell
lines. Specifically, we addressed the questions of what signal
transduction pathways are activated in leukemic cells treated with
TNF-
and whether the activated signal transduction pathway(s) is/are
involved in TNF-
-induced Mo7e cell proliferation. We found that
TNF-
transiently induced activation of NF-
B, but not the MAPK or
STAT signaling pathways, which are activated by IL-3, GM-CSF, and
thrombopoietin (TPO) in Mo7e and other cell lines. Pretreatment of Mo7e
cells with a synthesized membrane-permeable peptide, SN50, blocked the
TNF-
-induced translocation of NF-
B to the nuclei in a
dose-dependent manner. Pretreating Mo7e cells with SN50
specifically inhibited TNF-
-induced but not IL-3- or GM-CSF-induced
Mo7e cell proliferation. Thus, the data presented here provide direct
evidence that the activation and function of NF-
B is essential to
TNF-
-induced proliferation of Mo7e cells.
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EXPERIMENTAL PROCEDURES |
Reagents--
Recombinant human TNF-
, recombinant human
granulocyte/macrophage colony-stimulating factor (GM-CSF), and
anti-TNF-
antibody were obtained from R&D Systems (Minneapolis, MN).
IL-3, IL-6, and TPO were purchased from PeproTech (Rocky Hill, NJ).
[methyl-3H]Thymidine (specific activity 70-86
Ci/mmol), [32P]dATP (specific activity >3000
µCi/mmol), and [32P]cATP (specific activity >3000
µCi/mmol) were purchased from Amersham Pharmacia Biotech. Anti-goat
IgG antibody labeled with fluorescein isothiocyanate (FITC) was
purchased from Zymed Laboratories Inc. Antibodies
against human p50, p52, p65, and B-Rel NF-
B subunits were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal
anti-human I
B
and anti-phosphorylated I
B
antibodies were
purchased from New England Biolabs (Beverly, MA).
Cell Lines--
The human Mo7e megakaryoblastic leukemic cell
line, originally described by Avanzi et al. (35), was
maintained in Iscove's modified Dulbecco's medium (IMDM; Life
Technologies, Inc.) containing 10% fetal bovine serum, 1% glutamine,
and 5 ng/ml rhGM-CSF. The human Meg-01 megakaryoblastic leukemic cell
line, originally described by Ogura et al. (36), was
maintained in RPMI 1640 medium (Life Technologies, Inc.) with 10%
fetal bovine serum. The human leukemic HEL cell line, which has both
erythroid and megakaryocytic characteristics (14, 37, 38), was
maintained in IMDM with 10% horse serum. Both Meg-01 and HEL cell
lines were purchased from ATCC, and the Mo7e cell line was obtained
from Genetics Institute (Boston, MA). In experiments to detect effects
of cytokines, Mo7e cells were prepared by washing three times with
serum-free medium and were starved for 18 h in medium without
cytokine (14). Meg-01 and HEL cells were cultured in serum-free medium
with 1× Nutridoma HU (Roche Molecular Biochemicals) for 18 h
before cytokine treatments.
Synthesis of the Recombinant Membrane-permeable
Peptides--
SN50, a peptide that has been reported to have the
capacity to block the nuclear translocation of activated NF-
B (34,
39), contains membrane-permeable signal sequences of Kaposi's
fibroblast growth factor and the nuclear translocation motif
(VQRKRQKLMP) of human NF-
B p50. SN50mt, a mutant SN50, contains
membrane-permeable signal sequences of Kaposi's fibroblast growth
factor and the mutant nuclear translocation motif of human NF-
B p50
(39). Both SN50 and SN50mt were synthesized commercially (Genemed
Synthesis, South San Francisco, CA). The peptides were purified by
reverse phase high performance liquid chromatography, and the molecular weight of the purified peptides was verified by mass spectrometry analysis. To track the intracellular localization of the
membrane-permeable peptides in Mo7e cells, SN50 was labeled with FITC.
The synthesized peptides were dissolved in Me2SO to a final
concentration of 100 µg/µl and mixed directly with culture medium
(1:500-1000) before use.
Effects of TNF-
on Cell Growth--
To determine the effects
of TNF-
on cell proliferation, DNA synthesis was measured by
[3H]thymidine incorporation in freshly prepared cells.
The assays were performed in triplicate, using a total of 4 × 105 Mo7e or Meg-01 cells, or 2 × 105 HEL
cells. Mo7e cells in IMDM with 10% FCS, and Meg-01 and HEL cells in
medium with 1× Nutridoma HU, were cultured for 72 h in the
presence or absence of TNF-
or other cytokines. After that, the
cells were labeled with 4 µCi/ml [3H]thymidine for an
additional 4 h. The radioactivity incorporated into DNA (counts
per minute) was determined by a liquid scintillation counter according
to a previously described protocol (14). All assays for
[3H]thymidine incorporation were repeated at least three
times. Some of our data from [3H]thymidine incorporation
assays also were compared with those obtained from the Non-radioactive
Cell Proliferation Wst-1 kit (Roche Molecular Biochemicals). Both
methods yielded consistent results, but only the data from
[3H]thymidine incorporation assays are presented.
