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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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 FuGENE6^TM, 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 p42^MAPK (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).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

View larger version (87K): [in this window] [in a new window]   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.

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.

View larger version (78K): [in this window] [in a new window]   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.

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).

View larger version (42K): [in this window] [in a new window]   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.

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).

View larger version (16K): [in this window] [in a new window]   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.

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.

View larger version (28K): [in this window] [in a new window]   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.

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.

View larger version (31K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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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