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J Biol Chem, Vol. 274, Issue 40, 28286-28292, October 1, 1999
12-O-tetradecanoylphorbol-13-acetate-induced
Apoptosis Is Mediated by Tumor Necrosis Factor in Human
Monocytic U937 Cells*
Yasunari
Takada ,
Misao
Hachiya ,
Yoshiaki
Osawa ,
Yoshinori
Hasegawa§,
Koichi
Ando¶,
Yoshiro
Kobayashi**, and
Makoto
Akashi 
From the Division of Radiation Health, ¶ Space
and Particle Radiation Science Research Group, National Institute of
Radiological Sciences, Chiba, 263-8555 Japan, § First
Department of Internal Medicine, Nagoya University School of Medicine,
Nagoya 466-8550, Japan, and ** Laboratory of Molecular Immunology,
Department of Biomolecular Science, Faculty of Science, Toho
University, Funabashi 274-8510, Japan
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ABSTRACT |
12-O-tetradecanoylphorbol-13-acetate
(TPA), a phorbol ester that is known as a tumor promoter, induces
differentiation of myeloid cells and suppresses their proliferation. We
studied the regulation of apoptosis by TPA in human monocytic cell line
U937 cells that lack p53. Untreated U937 cells constitutively
underwent apoptosis, and TPA enhanced apoptosis in these
cells. Further studies showed that TPA increased production of tumor
necrosis factor- (TNF ) in U937 cells, and exogenously added
TNF induced apoptosis. Moreover, the induction of apoptosis by TPA
was blocked by anti-TNF antibody. Similar results were obtained in
the myeloblastic cell line KY821 cells. We also found that the
induction of apoptosis by TPA was increased in cells overexpressed with
TNF receptor 1 but not in control cells. Furthermore, TPA failed to
induce the production of TNF and apoptosis in cells with either
their protein kinase C or mitogen-activated protein kinase pathway
blocked. Our results indicate that TPA induces apoptosis, at least in
part, through a pathway that requires endogenous production of TNF in U937 cells. Our data also suggest that the induction of apoptosis by
TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNF is an autocrine-stimulating factor for the
induction of apoptosis in these cells.
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INTRODUCTION |
Apoptosis, a morphologically distinguished form of programmed cell
death, plays important roles not only during development and tissue
homeostasis but also in the pathogenesis of a variety of diseases
including cancer, autoimmune disease, viral infection, and
neurodegenerative disorder (1-6). Moreover, various chemicals and
drugs for treatment of cancers are known to destroy tumor cells through
apoptosis (7, 8). The precise mechanisms that control apoptosis have
not been elucidated; it appears that this form of cell death is
regulated by a genetic program involving both inducers and repressors
(6). The tumor suppressor p53 is one of the key proteins affecting the
fate of cells exposed to external insults; activation of p53 leads to
apoptosis or cell cycle arrest. Inactivation of p53 by either a
mutation of the gene or interaction with oncogenic viral or cellular
proteins is a common event in the development of malignancies (9). On the other hand, recent studies have found pathways of apoptosis independent of p53 (10).
Protein kinase C (PKC)1 plays
important roles in physiological events such as cell differentiation
and proliferation (11). PKC phosphorylates and activates Raf-1, which
leads to the activation of mitogen-activated protein kinase (MAPK) or
extracellular signal-regulated protein kinase (ERK) (12). By contrast,
studies have shown that proliferation of cells is also inhibited by PKC
activation; phorbol esters that can activate PKC have been shown to
inhibit the phosphorylation of retinoblastoma susceptibility gene
product (Rb) (13). 12-O-tetradecanoylphorbol-13-acetate (TPA) is a novel activator of PKC; previous studies (14-16) have reported that TPA induces apoptosis in MCF-7 breast cancer cells, U937 cells, and Jurkat T-lymphoma cells. TPA is also known to modulate
apoptosis induced by various agents (17, 18). However, the effects of
TPA on induction of apoptosis seem to depend upon the type of cells;
treatment of cells with inhibitors of PKC resulted in apoptosis in
lymphocytes and HL60 cells, and TPA blocked apoptosis induced by VP-16
or interleukin withdrawal (18-21). Studies have suggested that this
different response to TPA in apoptosis is, in part, due to the
existence of multiple PKC isotypes (22, 23).
Tumor necrosis factor- (TNF ) is a pleiotropic cytokine that was
originally identified on the basis of its ability to induce hemorrhagic
necrosis in murine transplantable tumors and of its direct cytotoxic
effects against transformed cells lines in vivo (24-26).