MAP Kinase Kinase Activity and Western Blotting
Analysis--
The functional activity of MAP kinase kinase (MEK) was
detected by the kinase-induced phosphorylation of a kinase-defective p42mapk (K52R) labeled with [32P]ATP,
following a published protocol (14). For the detection of tyrosine
phosphorylation of MAPK (mitogen-activated protein kinase), cells
treated with or without cytokines were boiled in SDS buffer. Total
cellular proteins (30 µg) were loaded into each lane and subjected to
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The separated proteins were transferred to PVDF membranes
and probed with an anti-PhosphoPlus MAPK antibody (New England
Biolabs), which reacts only with MAPK that is phosphorylated on Thr-202
and Tyr-204, or with an anti-total (unphosphorylated and
phosphorylated) MAPK antibody as a control. Phosphorylation of STAT1
and STAT3 was examined by 10% SDS-PAGE Western blotting with specific
anti-phosphorylated-STAT1 and anti-phosphorylated-STAT3 antibodies (New
England Biolabs), according to the methods suggested by the
manufacturer. For comparison, total STAT1 and STAT3 (both phosphorylated and unphosphorylated) also were examined with anti-STAT1 and -STAT3 antibodies. Western blots were visualized with enhanced chemiluminescence.
Preparation of Nuclear Extracts and Electrophoretic Gel Mobility
Shift Assays--
Preparation of nuclear extracts and gel mobility
shift assays were performed according to methods described previously
(40, 41). The sequence of the NF-
B-binding oligonucleotide used as a
radioactive DNA probe was 5'-TCGACAGAGGGGACTTTCCGAGAGGC-3'. Equal
amounts of nuclear proteins (5-10 µg) for each sample were incubated
with 1 ng of 32P-labeled probe. The DNA binding reaction
was performed at room temperature in a volume of 25 µl, which
contained binding buffer (10 mM Tris-HCl, pH 7.5, 1 µM EDTA, 100 mM NaCl, 20 µg/ml bovine serum
albumin, and 0.2% Nonidet P-40, 1.8 µg/ml salmon sperm DNA), 1 ng of
3'-labeled probe, and 5-10 µg of nuclear proteins. After incubation
for 15 min, the samples were electrophoresed on native 6% acrylamide,
0.25× Tris borate-EDTA gels. The gels were dried and exposed to x-ray
film. Competition was performed by adding 50-100 M excess
of each unlabeled DNA fragment along with the 32P-labeled
probe. For gel mobility supershift assays, nuclear extracts were
co-incubated with the indicated specific anti-NF-
B subunit antibodies and 32P-labeled oligonucleotide probes. The
DNA-protein complexes and unbound probe were separated
electrophoretically on 6% native polyacrylamide gels in 0.25× TBE
buffer (44.5 mM Tris, pH 8.0, 1 mM EDTA, and
44.5 mM boric acid). The gels were fixed and dried, and the
DNA-protein complexes were visualized by autoradiography at
70 °C
with Kodak X-Omat film and a DuPont Cranex Lightning Plus intensifying screen.
Flow Cytometry Analysis--
A FACScan flow cytometer was used
to examine Mo7e cell cycle status, intracellular localization of SN50
peptides, and apoptosis. Mo7e cells were incubated with 50 µg/ml
FITC-labeled SN50 peptide for various periods or with various amounts
of FITC-SN50 for 30 min to investigate the intercellular incorporation
of the peptides. The status of apoptosis was analyzed by incubating
cells with FITC-labeled annexin V and propidium iodide (PI), following
the manufacturer's suggested procedure. For the analysis of cell cycle status and cellular DNA ploidy, the cells were fixed with methanol and
stained with PI according to the published method (14).
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RESULTS |
TNF-
Induces Activation of NF-
B in Human Leukemic Cell
lines--
When the electrophoretic gel mobility shift assay (EMSA)
was used to examine the activation of NF-
B in Mo7e, HEL, and K562 human leukemic cell lines, no detectable activation of NF-
B was observed in the cells without cytokine exposure (Fig.
1a, CTL). However,
exposure of the cells to 5 ng/ml TNF-
for 30 min significantly induced the activation of NF-
B in Mo7e, HEL, and K562 cells (Fig. 1a, TNF). The Mo7e cells were used to further
investigate the dynamics of the activation of NF-
B. When Mo7e cells
were treated with 5 ng/ml TNF-
for 0-120 min, the activation of
NF-
B was first detected within 5 min, attained a maximal level at 30 min, and declined thereafter (Fig. 1b). Supershift EMSA was
then used to examine the components of the DNA-protein complexes. The
results showed that both anti-p50 and anti-p65 antibodies induced a
supershift of the DNA-protein complex in nuclear extracts from Mo7e,
HEL, and K562 cells treated with 5 ng/ml TNF-
for 30 min (Fig.
