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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2369-2379
By
From the Division of Medical Oncology and Hematology, the Department
of Internal Medicine and the Department of Biochemistry and Molecular
Biology, University of South Florida College of Medicine, and Molecular
Oncology and Hematologic Malignancy Programs, H. Lee Moffitt Cancer
Center and Research Institute, Tampa, FL.
The factor-independent Dami/HEL and Meg-01 and factor-dependent Mo7e
leukemic cell lines were used as models to investigate JAK/STAT signal
transduction pathways in leukemic cell proliferation. Although Dami/HEL
and Meg-01 cell proliferation in vitro was independent of and
unresponsive to exogenous cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), and tumor necrosis factor-
SURVIVAL, PROLIFERATION, and
differentiation of normal hematopoietic cells are controlled by a
number of growth factors that interact with members of the
hematopoietic growth factor receptor family or with one of several
receptor tyrosine kinases. In contrast, leukemic cells or leukemic cell
lines frequently grow in a factor-independent manner. The mechanisms
that result in the conversion of factor-dependent cells to
factor-independent cells are poorly understood. Some investigators have
reported that mutations of growth factor receptor(s) or signal
transduction proteins cause constitutive activation of signal
transduction pathways and induce growth factor-independent cell
proliferation.1-4 Autocrine growth factor-stimulated growth
of human leukemic cells or cell lines due to aberrant cytokine
production by the malignant cells has been detected
infrequently.5-7
The signal transduction pathways triggered by growth factor ligands
binding to cell surface receptors are involved in the reversible
phosphorylation of receptors, cytosolic nonreceptor kinases, and other
cellular proteins. Among the many known signal transduction pathways,
the JAK (Janus kinase)/STAT (signal transducer and activator of
transcription) cascade and the MAPK (mitogen-activated protein kinase)
cascade are activated after exposure of megakaryocytic cells to
cytokines.8-12 Although the significance of
cytokine-induced activation of these signaling pathways remains under
active investigation, several reports have shown that phosphorylation
and activation of JAK and STAT proteins appears to play a critical role
in thrombopoietin-induced signal transduction.8,13,14 JAK
proteins are members of a family of cytoplasmic kinases, which is known
to have four members: TYK2, JAK1, JAK2, and JAK3. JAK2 is activated in
normal megakaryocyte precursors and megakaryocytic cell lines by
several specific hematopoietic cytokines and has been reported to be
required for megakaryocytic cell proliferation.14,15 The
STATs, of which at least seven distinct family members and several
alternative RNA splicing-derived variants have been described so far,
are cytosolic transcription factors, which are tyrosine-phosphorylated
after ligand activation.16 They dimerize and translocate
into the nucleus, where they bind to specific DNA sequences and
activate gene transcription. Originally, STAT5, which was described as
a transcription factor activated by prolactin and required for the
induction of transcription of milk caseins, now has been identified as
two closely related proteins, STAT5a and STAT5b.17 Recent
studies indicate that they are widely expressed in many
tissues.18 STAT5 is known to be activated by the JAKs and
Bmx tyrosine kinases,19,20 and the activated STAT5 dimers
recognize and bind to a specific palindromic DNA sequence,
TTCNNNGAA.16 This oligonucleotide sequence is found not
only in the In this study, we have investigated the activation of the JAK2/STAT5
signal transduction pathway in growth factor-dependent and
factor-independent megakaryocytic cell lines. We found that the
JAK2/STAT5 signaling pathway is activated constitutively in factor-independent Dami/HEL and Meg-01 cells, but is transiently cytokine-induced in factor-dependent Mo7e cells. The constitutive activation of the JAK2/STAT5 signaling pathway appears to correlate with the factor-independence of Dami/HEL and Meg-01 cell lines. Although BCR/ABL oncogene transcripts are detected in Meg-01 cells, Dami/HEL cells do not express the BCR/ABL oncogene. Constitutive phosphorylation of Raf-1 occurs in Dami/HEL, but not Meg-01 cells. Thus, constitutive activation of JAK/STAT5 and proliferation may be
triggered by different mechanisms in Dami/HEL and Meg-01 cells.
Reagents.
Recombinant human thrombopoietin (TPO), interleukin-3 (IL-3), IL-6,
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
epidermal growth factor (EGF) were purchased from PeproTech (Rocky
Hill, NJ). Anti-IL-3 and anti-TPO neutralizing antibodies were
purchased from R & D Systems (Minneapolis, MN). Antiphosphotyrosine antibody-agarose and anti-JAK2 antiserum were purchased from Upstate Biotechnology (Lake Placid, NY). Phosphotyrosine Western blotting kit
(chemiluminescence) was purchased from Boehringer Mannheim Biochemicals
(Indianapolis, IN). Antigoat IgG and antimouse IgG antibodies labeled
with fluorescein isothiocyanate were purchased from Zymed (South San
Francisco, CA). Anti-STAT1, -STAT2, -STAT3, -STAT4, -STAT5 -STAT6, and
anti-Raf-1 antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). PhosphoPlus p44/p42 MAPK antibody kit containing
antitotal and anti-T202/Y204 phosphorylated p42/p44 MAPK antibody were
purchased from New England Biolabs (Beverly, MA).