TNF plays a physiological role in oncogeny and in the control of
major inflammatory and immune reactions affecting both myelocytic and
nonmyelocytic cells (27). It is primarily produced by activated
macrophages and exerts its effect through two distinct cell surface
receptors, denoted TNFR1 and TNFR2. These two TNF receptors are
present on all nucleated cell types (28, 29). TNF preferentially
kills transformed cells in vivo although it is not cytotoxic
to normal diploid cells (26, 28, 29). The mechanisms underlying the
cytotoxic effects of TNF are not completely understood. However, it
has been shown that cytotoxicity of TNF is mediated through mainly
TNFR1, which has a 70-amino acid region in the cytoplasmic domain
termed the "death domain" (30). Further studies have found that
inhibition of DNA topoisomerase, production of reactive oxygen
intermediates in the mitochondria, and induction of proteases appears
to be involved (31-33). It has been reported that the cytotoxic
effects of TNF on neoplastic cells are synergistically enhanced by
TPA in vitro although the molecular mechanisms underlying this synergism are not known (34).
In this study, we examined the cytotoxic effects of TPA on a human
monocytic cell line, U937, and a human myeloblastic cell line, KY821.
Our results demonstrate that TPA induces apoptosis through TNF
production in U937 and KY821 cells.
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MATERIALS AND METHODS |
Cells and Cell Culture--
U937, derived from a diffuse human
histiocytic lymphoma (American Type Tissue Culture Collection,
Manassas, VA) and KY821, derived from human myeloblastic leukemia cells
(kindly provided by Dr. K. Kishi, Niigata University School of
Medicine, Niigata, Japan) (35) were cultured in MEM (Cosmo Bio Co.
Ltd., Tokyo, Japan). The medium was supplemented with 7% fetal calf
serum (Mitsubishi Kasei Co, Tokyo, Japan). The cells were incubated at
37 °C in a humidified atmosphere with 5% CO2.
Reagents--
A monoclonal TNF antibody and a neutralizing
polyclonal rabbit antibody against human TNF were purchased from
Genzyme Co. (Cambridge, MA). 10 µl of this antibody neutralizes >100
units/ml TNF in L929 cell cytotoxic assay. The neutralizing antibody
against IL-1 was a polyclonal rabbit anti-human IL-1 antibody and
was kindly provided by Dr. T. Nishida (Otsuka Pharmaceutical Co. Ltd. Tokushima, Japan). This antibody neutralized more than 100 units of
IL-1 at a final dilution of 1:100 in the thymocyte co-stimulating assay. The p53 monoclocal antibody PAb1801 (Ab-2) was purchased from
Oncogene Science (Cambridge. MA). The polyclonal antibody against human
Fas was from Calbiochem. TPA was purchased from Sigma. A selective PKC
inhibitor, GF-109203X (36), and a selective inhibitor of MAPK kinase
(MEK), PD-98059 (37), were purchased from RBI (Natick, MA) and BIOMOL
(Plymouth Meeting, PA), respectively.
Analysis of DNA Fragmentation--
DNA was isolated from cells
treated with or without TPA as described previously (38). Cells were
washed with phosphate-buffered saline without Ca2+ or
Mg2+ (PBS ) twice and then lysed in buffer
containing 0.1 M Tris-HCl, pH 7.4, 50 mM NaCl,
10 mM EDTA, 0.2% SDS, and incubated with 1 mg/ml proteinase K (Sigma) overnight at 37 °C. After extraction by
phenol/chloroform and precipitation in ethanol, the precipitates were
treated with RNase A (Sigma, 56 µg/ml) at 37 °C for 2 h in TE
buffer (10 mM Tris-NaCl, pH 8.0, 1 mM EDTA).
The DNA was extracted with phenol/chloroform and resuspended in TE
buffer. The same amount of DNA for each sample was loaded and
electrophoresed in a 2% agarose gel with TAN buffer (1.6 M
Tris-HCl, pH 7.2, 0.8 M NaOAc, 40 mM EDTA) and the gel was stained with ethidium bromide.
Quantitative Analysis of Apoptosis--
Cells were harvested,
then fixed with 1% glutaraldehyde in PBS at room
temperature overnight. Samples were washed with PBS and
stained with 8 µg/ml of DNA-binding dye Hoechst 33258 (39). Cells
were examined under an Axioplan microscope (Carl Zeiss, Oberkochen,
Germany) and cells characterized by nuclear fragmentation and chromatin
condensation were defined as apoptotic cells.
Isolation and Blotting of RNA--
Total RNA from cells was
obtained by the guanidinium/hot phenol method (40, 41). Cells were
lysed in a guanidinium isothiocyanate mixture (4 M
guanidinium isothiocyanate, 50 mM Tris-HCl, pH 7.6, 20 mM EDTA, 2% sodium lauryl sarcosinate, and 140 mM 2- -mercaptoethanol). The lysed cells were treated
with proteinase K, and then their total RNA was extracted by
phenol/chloroform. After denaturation at 65 °C, RNA was
electrophoresed in a 1% agarose-formaldehyde gel and transferred to a
nylon membrane filter (Hybond-N; Amersham Pharmacia Biotech) (42).