2, a and b),
indicating that the TNF-
-induced DNA-protein complexes in these
cells contained both p50 and p65 NF-
B subunits. When the nuclear
extracts from cells treated with TNF-
were incubated with anti-p52,
anti-RelB, or anti-c-Rel antibodies, no supershift was observed (data
not shown). Furthermore, the formation of the NF-
B-DNA complex was
inhibited competitively by an unlabeled NF-
B DNA probe in a
dose-dependent manner. A 100-fold molar excess of
nonradioactive DNA containing the NF-
B binding site completely prevented binding of the activated NF-
B to the
32P-labeled NF-
B-binding probe (Fig. 2c), but
did not affect the nonspecific DNA-protein complexes (Fig. 2,
bands marked as NS). The results, therefore,
demonstrate clearly that the NF-
B p65/p50 heterodimer is activated
in these leukemic cell lines in response to TNF-
.

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Fig. 1.
Effects of TNF-
treatment on the activation of NF- B in
leukemic cell lines. a, TNF- -induced activation of
NF- B in Mo7e, HEL, and K562 leukemic cell lines. Nuclear extracts
were prepared from Mo7e, HEL, and K562 cells treated without TNF-
(CTL) or with 5 ng/ml TNF- (TNF) for 30 min.
Equal amounts of nuclear proteins were incubated with
32P-labeled NF- B-binding probe. b, time
course of NF- B activation in Mo7e cells treated with TNF- .
Nuclear extracts were prepared from the cells exposed to 5 ng/ml
TNF- for 0-120 min. EMSAs were used to examine the activation of
NF- B. The DNA-protein complexes and unbound probe were separated on
6% native polyacrylamide gels. Similar results were obtained in three
separate experiments, and one is displayed here. The autoradiographs
show the location of NF- B and the nonspecific DNA-binding band
(NS).
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Fig. 2.
Identification of the components of activated
NF- B in the DNA-protein complex by EMSA.
a, nuclear extracts from Mo7e, HEL, and K562 cells treated
with 5 ng/ml TNF- were incubated with 32P-labeled
NF- B-binding probe alone (lanes 1-3), with
radioactive probe and anti-NF- B p65 subunit antibody
(lanes 4-6). b, nuclear extracts from
Mo7e, HEL, and K562 cells treated with 5 ng/ml TNF- were incubated
with 32P-labeled NF- B-binding probe alone
(lanes 1-3), with radioactive probe and
anti-NF- B p50 subunit antibody (lanes 4-6).
NS indicates the nonspecific DNA-binding band, and the arrow
indicates the supershift complexes. c, nuclear extracts from
Mo7e cells treated without TNF- (lane 1) or
with 5 ng/ml TNF- for 30 min were incubated with
32P-labeled NF- B-binding probe alone (lane
2) or 32P-labeled probe and the indicated -fold
molar excess of unlabeled probe containing the NF- B binding site
(lanes 2-6) to determine the specificity of the
binding of activated transcriptional factors to the NF- B-binding
probe. The DNA-binding complexes were separated in 6% native
polyacrylamide gels. Similar results were obtained in three separate
experiments. The autoradiograph shows the location of NF- B and the
nonspecific band (NS).
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TNF-
Treatment Results in a Rapid Translocation of NF-
B to
the Nucleus of Mo7e Cells--
We then examined the effect of TNF-
treatment on the localization of NF-
B in Mo7e cells by Western
blotting with specific antibodies against the p65 NF-
B subunit.
Before stimulation, p65 was detected exclusively in the cytoplasmic
lysates (Fig. 3a,
lane 1). When Mo7e cells were treated with 5 ng/ml TNF-
for 5-120 min, the level of cytoplasmic NF-
B p65
declined gradually (Fig. 3a, lanes
2-7). When nuclear extracts were used in Western blotting
to analyze the level of nuclear NF-
B p65, there was no detectable
NF-
B p65 in the nucleus of Mo7e cells without TNF-
exposure (Fig.
3b, lane 1). However, NF-
B was
detectable in the nucleus within 5 min, and the level of nuclear
NF-
B p65 increased markedly in cells exposed to TNF-
, with a
maximal level within 30-60 min of the treatment (Fig. 3b,
lanes 4-7). When nonimmune immunoglobulins were
used as the first antibody, no NF-
B p65 was detected in the nuclear
extracts from Mo7e cells (data not shown). The results from Western
blotting analysis of cytoplasmic lysates and nuclear extracts confirm
and extend the EMSA findings of activation and nuclear translocation of
NF-
B in Mo7e cells treated with TNF-
.

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Fig. 3.
Activation and localization of
NF- B in the nuclear extracts and cytoplasmic
lysates of Mo7e cells. Cytoplasmic lysates (a) and
nuclear extracts from Mo7e cells (b) treated without or with
5 ng/ml TNF- for the indicated time. Equal amounts of proteins were
separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed
with anti-NF- B p65 antibody. Similar results were obtained in two
separate experiments.
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Degradation of I
B
Is Associated with the Activation of
NF-
B in Mo7e Cells--
A common feature of the regulation of
NF-
B is its sequestration in the cytoplasm as an inactive complex
with I
B
in the basal state. To investigate the role of I
B
in the activation of NF-
B by TNF-
, phosphorylation and the total
level of I
B
were analyzed by Western blotting, and the results
were compared with the dynamics of NF-
B activation in Mo7e cells
after TNF-
stimulation. When nuclear extracts from Mo7e cells
treated with TNF-
were analyzed by Western blotting with an antibody
specific for phosphorylated I
B
, the phosphorylation of I
B
became detectable in cells treated with 5 ng/ml TNF-
for 1 min,
reached the maximum level at 5 min, and then declined rapidly (Fig.
4a, lanes
2-6). Western blotting analysis of cytoplasmic lysates
showed that the rapid decline of nuclear I
B
in TNF-
-treated
Mo7e cells was due to a decrease of total I
B
rather than
dephosphorylation of I
B
, since the level of total I
B
(phosphorylated and unphosphorylated) also was decreased markedly upon
TNF-
treatment (Fig. 4b). Comparison of these results to
those in Fig. 3, which showed that the activation of NF-
B induced by
TNF-
was first detected within 5 min and maximal at 30 min, indicate
clearly that the degradation of I
B
coincides with the activation
and nuclear translocation of NF-
B in Mo7e cells.

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Fig. 4.
Effects of TNF- on
the phosphorylation and degradation of
I B in Mo7e
cells. Total cellular proteins (40 µg/ml) from Mo7e cells
treated without TNF- (lane 1) or with 5 ng/ml
TNF- for 1-60 min were subjected to SDS-PAGE, transferred to PVDF
membrane, and probed with anti-human phosphorylated-I B antibody
(a) and anti-total I B antibody (b).
P-I B and T-I B
indicate the locations of phosphorylated I B and total I B ,
respectively. Similar results were obtained in two separate
experiments.
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TNF-
Specifically Induces the Activation of NF-
B, but Not
MAPK or STAT5, Which Are Activated by IL-3 and GM-CSF--
MAPK and
STAT signal transduction pathways have been reported to be activated by
several hemopoietic growth factors and to be involved in growth
factor-induced Mo7e cell proliferation (14, 42). To investigate whether
these signal transduction pathways are activated by TNF-
and whether
they are involved in TNF-
-induced cell proliferation, we examined
the effect of TNF-
on the activation of MAPK and STAT signal
transduction pathways. When Mo7e cells were treated with 5 ng/ml IL-3
or GM-CSF for 30-60 min, no significant activation of NF-
B was
observed (Fig. 5a,
lanes 3-6), although TNF-
treatment induced
significant activation of NF-
B (Fig. 5a, lane
2). When the activation of the STAT5 signal transduction pathway in Mo7e cells was examined by EMSA, the results, which were
similar to those that we reported previously (14), showed that STAT5
activation was observed in Mo7e cells treated with GM-CSF, IL-3, TPO,
and IL-6, but not in cells treated with 5 ng/ml TNF-
for 10-60 min
(data not shown). When the activation of STAT1 and STAT3 was examined
by EMSA, supershift EMSA, or Western blotting with specific
anti-phosphorylated STAT1 or STAT3 antibodies, no significant
activation or phosphorylation of STAT1 or STAT3 was observed in Mo7e
cells treated with 5 ng/ml TNF-
, IL-3, or GM-CSF for 10-60 min
(data not shown). However, a significant activation of STAT1 and STAT3
was detected in control SK-BR-3 cells treated with 2 ng/ml epidermal
growth factor for 30 min, which we reported previously as the positive
control for detecting activation of STAT1 and STAT3 (42).

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Fig. 5.
Effects of TNF- ,
IL-3, and GM-CSF on the activation of NF- B and
MEK in Mo7e cells. a, nuclear extracts from Mo7e cells
treated without cytokine (lane 1), with 5 ng/ml
TNF- for 30 min (lane 2), 5 ng/ml IL-3 for 30 and 60 min (lanes 3 and 4), and 5 ng/ml GM-CSF for 30 and 60 min (lanes 5 and
6), respectively, were incubated with
32P-labeled NF- B-binding probe. Similar results were
obtained in two separate experiments. The autoradiograph shows the
location of NF- B and the nonspecific band (NS).
b, whole cell lysates were prepared from Mo7e and HEL cells
treated without cytokine (CTL), with 5 ng/ml GM-CSF
(GM) or 5 ng/ml TNF- (TNF) for 10 min. MEK
activity was detected by the kinase-induced phosphorylation of a
kinase-defective p42mapk (K52R) labeled with
[32P]ATP. No MEK activation was observed in cells treated
with TNF- for 5, 10, or 15 min, and only data from 10 min treatments
are displayed here. Similar results were obtained from two separate
experiments for each time point. Phosphorylation of MAPK p42 is used as
a read-out to detect the activation and functional activity of
MEK.