Filters were hybridized with 32P-labeled probe for 16-24 h
at 42 °C, in 50% formamide, 0.1% SDS, 10% dextran sulfate, 100 µg/ml salmon sperm DNA, 2× SSC (1× SSC, 150 mM NaCl, 15 mM sodium citrate), and 5× Denhardt's solution. Filters
were washed to a stringency of 0.1× SSC at 65 °C and exposed to
x-ray film (RX, Fuji Photo Film Co. Ltd., Kanagawa, Japan). Autoradiograms were developed at different exposures.
DNA Probes--
Human TNF cDNA probe (0.8 kb,
EcoRI) and IL-1 cDNA were from pSPl 42-2 and PA 26, respectively (43, 44), and the p53 cDNA was purified from the
pR4-2 plasmid (0.5 kb, NcoI). For TNFR1 probe, the two
20-mer oligodeoxynucleotide, the forward primer (5'-TCCCCTCCCACCTTCTCTCC-3') and the reverse primer
(5'-GCCCACCAGCCCACTCTTCC-3') were synthesized according to the
nucleotide sequence of human TNFR1 DNA (45). The 333-base pair DNA
fragment corresponding to exon 1 of the human TNFR1 gene was amplified
from genomic DNA of human granulocytes by polymerase chain reaction in
a Thermal Cycler MP (Takara, Tokyo, Japan) using EXTaq DNA-polymerase
(Takara). The resulting polymerase chain reaction product was subcloned into pCR 2.1 vector (Invitrogen, Inc., Carlsbad, CA). DNA sequence analysis of the cloned DNA confirmed that it contained the complete sequence for exon 1 of human TNFR1 DNA (45). For Northern blotting, a
0.3-kb DNA fragment was excised by digesting the exon 1 fragment with
EcoRI. These probes were 32P-labeled by the
random priming method (46). The specific activity was approximately
2 × 108 cpm/µg of DNA.
Western Blot Analysis--
Cells were lysed in buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaN3,
0.1% SDS, 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin,
1% Nonidet P-40, and 0.5% sodium deoxycholate). After debris had been
removed by centrifugation, protein concentrations were measured by the
method of Bradford (Bio-Rad) (47). Equal amounts of protein were
dissolved in SDS-polyacrylamide gel electrophoresis sample loading
buffer and electrophoresed in a polyacrylamide gel (12%). On
transferral to a polyvinylidene difluoride membrane (Immobilon,
Millipore, Bedford, MA), immunoblotting was performed using rabbit
anti-human TNF antibody (10 µg/ml, final concentration). After
washing the blots, alkaline phosphatase-conjugated goat anti-rabbit
immunoglobulin G (IgG) diluted 1:2,000 was added. Immunoreactivity on
blots was detected by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Life Technologies, Inc.) staining.
Construction of Expression Vectors--
A plasmid bearing the
complete coding region of human 55-kDa TNF receptor (TNFR1 in pUC19)
was provided by Dr. H. Loetscher (F. Hoffmann-La Roche Ltd., Basel,
Switzerland). An expression vector of human TNFR1 was constructed as
described previously (48). Briefly, TNFR1 cDNA was cloned in the
pRC/RSV eukaryotic expression vector (Invitrogen, Carlsbad, CA) that
contains Rous sarcoma virus long terminal repeat (RSV-LTR) and the
neomycin resistance gene (Neo) expressed under the SV40 promoter. For
control, N-terminal deletion mutant of TNFR1 was generated by digestion of a TNFR1-expression vector with HindIII, and subsequent
self-ligation (Fig. 5A). In this vector, approximately 240 amino acids were removed from the N-terminal end, resulting in lack of
extracellular domain and transmembrane domain sequences.
DNA Transfection and Selection of Transformed Cells--
2 µg
of plasmid DNA was introduced into 5 × 105 of U937
cells using FuGENE6TM, lipofection reagent (Roche Molecular
Biochemicals). 48 h after exposure to DNA, cells were cultured on
two-layer soft agar in selection medium supplemented with 0.5 mg/ml of
G418 for 4 weeks. Single colonies of G418-resistant cells were
isolated, cultured in complete medium supplemented with 0.5 mg/ml of
G418 and then screened for levels of the expression of TNFR1 mRNA
by Northern blotting using a TNFR1 cDNA probe as indicated in Fig.
5A. These transformants were maintained in complete medium
containing 0.3 mg/ml of G418.