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When functional MEK activation was investigated in Mo7e, HEL, or Meg-01
human leukemic cell lines after exposure to TNF-
, we found no
significant activation of MEK in any of these cell lines exposed to 5 ng/ml TNF-
for 5-30 min (data not shown). For example, in Mo7e or
HEL cells treated with 5 ng/ml TNF-
for 10 min, no significant MEK
activation was observed (Fig. 5b, Mo7e and
HEL, TNF). However, activation of MEK was
observed in these cells after exposure to 5 ng/ml GM-CSF for 10 min
(Fig. 5b, GM). In separate experiments to examine
phosphorylation of p44/p42 MAPK, our data also showed that
phosphorylation of MAPK was barely detectable (not different from
untreated control cells) in all of these cell lines after exposure to 5 ng/ml TNF-
for 5-30 min, but increased phosphorylation was readily
detectable in cells treated with 5 ng/ml IL-3 or GM-CSF for 10 min and
reached the maximal level around 30 min (data not shown).
SN50 Peptide Inhibits Nuclear Translocation of Activated
NF-
B--
SN50 is a synthetic peptide containing signal sequences
of Kaposi's fibroblast growth factor and the nuclear localization sequence of NF-
B p50. It has been reported to have the capacity to
specifically block the nuclear translocation of activated NF-
B (39).
To investigate the membrane permeability, dynamics of cellular uptake,
and intracellular incorporation of the synthesized peptide,
FITC-labeled SN50 was incubated with Mo7e cells. Flow cytometry
analysis showed that the peptide penetrated into Mo7e cells rapidly.
When the cells were incubated with 50 µg/ml amounts of the peptide,
the maximal level of FITC-SN50 was detected within 15 min and longer
incubation with FITC-labeled peptide did not further increase
fluorescence (Fig. 6a). When
Mo7e cells were incubated with 1-100 µg/ml FITC-SN50 for 30 min, a
dose-dependent intracellular accumulation of SN50 was
observed (Fig. 6b). Furthermore, our data showed that SN50
is quite stable after penetrating into the cells, with a half-life more
than 12 h (data not shown).

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Fig. 6.
Cellular uptake of SN50 in Mo7e
cells. The cells were incubated with 50 µg/ml FITC-labeled SN50
peptide for 0-60 min (a) and 1-100 µg/ml FITC-SN50 for
30 min (b). The cells were washed three times and analyzed
by flow cytometry. The solid line
peaks represent untreated control cells, and
broken line peaks indicate the cells
treated with various amounts of SN50. Similar results were obtained in
three separate experiments.
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When Mo7e cells were pretreated with various amounts of SN50 for 15 min
and then incubated with 5 ng/ml TNF-
for an additional 30 min, there
was a marked SN50 dose-dependent reduction of
TNF-
-induced NF-
B-binding activity in Mo7e nuclei, as determined
by EMSA. For example, in cells treated with 50-100 µg/ml SN50,
the nuclear NF-
B-DNA complexes were almost undetectable (Fig.
7a). When Western blotting
with a specific anti-NF-
B p65 antibody was used to analyze effects
of SN50 on nuclear translocation of NF-
B in Mo7e cells treated with
TNF-
, the results showed that there was a marked SN50
dose-dependent reduction of the amount of nuclear NF-
B
p65. For example, in cells treated with 5 ng/ml TNF-
and 100-200
µg/ml SN50, almost no nuclear NF-
B P65 subunit was detected (Fig.
7b). However, no significant decrease of the level of
nuclear NF-
B p65 was observed in nuclear extracts from the cells
treated with 5 ng/ml TNF-
and 10-200 µg/ml SN50mt, a
nonfunctional mutant of SN50 (Fig. 7c). These results
demonstrate the specific inhibitory effect of SN50 on the nuclear
translocation of activated NF-
B.

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Fig. 7.
Effects of SN50 and SN50mt on
TNF- -induced activation and translocation of
NF- B. a, identification of the
effect of SN50 on the activity of NF- B by EMSA. Nuclear extracts
were prepared from cells treated without TNF- (lane
1) or with 5 ng/ml TNF- alone (lane
2) or 5 ng/ml TNF- plus 1-100 µg/ml SN50
(lanes 3-6). The DNA-protein complexes and
unbound probe were separated on 6% native polyacrylamide gels. The
autoradiographs show the location of NF- B and the nonspecific
DNA-binding band (NS). Similar results were obtained in two
separate experiments. b, the effect of SN50 on the level of
NF- B p65 was analyzed by Western blotting in nuclear extracts from
Mo7e cells treated without TNF- (lane 1), with
5 ng/ml TNF- alone (lane 2), or 5 ng/ml
TNF- plus indicated amounts of SN50 (lanes
3-6). c, the effect of SN50mt on the level of
NF- B p65 was analyzed by Western blotting in nuclear extracts from
Mo7e cells treated without TNF- (lane 1) or
with 5 ng/ml TNF- alone (lane 2) or with 5 ng/ml TNF- plus indicated amounts of SN50mt (lanes
3-6). Western blotting was performed on 10% SDS-PAGE, and
the blots were probed with anti-NF- B p65 antibody. Similar results
were obtained in two separate experiments.
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SN50 Specifically Inhibits TNF-
-induced Survival and
Proliferation of Mo7e Cells--
To investigate the role of NF-
B
activation in TNF-
-induced Mo7e cell proliferation, SN50 and SN50mt
were used to examine the effect of SN50-induced inhibition of NF-
B
nuclear translocation on TNF-
-induced Mo7e cell survival and
proliferation. When Mo7e cells were treated with various amounts of
SN50 with 1 ng/ml TNF-
, TNF-
-induced Mo7e cell proliferation was
inhibited in a dose-dependent manner. For example, treating
Mo7e cells with 1 ng/ml TNF-
and 50 µg/ml SN50 completely blocked
TNF-
-induced cell proliferation (Fig.
8a, lane
5). In contrast, incubation of Mo7e cells with the same
concentration of SN50mt showed no effects on TNF-
-induced cell
proliferation (data not shown). Exposure of Mo7e cells to 50 µg/ml
SN50 alone did not show significant effects on cell proliferation (Fig.
8a, lane 7).

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Fig. 8.
Effects of SN50 on DNA synthesis in
TNF- - or GM-CSF-treated Mo7e cells.
a, the cells were treated without cytokine (lane
1), with 1 ng/ml TNF- alone (lane
2), 50 µg/ml SN50 alone (lane 7), or
1 ng/ml TNF- plus 1-100 µg/ml SN50 (lanes
3-6) for 72 h and labeled with 4 µCi/ml
[3H]thymidine for additional 4 h. b, Mo7e
cells were pretreated without cytokine (lane 1),
or with 1 ng/ml GM-CSF alone (lane 2) or 1 ng/ml
GM-CSF plus 1-100 µg/ml SN50 (lanes 3-6) for
72 h and labeled with 4 µCi/ml [3H]thymidine for
an additional 4 h. The [3H]thymidine incorporation
was determined from triplicate samples and expressed as the mean ± S.E. of counts per minute.
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|
We then investigated the effect of these peptides on the proliferation
of Mo7e cells promoted by GM-CSF and IL-3, which stimulate Mo7e
proliferation via the activation of MAPK and STAT5 signaling pathways.
The results showed that incubation of Mo7e cells with 1-100 µg/ml
SN50 with 1 ng/ml GM-CSF or IL-3 for 72 h showed no significant
effects of SN50 on [3H]thymidine incorporation promoted
by GM-CSF (Fig. 8b) or IL-3 (data not shown). SN50mt also
did not show the inhibitory effect on GM-CSF- or IL-3-induced Mo7e cell
proliferation (data not shown).
When flow cytometric analysis was used to analyze the effect of
SN50-induced inhibition of nuclear translocation of NF-
B on cell
cycle status, the results showed that TNF-
treatment enhanced the
progression of Mo7e cells into S-phase of the cell cycle, but
preincubating Mo7e cells with 100 µg/ml SN50 for 3 days blocked
TNF-
-induced progression of the cells into S-phase (Fig.
9a). SN50mt (100 µg/ml) had
no inhibitory effect on TNF-
-induced progression of Mo7e cells into
S-phase (Fig. 9a, TNF+SN50mt). The effect of SN50
on cell death was also investigated with a flow cytometry-based
technique combined with FITC-labeled annexin V and PI staining. The
results showed that significant numbers of Mo7e cells spontaneously
died via apoptosis in cultures without any exogenous cytokines for 5 days (Fig. 9b, CTL). Incubation of the cells with
5 ng/ml TNF-
promoted the survival of Mo7e cells by preventing their
apoptosis (Fig. 9b, TNF) and exposure of the
cells to 100 µg/ml SN50 alone did not show significant effects on
cell growth (data not shown). When Mo7e cells were co-incubated with
various amounts of SN50 and 5 ng/ml TNF-
for 5 days, TNF-
-induced
protection of Mo7e cells against apoptosis was inhibited in a
dose-dependent manner. For example, incubating the cells
with 5 ng/ml TNF-
plus 100 µg/ml SN50 blocked TNF-
-induced anti-apoptosis effects (Fig. 9b, TNF+SN50).
However, when the cells were co-incubated with 5 ng/ml TNF-
and 100 µg/ml SN50mt for 5 days, the protective effect of TNF-
against
apoptosis was still observed (Fig. 9b,
TNF+SN50mt). Comparison of these results of cell survival
and proliferation with that of the effect of SN50 on the nuclear
translocation of NF-
B demonstrates that the activation and nuclear
translocation of NF-
B is essential for TNF-
-induced cell survival
and proliferation.

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Fig. 9.
Effects of SN50 and SN50mt on
TNF- -induced survival and proliferation of
Mo7e cells. a, the cells, treated without TNF-
(CTL) or with 5 ng/ml TNF- alone, 5 ng/ml TNF- plus
100 µg/ml SN50 (TNF+SN50), or 5 ng/ml TNF plus 100 µg/ml
SN50mt (TNF+SN50mt) for 3 days, were fixed and stained with
PI to examine the cell cycle status by flow cytometry analysis. Similar
results were obtained in three separate experiments. b, Mo7e
cells, treated as above for 5 days, were incubated with PI and
FITC-labeled annexin V to determine the apoptotic status by flow
cytometry. The percentages in the lower right and
upper right panels indicate the
earlier and later apoptotic cells, respectively, and M1
indicates the percentage of total apoptotic cells (lower
right and upper right). Three
experiments had similar results, and one is displayed here.