MEK Kinase Assay--
MEK kinase activity was determined by
in vitro phosphorylation of MAPK proteins (49, 50). Cells
were lysed in ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM
Na3VO4, 1 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride) and sonicated. Debris were removed by
centrifugation, and the supernatants were subjected to
immunoprecipitation with a rabbit polyclonal MEK1/2 (Ser-217/221)
antibody (Phospho-MEK1/2, New England Biolabs, Inc, Beverly, MA), which
is specific for phosphorylated MEK1/2 at Ser-217/221. After each
immunoprecipitation mixture was incubated with protein A-Sepharose
beads (Amersham Pharmacia Biotech) at 4 °C for 3 h, the immune
complex was washed twice in the lysis buffer and twice in kinase buffer
(25 mM Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2). The beads were resuspended in the kinase buffer supplemented with 200 µM ATP and incubated at 30 °C
for 30 min in the presence of 2 µg of an inactive recombinant
p42MAPK (ERK2) as substrate (New England
Biolabs). Reactions were terminated by adding SDS-polyacrylamide gel
electrophoresis sample loading buffer and boiled for 5 min. After
centrifuged, the supernatants were electrophoresed in a 12% acrylamide
gel. Phosphorylated MAPK (ERK2) was detected by immunoblotting using a
mouse monoclonal antibody against p44/42 MAPK (ERK1 and ERK2) activated
by phosphorylation at Thr-202 and Tyr-204. (IgG, Phospho-p44/42 MAPK,
New England Biolabs).
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RESULTS |
TPA Induces Apoptosis in U937 Cells--
Cells were treated with
various concentrations of TPA. After 8 h, DNA was extracted from
cells and electrophoresed in 2% agarose gel. Analysis of genomic DNA
revealed a fragmentation pattern, characteristic of apoptosis, upon
treatment with as low a concentration as 0.02 nM of TPA and
distinct nucleosome ladders at 0.2 or 2 nM of TPA (Fig.
1, left panel).

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Fig. 1.
TPA induces apoptosis in U937 cells.
Left panel, dose-dependent effect of TPA on DNA
fragmentation. Cells were cultured for 8 h with TPA at various
concentrations. Genomic DNA (5 µg/lane) was prepared and
electrophoresed in a 2% agarose gel as described under "Materials
and Methods." Right panel, time-dependent
effect of TPA on DNA fragmentation. Cells were cultured with 2 nM TPA for various durations.
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To determine the kinetics of apoptosis by TPA, cells were treated with
2 nM of TPA and harvested sequentially at several different time points. DNA fragmentation was observed within 2 h after
treatment with TPA and maximum induction of apoptosis occurred at
8 h (Fig. 1, right panel).
Induction of TNF mRNA Expression by TPA in U937 and KY821
Cells--
To study the effect of TPA on TNF expression, we
performed Northern blot analysis of total RNA using
32P-labeled TNF cDNA probe (Fig.
2). U937 and KY821 cells were treated
with TPA at a concentration of 2 nM and harvested
sequentially at different time points. The time course study showed
that TNF mRNA expression was induced after 1 h with a peak at
4 h or 8 h after treatment in U937 cells (Fig. 2, left
panel). On the other hand, TPA slightly increased the levels of
TNFR1 mRNA at detectable levels. TPA also increased levels of
IL-1 RNA in these cells; an increase was detected at 4 h after
exposure to TPA, and thereafter the induction of IL-1 mRNA
occurred almost in a time-dependent fashion.

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Fig. 2.
Induction of TNF mRNA expression by TPA in U937 and KY821 cells. Cells
were cultured with TPA at a concentration of 2 nM for
various durations. Total RNA (30 µg/lane) was prepared, analyzed by
formaldehyde-agarose gel electrophoresis, and transferred to a nylon
membrane. Hybridization was performed with 32P-labeled
TNF cDNA (1.7 kb), TNFR1 cDNA (2.1 kb), and IL-1 cDNA
(1.6 kb). The bottom panel shows the ethidium
bromide-stained formaldehyde gel before Northern blotting; levels of 28 S and 18 S ribosomal RNA were comparable in each lane.
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We also studied the levels of these mRNAs in TPA-treated KY821
cells (Fig. 2, right panel). The results obtained were
similar to those in U937, although levels of TNFR1 were markedly
increased and an increased level of IL-1 mRNA was observed at
1 h in KY821 cells.
TPA Induces TNF Production in U937 Cells--
We determined
whether TPA affected translation of TNF in U937 cells (Fig.
3A). The U937 cells were
cultured for 8 h with TPA at different concentrations, and the
levels of TNF in the culture supernatants were determined by Western
blotting using a TNF polyclonal antibody (Fig. 3A).
Supernatants from untreated U937 cells did not contain TNF protein
at a detectable level. An increased level of TNF was observed at a
concentration of 0.02 nM, and TPA stimulated the production
in a dose-dependent manner. The level of TNF in cells
treated with 20 nM TPA was approximately 10 times that of
cells treated with 0.02 nM TPA. Studies of TNF
production using enzyme-linked immunosorbent assay were also performed.