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 |
DISCUSSION |
We previously reported that TNF-
stimulated the proliferation
of the Mo7e human leukemic cell line (14). In addition to TNF-
,
numerous other hemopoietic growth factors, including GM-CSF, IL-3,
IL-6, and TPO, also support the survival and proliferation of Mo7e
cells (14, 35). Considering our previous report and present data, we
conclude that the signaling for TNF-
-induced Mo7e cell proliferation
occurs via a different mechanism from that of other cytokines.
Cytokines like IL-3, GM-CSF, IL-6, and TPO mainly cause activation of
MAPK and JAK2/STAT5 signal transduction pathways (42), but TNF-
specifically induces activation of NF-
B in Mo7e cells. Our data are
also consistent with prior reports that phosphorylation and degradation
of I
B
play an important role in the activation of NF-
B.
TNF-
rapidly induces the degradation of I
B
, and then the
release of activated NF-
B, which translocates to the nucleus of Mo7e
cells. Western blotting analysis further showed that the nuclear
translocation of NF-
B correlated well with TNF-
-induced binding
of nuclear NF-
B to the NF-
B-binding DNA probe, as detected by
EMSA. Using a specific inhibitory peptide (SN50) to block the nuclear
translocation of activated NF-
B, we provide clear evidence showing
the direct linkage of TNF-
-induced NF-
B activation and Mo7e cell
survival and proliferation.
TNF-
is an extremely pleiotropic factor, which can induce growth
inhibition or death in some malignant cell lines but promotes the
survival and proliferation of other cell lines (1, 3-5). The different
mechanisms responsible for its pleiotropic functions are not well
understood. In this study, we examined TNF-
-induced signal
transduction pathways in Mo7e, Meg-01, K562, and HEL leukemic cell
lines. We found that TNF-
-induced activation of signal transduction pathways is very similar in all these cell lines, although they behave
differently in response to other cytokines. In Mo7e cells, TNF-
induces the activation of NF-
B but not MAPK or STAT signal transduction pathways. In HEL and Meg-01 cells, although there is
constitutive activation of STAT5, as we reported previously (42),
TNF-
-induced activation of NF-
B was observed (Figs. 1, 2, and 5).
The activation of NF-
B is specifically derived from the binding of
TNF-
to TNF receptors, as the activation can be blocked by
anti-TNF-
neutralizing antibody (data not shown). Based on the
published observation that TNF-
stimulates a number of other human
leukemic cell lines such as CMK, HU-3, and M-MOK megakaryocytic
leukemic cell lines (3-5, 14), the role of TNF-
-induced activation
of NF-
B in preventing apoptosis and promoting cell proliferation is
likely to be a more general phenomenon.
It has been reported that TNF-
treatment activates MAPK and STAT
signal transduction pathways in several human cell lines (20-23, 43).
TNF-
-induced MAPK activation was also reported to be involved in
TNF-
-induced apoptosis (27, 31). The data presented here show that
TNF-
treatment did not activate STAT signal transduction pathways in
the Mo7e cell line, although the functional STAT5 signaling pathway was
detected in this cell line treated with IL-3, GM-CSF, or TPO. Our data
also indicated that TNF-
treatments failed to activate STAT1 and
STAT3 in Mo7e cells, confirmed by either Western blotting, EMSA, or
supershift EMSA analyses (data not shown). When the DNA probes, which
bind to STAT2, STAT4, and STAT6, were used in EMSA to examine the
activation of these STATs, no significant DNA-protein complexes were
detected in Mo7e cells treated with 1-5 ng/ml TNF-
for 5-30 min
(data not shown). Furthermore, we provided clear evidence showing that TNF-
treatment also failed to activate the MAPK signal transduction pathway. If the activated MAPK signal transduction pathway is involved
in TNF-
-induced apoptosis in other reported cell lines (27, 31), the
lack of TNF-
-induced MAPK activation may be one of the reasons why
TNF-
promotes cell survival and proliferation, rather than apoptosis
in Mo7e cells.
There is considerable evidence that the activation of NF-
B promotes
survival and proliferation of certain cell types. In RelA (p65)
knockout mice, Beg et al. (44) showed that a considerable degree of apoptosis occurred in hepatocytes in RelA-deficient embryos.
They also reported that TNF-
induced apoptosis of fibroblasts and
macrophages derived from RelA knockout mice, and transfection of RelA
into the cells rescued them from TNF-
-induced apoptosis (45). Wang
et al. (46) and Antwerp et al. (47) independently demonstrated the anti-apoptotic role of NF-
B using cell lines stably transfected with dominant negative I
B
. Liu et
al. (30) also showed that three different responses to TNF-
were mediated by activation of the TNF receptor complex: activation of
JNK, activation of NF-
B, and induction of apoptosis. The activation of NF-
B protected the cells against TNF-
-induced apoptosis. Ozaki
et al. (48) also reported that NF-
B inhibitors
pyrrolidine dithiocarbamate (PDTC) and chloromethyl ketone stimulated
apoptosis of mature rabbit osteoclasts, which resulted in the
inhibition of bone resorption. These findings, together with the
present report, indicate that the NF-
B signaling cascade is closely
involved in the prevention of apoptosis of cells.