The results obtained were almost identical with those described above
(data not shown). In parallel, cells were lysed and determined for
expression of Fas or p53 (Fig. 3B). However, TPA did not
affect expression of Fas or induce production of p53 in these cells.
Northern blot analysis also showed that p53 expression was not detected
in U937 cells, and TPA did not induce p53 in these cells (Fig.
3C).

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Fig. 3.
TPA induces TNF production in U937cells. Panel A, cells were cultured
for 8 h with TPA at various concentrations. Once harvested, the
culture supernatants were electrophoresed in a 12% SDS-polyacrylamide
gel and transferred to a polyvinylidene fluoride membrane. Western
blotting was performed using anti-TNF antibody. Panel B,
cells were lysed as described under "Materials and Methods" and
effects of TPA on Fas and p53 expression were determined by Western
blot analysis. As positive control for p53, a hepatoma cell line
SK-HEP-1 was used (last lane). Panel C, cells
were cultured with different concentrations of TPA for 4 h.
Northern blotting was performed with 32P-labeled p53
cDNA (2.8 kb). SK-HEP-1, which is a hepatoma cell line, was used a
positive control.
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TPA Induces Apoptosis through Production of TNF --
To
investigate the mechanisms of apoptosis induced by TPA, U937 cells were
preincubated with a neutralizing antibody against human TNF for 30 min, which neutralizes 1,000 units/ml of TNF (Fig.
4A). Then 2 nM TPA
was added, and the cells were cultured in the presence of anti-TNF
antibody. After 8 h, cells were harvested, and quantitative
analysis of apoptosis by Hoechst staining was performed using
cells cultured in medium alone as a control. Neutralization of the
endogenous TNF with anti-TNF antibody almost completely blocked
the induction of TPA-induced apoptosis. In parallel, cells were
cultured with TNF for 8 h. Exogenously added TNF induced apoptosis in a dose-dependent fashion (Fig. 4B).
TPA also induced expression of IL-1 in U937 cells as shown in Fig.
2. However, pretreatment of the cells with anti-IL-1 antibody for 30 min failed to inhibit the induction of apoptosis by TPA in these cells (Fig. 4C). Treatment of KY821 cells with anti-TNF
antibody also inhibited apoptosis induced by TPA (Fig.
4D).

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Fig. 4.
TNF is required for
TPA-induced apoptosis. Panels A and C, U937 cells
were treated for 8 h with TPA at a concentration of 2 nM. In parallel, cells were pretreated for 1 h with
either anti-TNF antibody that neutralizes 1,000 units/ml of TNF (panel
A) or anti-IL-1 antibody that neutralizes 100 units/ml of
IL-1 (panel C). After adding 2 nM of TPA,
cells were further cultured for 8 h in the presence of either
antibody. Panel B, U937 cells were cultured for 8 h
with different concentrations of TNF . Panel D, KY821
cells were treated with TPA with or without anti-TNF antibody as
described in panel A. Quantitative analysis of apoptosis was
performed by staining with Hoechst 33258.
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Apoptosis Induced by TPA in TNFR1 Gene-transfected U937
Cells--
To further clarify the role of TNF in apoptosis induced
by TPA, TNFR1 gene was stably transfected into U937 cells (Fig.
5A). The level of TNFR1
expression was determined by Northern blot analysis. We obtained
several clones with different levels of TNFR1 mRNA. Fig.
5B shows one of these clones, and this clone had a 6-fold
higher level of TNFR1 mRNA as compared with that of untreated U937
cells or control-transfected cells. We studied apoptosis in cells
overexpressed with TNFR1. Control-transfected cells and cells
transfected with TNFR1 were exposed to 2 nM of TPA for
8 h; quantitative analysis of apoptosis was performed by Hoechst
staining (Table I). Transfection of cells
with TNFR1 resulted in induction of apoptosis as compared with
untreated cells (p < 0.05). Furthermore, cells
overexpressed with TNFR1 underwent apoptosis following exposure to TPA
more extensively as compared with that in control-transfected
cells.

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Fig. 5.
TPA-induced apoptosis in U937 cells highly
expressing TNFR1. Panel A, structure of the expression
vectors. The full-length TNFR1 cDNA was ligated into
BstXI site of the pRC/RSV expression vector containing Rous
sarcoma virus-long terminal repeat (RSV-LTR) and the
neomycin resistance gene (Neo) expressed under the SV40
promoter (SV40). As control, N-terminal deletion mutant of a
TNFR1 gene was introduced into cells. The dotted region
indicates deleted sequences. Panel B, Northern blot analysis
of cells transfected with an expression vector of TNFR1. Levels of
TNFR1 mRNA were determined using a TNFR1 cDNA probe as
indicated. The bottom panel shows the ethidium
bromide-stained formaldehyde gel. Panel C, levels of
overexpressed TNFR1 in U937 cells. Levels of TNFR1 mRNA were
quantified by normalization to the amount of Neo specific
transcripts.