In contrast, several reports demonstrated that activation of NF-
B
induces apoptosis in certain cells (32-34). Bessho et al. (33) showed that treatment of human leukemia cells and thymocytes with
PDTC, an antioxidant inhibitor of NF-
B activation, prevented their
apoptosis. In addition, radiation or agents such as lipopolysaccharides and TNF-
triggered the hydrolysis of membrane phospholipids, which
produced ceramide, which in turn activated NF-
B and induced apoptosis (23). Although there is some controversy in the literature, it appears at present that the activation of NF-
B generally mediates survival signals that protect cells from apoptosis in certain cell
lines; however, under some circumstances, activation of NF-
B may
also promote apoptosis.
To investigate the role of activation of NF-
B in TNF-
-induced
cell proliferation, one approach is to use specific reagents to block
either the activation of NF-
B or phosphorylation and degradation of
I
B
by antisense oligonucleotides, proteasome inhibitors, or
transfected dominant-negative I
B
. Proteasome inhibitors
N-acetyl-L-cysteine and PDTC were reported to
inhibit the degradation of I
B
, which then blocked the activation
of NF-
B. When these chemical reagents were used in our study in attempt to block the activation of NF-
B, we found that these reagents inhibited TNF-
-induced Mo7e cell proliferation. However, PDTC showed extreme cytotoxic effects on Mo7e cell growth at the reported concentration of 50-100 µM (33, 48), and no
difference of the effect of PDTC on TNF-
- and GM-CSF-induced Mo7e
cell proliferation was observed at lower concentration (data not
shown). N-Acetyl-L-cysteine inhibited not only
TNF-
-induced, but also GM-CSF- and IL-3-induced Mo7e cell
proliferation (data not shown). As it is known that GM-CSF and
IL-3-induced Mo7e cell proliferation occurs via different signal
transduction pathway(s) (Figs. 3, 5, and 8), the specificity of the
chemical-induced inhibition of TNF-
-induced cell proliferation is
open to serious questions.
Lin et al. reported a technique called peptide-induced
membrane translocation using a membrane-permeable peptide (SN50)
containing signal sequences of Kaposi's fibroblast growth factor and
nuclear translocation sequences of NF-
B p50 to block the nuclear
translocation of NF-
B (39). The ability of SN50 to be imported into
intact cells seems to be dependent on a hydrophobic region and was
verified by its inaccessibility to extracellular proteases and by
confocal laser scanning microscopy. Liu et al. later
reported that the hydrophilic region may also be involved in the
SN50-induced membrane translocation (49). Subsequent studies
demonstrated that SN50 specifically blocked the nuclear translocation
of NF-
B, but did not affect the activity of AP-1, SP-1 factor, and
OCT-1 transcriptional factors (34). The synthesized SN50 peptide worked
very effectively and specifically in our study to block TNF-
-induced
activation and nuclear translocation of NF-
B in the Mo7e cell line.
Pretreating Mo7e cells with SN50 significantly reduced the level of
NF-
B binding to the radioactive probe containing NF-
B binding
site, as revealed by EMSA. Western blotting analysis further
demonstrated that the reduced binding of NF-
B to the NF-
B-binding
probe in the nuclear extracts from Mo7e cells treated with TNF-
was
due to the blocking of nuclear translocation of activated NF-
B,
rather than the inactivation of NF-
B (Fig. 7). Furthermore, our data showed that SN50 and SN50mt were not cytotoxic within the concentration range used in our experiments (Fig. 8 and data not shown). These results suggested that the cell-permeable synthetic peptide SN50 can be
used to block the nuclear translocation of activated NF-
B specifically.
Using SN50 to block the nuclear translocation of activated NF-
B, we
demonstrated that NF-
B has an essential role in TNF-
-induced Mo7e
cell proliferation, thus confirming that NF-
B has an anti-apoptosis function in Mo7e cells stimulated by TNF-
. Our experiments
demonstrated a direct linkage of activated NF-
B and TNF-
-induced
cell proliferation. Furthermore, we find that TNF-
treatment not
only prevents apoptosis but also actually stimulates DNA synthesis and
promotes the progression of Mo7e cells into S-phase (Fig. 9). Thus, the
Mo7e cell line seems to be a good model system to investigate the
signal transduction pathways involved in TNF-
-induced cell proliferation.
Studying the control mechanisms of survival and proliferation of
leukemia cells would be important for understanding the regulation of
leukemia cell survival, not only in vitro, but also possibly in vivo. Treatment of Mo7e cells with TNF-
for 1 h
could support their survival, which was evaluated 32-48 h later (data
not shown). This indicates that NF-
B stimulates expression of gene
products that have long term effects on the survival of Mo7e cells.
Identification of such activated survival and proliferation-promoting
proteins in Mo7e cells will be an important focus of future studies.
Further studies are required to determine a more detailed mechanism of the involvement of NF-
B in the survival and proliferation of Mo7e
cells and the expression of specific gene products promoted by TNF-
that exert anti-apoptotic and pro-proliferative effects.