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Table I
TPA-induced apoptosis in U937 cells transfected with TNFR1 gene
Cells were treated with 2 nM of TPA for 8 h, and
apoptosis was determined by Hoechst staining. The data represent the
mean ± S.D. of results from three independent experiments.
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Role of the PKC and MEK Pathways in Induction of Apoptosis by
TPA--
Many extracellular stimuli including growth factors and
stresses result in activation of phosphorylation cascades using MAPK. To examine the role of MAPK cascade in the production of TNF and the
induction of apoptosis by TPA, we used a specific inhibitor of MAPK
kinase (also known as MEK), PD-98059. This compound is specific for
MEK; it inhibits both the phosphorylation and activation MAPKs with no
inhibitory activity against a number of other serine/threonine and
tyrosine kinases (37). To examine the ability of the compound to
inhibit MEK kinase activity induced by TPA, U937 cells were preincubated with either 100 µM of PD-98059 or 10 µM of GF-109203X, a specific inhibitor of PKC (36), for
1 h and then cultured with 20 nM of TPA for 8 h
(Fig. 6A). Activation of MEK
kinase by TPA was completely blocked by pretreatment with these
compounds. We next determined whether these compounds inhibit the
production of TNF (Fig. 6B) and apoptosis (Fig.
6C) induced by TPA. Pretreatment with PD-98059 attenuated by
more than 90% the production of TNF induced by TPA. Treatment with
GF-109203X almost completely inhibited the TPA-induced production of
TNF in U937 cells. In parallel, we studied apoptosis in these cells.
Cells were harvested and analyzed for apoptosis by Hoechst staining.
Preincubation of cells with either GF-109203X or PD-98059 almost
completely inhibited the induction of apoptosis by TPA.

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Fig. 6.
Role of PKC or MEK pathway in apoptosis
induced by TPA. Panel A, inhibitory effect of PD-98059 or
GF-109203X on MEK1/2 kinase activity induced by TPA in U937 cells. U937
cells were incubated with either 100 µM PD-98059 or 10 µM GF-109203X for 1 h, and then cultured with 20 nM of TPA for 8 h. Cell extracts were
immunoprecipitated with a phosopho-MEK1/2 antibody, and MEK was assayed
with a recombinant p42 MAPK (ERK2) protein as a substrate. Kinase
reactions products were separated on a 12% SDS-polyacrylamide gel,
transferred to a polyvinylidene fluoride membrane, and then visualized
by immunoblotting with a phospho-p44/42 MAPK (ERK1/ERK2) antibody.
Panel B, effect of inhibition of PKC or MEK on production of
TNF . After cell lysis, 20 µg of whole cell protein was
electrophoresed in a 12% SDS-polyacrylamide gel and transferred to a
polyvinylidene fluoride membrane. Western blotting was performed using
anti-TNF antibody. Panel C, apoptosis induced by TPA in
cells with MEK or PKC pathway blocked. In parallel, apoptosis was
determined by Hoechst staining. The data are mean ± S.D. from the
results of three experiments.
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DISCUSSION |
The development and maintenance of tissues are achieved by several
dynamically regulated processes that include cell proliferation, differentiation, and programmed cell death (51, 52). When cells are
exposed to external insults, negative regulation of the cell cycle or
apoptosis occurs; cells which fail to repair DNA are eliminated through
apoptosis (53, 54). In this study, we have investigated the mechanisms
of apoptosis by a potent stimulator of PKC, TPA in the human monocytic
cell line U937 and the myeloblastic cell line KY821. Our results show
that TPA induces apoptosis, at least in part, through a pathway which
requires endogenous production of TNF in these cells.
Our study showed that TPA stimulated production of TNF in these
cells, and pretreatment of cells with anti-TNF neutralizing antibody
abrogated apoptosis induced by TPA. Moreover, exogenously added TNF
also induced apoptosis in U937 cells. TPA also increased levels of
IL-1 mRNA in U937cells. IL-1 is a cytokine, which is also
produced by cells of monocytic lineage, and TNF and IL-1 share a
number of biological properties although they are molecularly distinct
cytokines (55). However, treatment with anti-IL-1 antibody did not
affect apoptosis induced by TPA. TNF induces apoptosis through
binding to TNFR1 (56). TNFR1 belongs to the same receptor family as Fas
(APO/CD95) which is known to trigger apoptosis in a number of different
cell types upon ligand binding (57). Studies have shown that both
receptors contain an approximately 70-amino acid domain in the
cytoplasmic region known as the "death domain," which is required
for the cytotoxic effects (58, 59). In this study, TPA increased levels
of TNFR1 mRNA in U937 and KY821 cells. Moreover, the overexpression
of TNFR1 enhanced the apoptosis induced by TPA in U937 cells whereas
TPA did not affect levels of Fas. The cytotoxic effect of TNF on
neoplastic cells has been shown also to be synergistically enhanced by
TPA in vitro (34). Taken together, these results suggest
that endogenous production of TNF plays a crucial role in apoptosis
of these cells.
Death domain receptors including TNFR1 are constitutively expressed in
various types of cells; these receptors are maintained in inactive
state in the absence of ligands (60, 61). Prior studies (60) have
suggested that it is difficult to generate stable cell lines
overexpressing death domain receptors because the overexpression leads
to receptor aggregation and constitutive apoptosis. We stably
transfected U937 cells with a human TNFR1 expression vector and
obtained several clones with different levels of TNFR1 mRNA. The
most highly TNFR1-overexpressing U937 cells showed an approximately
6-fold TNFR1 level, as compared with that of control-transfected cells,
with a slightly but significantly elevated basal level of apoptosis. A
recent study has found a protein that protects against
ligand-independent signaling by TNFR1 and other death domain receptors
(60). This protein, silencer of death domain (SODD), inhibits the
intrinsic self-aggregation properties of the death domain and maintains
TNFR1 in an active monomeric state; TNF reduces levels of silence of
death domain bound to TNFR1 in U937 cells (60). The mechanism
responsible for the overexpression of TNFR1 in our U937 cells remains
to be elucidated but may involve the expression of silence of death domain at a high level in these U937 cells.
Recent studies have demonstrated that an activation of PKC-modulated
apoptosis though results are contradictory; TPA induces apoptosis in
certain cells including U937 cells but inhibits apoptosis induced by
various agents including ceramide (18, 21-23, 62-64). Other studies
have also reported that activation of PKC by TPA prevented apoptosis
induced by TNF in U937 cells (65, 66). These results suggest that
whether apoptosis is induced in response to TPA depends on the type of
cell. In this study, TPA induced apoptosis in U937 cells; these results
were consistent with previous studies (15, 23). We also showed that TPA
stimulated MAPK activity and inhibition of PKC or MAPK pathway almost
completely prevented the production of TNF and the induction of
apoptosis by TPA. Activation of the MAPK signaling pathway via PKC is
an important mechanism for several biological events such as apoptosis, and PKC regulates the MAPK pathway, alone or with other mechanisms (67,
68). Our results indicate that activation of the PKC and MAPK-signaling
cascade leads to the accumulation of TNF , which is required for the
induction of apoptosis by TPA. However, much evidence accumulated
suggests that TPA and TNF exert opposing effects on the induction of
apoptosis in U937 cells (60).
TNF stimulates sphingomyelin degradation with the subsequent
formation of ceramide, and ceramide induces apoptosis in leukemia cells
(69-75). Moreover, there are reports that PKC activation is
antagonistic to ceramide-induced apoptosis (64, 75, 76). Also TPA has
been shown to activate sphingosine kinase, leading to increased
concentrations of sphingosine-1-phosphate (SPP), which is a metabolite
of ceramide and suppresses ceramide-mediated apoptosis (65, 77).
Furthermore, it has been demonstrated that blocking the MAPK pathway
prevented the cytoprotective effect of SPP on TNF -induced
apoptosis in U937 cells (65). SPP stimulates MAPK activity and
activation of MAPK promotes cell survival (77, 78). Taken together,
these studies suggest that activation of MAPK may stimulate different
pathways responsible for the opposing effects on apoptosis and cell survival.
Leukemia cell lines including U937 and HL60 promyelomatic cells consist
of phenotypically and biologically heterogeneous populations of cells
that arrest at different stages of differentiation (79). A recent
report has shown that certain PKC isotypes were down-regulated in
differentiated cells (23). Although we provided evidence that TPA
induced apoptosis through production of TNF in U937 cells, the
mechanism for the discrepancy between studies by other groups and us
remains unclear. Studies suggest that the balance between the
intracellular levels of ceramide and SPP is critical for the fate of
cells (65, 66). Considering these studies, the ceramide/SPP rheostat
may be characteristic of cell type and, also, the
differentiation-stage. Furthermore, the levels of products of PKC
activation, TNF , and SPP may be varied in each U937 cell line,
leading to opposing effects on programmed cell death.
Studies have shown that U937 cells have no wild type p53 transcripts or
protein; a point mutation that converts G into A at the first base of
intron 5 results in a partial deletion of 46 bases from the p53
mRNA (80, 81). Our Western blot detected no p53 protein in U937
cells, and TPA did not induce p53. TNF -induced apoptosis can occur
in cells deficient for p53 or with mutated p53 (82). Thus, TNF may
be of critical importance for apoptosis in U937 cells that lack
functional p53. Furthermore, in this study, untreated U937 cells
constitutively underwent apoptosis to an extent. Moreover, the
constitutive apoptosis was augmented by the overexpression of TNFR1 in
these cells. Whereas the TNF protein in the supernatant of untreated
cells was below the level of undetectability, enzyme-linked
immunosorbent assay showed that cell lysates of untreated U937 cells
contained TNF (0.39 µg/mg protein and 20 pg/106
cells). Endogenously produced TNF may induce apoptosis, in part, through autocrine loops in the endoplasmic reticulum.
 |
ACKNOWLEDGEMENT |
We thank Dr. H. Loetscher (F. Hoffmann-La
Roche Ltd., Basel, Switzerland) for a plasmid containing the complete
coding region of human 55 kDa TNF receptor (TNFR1 in pUC19). We also
thank Sayuri Nakayama and Misayo Tanaka for secretarial assistance and
Ikuko Furusawa for technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by a Grant-in-aid for
Science Research from the Japanese Ministry of Education, Science,
Culture, and Sports, Japan (No. 10670979).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.

To whom correspondence should be addressed: Division of
Radiation Health, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-city, CHIBA, 263-8555 Japan. Tel.:
81-43-206-3122; Fax: 81-43-284-1736; E-mail: akashi@nirs.go.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PKC, protein kinase
C;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
TNF , tumor
necrosis factor- ;
IL-1 , interleukin-1 ;
TNFR1, TNF receptor 1;
MAPK, mitogen-activated protein kinase;
ERK, extracellular
signal-regulated protein kinase;
MEK, mitogen-activated/extracellular-regulated kinase;
kb, kilobase(s);
SPP, sphingosine-1-phosphate;
Rb, retinoblastoma susceptibility gene
product.
 |
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September 1, 2003;
116(17):
3491 - 3501.
[Abstract]
[Full Text]
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L. Cartee, S. C. Maggio, R. Smith, H. M. Sankala, P. Dent, and S. Grant
Protein Kinase C-dependent Activation of the Tumor Necrosis Factor Receptor-mediated Extrinsic Cell Death Pathway Underlies Enhanced Apoptosis in Human Myeloid Leukemia Cells Exposed to Bryostatin 1 and Flavopiridol
Mol. Cancer Ther.,
January 1, 2003;
2(1):
83 - 93.
[Abstract]
[Full Text]
[PDF]
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S. Wang, Z. Wang, and S. Grant
Bryostatin 1 and UCN-01 Potentiate 1-beta -D-Arabinofuranosylcytosine-Induced Apoptosis in Human Myeloid Leukemia Cells through Disparate Mechanisms
Mol. Pharmacol.,
January 1, 2003;
63(1):
232 - 242.
[Abstract]
[Full Text]
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O. Sordet, C. Rebe, S. Plenchette, Y. Zermati, O. Hermine, W. Vainchenker, C. Garrido, E. Solary, and L. Dubrez-Daloz
Specific involvement of caspases in the differentiation of monocytes into macrophages
Blood,
December 15, 2002;
100(13):
4446 - 4453.
[Abstract]
[Full Text]
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J.-M. Lai, S. Wu, D.-Y. Huang, and Z.-F. Chang
Cytosolic Retention of Phosphorylated Extracellular Signal-Regulated Kinase and a Rho-Associated Kinase-Mediated Signal Impair Expression of p21Cip1/Waf1 in Phorbol 12-Myristate-13- Acetate-Induced Apoptotic Cells
Mol. Cell. Biol.,
November 1, 2002;
22(21):
7581 - 7592.
[Abstract]
[Full Text]
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L. Cartee, R. Smith, Y. Dai, M. Rahmani, R. Rosato, J. Almenara, P. Dent, and S. Grant
Synergistic Induction of Apoptosis in Human Myeloid Leukemia Cells by Phorbol 12-Myristate 13-Acetate and Flavopiridol Proceeds via Activation of Both the Intrinsic and Tumor Necrosis Factor-Mediated Extrinsic Cell Death Pathways
Mol. Pharmacol.,
June 1, 2002;
61(6):
1313 - 1321.
[Abstract]
[Full Text]
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X. Hu, W. E. Janssen, L. C. Moscinski, M. Bryington, A. Dangsupa, N. Rezai-Zadeh, B. A. Babbin, and K. S. Zuckerman
An I{kappa}B{alpha} Inhibitor Causes Leukemia Cell Death through a p38 MAP Kinase-dependent, NF-{kappa}B-independent Mechanism
Cancer Res.,
August 1, 2001;
61(16):
6290 - 6296.
[Abstract]
[Full Text]
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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