[Methyl-3H]-thymidine ([3H]-TdR; specific
activity, 70 to 86 Ci/mmol) and [32P]-deoxyadenosine
triphosphate (dATP) (specific activity, >3,000 µCi/mmol) were purchased from Amersham Life Science (Arlington Heights, IL).
Cell lines.
Human megakaryocytic leukemic cell line, Dami, originally described by
Greenberg et al,25 was obtained from American Type Culture
Collection (ATCC, Rockville, MD) and was maintained in Iscove's Modified Dulbecco's Medium (IMDM; GIBCO-BRL, Grand Island, NY) containing 10% horse serum. Recently, ATCC and DSMZ (German Collection of Microorganisms and Cell Cultures) have determined that
all samples of the Dami cell line that were available for them to
analyze were genetically and karyotypically identical to the human
erythroleukemia cell line (HEL) described previously,26 which has both erythroid and megakaryocytic
characteristics.27 For clarity and for comparison to prior
reports, we are designating this cell line as Dami/HEL in this report.
Human megakaryoblastic leukemic cell line, Meg-01, originally described
by Ogura et al,28 was obtained from ATCC and was maintained
in RPMI 1640 medium (GIBCO-BRL) with 10% heat-inactivated fetal bovine
serum (FBS). Human factor-dependent megakaryoblastic leukemic cell
line, Mo7e, originally described by Avanzi et al,29 was
obtained from Genetics Institute (Boston, MA) and was maintained in
IMDM with 10% FBS, 1% L-glutamine and 5 ng/mL GM-CSF. All of the
cells were incubated at 37°C in a fully humidified atmosphere with
5% CO2 in medium alone or in the presence of various
concentrations of cytokines. In experiments to detect the effects of
known cytokines or potential cytokines in Dami/HEL or Meg-01
conditioned media, Mo7e and TF-1 cells were prepared by washing cells
three times with medium and starved for 18 hours in medium without
growth factors before cytokine or conditioned medium treatments, as
described by Dusanter-Fourt et al.30 Dami/HEL cells were
cultured in IMDM without serum, but with 1X Nutridoma-HU (Boehringer
Mannheim Biochemicals), and Meg-01 cells in RPMI 1640 with 1X
Nutridoma-HU in experiments in which cytokine production and specific
cytokine effects were being studied.
Effects of cytokines on cell growth.
To determine the proliferative response of cells to cytokines, DNA
synthesis was measured by [3H]-thymidine (TdR)
incorporation. The assays were performed in triplicate, using 4 × 105 cells for Mo7e and Meg-01 or 2 × 105
cells for Dami/HEL cells. Mo7e cells in IMDM with 10% FBS and Dami/HEL
and Meg-01 cells in medium with 1X Nutridoma-HU were cultured for 72 hours without or with cytokines. Subsequently, the cells were labeled
with 2 µCi/mL of [3H]-TdR for an additional 4 hours.
Incorporation of [3H]-TdR into newly synthesized DNA
(counts per minute; cpm) was determined by liquid scintillation
counting, according to a previously described protocol.31
Immunoprecipitation and Western blotting.
Unstimulated or cytokine-stimulated cells were lysed in modified
radioimmunoprotection assay (RIPA) buffer with 1% NP-40
and extracts were immunoprecipitated as described.30 The
detergent-soluble proteins were incubated with antiphosphotyrosine
agarose for 3 hours at 4°C with shaking. The immunoprecipitates
were washed four times with modified RIPA buffer, lysed in sodium
dodecyl sulfate (SDS) buffer and separated by 7.5% nonreducing
SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were then
transferred to nitrocellulose membrane and probed sequentially with
rabbit anti-JAK2 antibody and antirabbit IgG antibody labeled with
peroxidase. Phosphorylated JAK2 was visualized with enhanced
chemiluminescence techniques according to the manufacturer's
recommended procedure. For the detection of phosphorylation of Raf-1, a
band shift assay was used in Western blot using an anti-Raf-1 antibody
(Santa Cruz Biotechnology) following a previously reported
procedure.33 For detecting the phosphorylation of cellular
proteins, cells treated with or without cytokines were lysed in SDS
buffer, and 40 to 80 µg of cellular proteins was loaded in each
sample lane, separated by 10.5% SDS-PAGE. For detecting total and
phosphorylated MAPK, the SDS-PAGE-separated proteins were transferred
to polyvinylide difluoride (PVDF) membranes and probed
with antitotal and antiphosphorylated p42/p44 MAPK, with
antiphosphorylated STAT3 antibody (New England Biolabs), or with
antiphosphotyrosine antibody (Boehringer Mannheim).
Preparation of nuclear extracts and gel mobility shift assays.
Preparation of nuclear extracts and electrophoretic gel mobility shift
assay (EMSA) was performed according to methods described previously.34 Briefly, equal amounts of nuclear proteins (5 to 10 µg) for each sample were incubated for 30 minutes at 30°C with 10,000 dpm of [32P]-labeled double-strand DNA
fragment (IRF-1 GAS; 5'-GCCTGATTTCCCCGAAATGACGGCA) containing the
interferon- Reverse transcription-polymerase chain reaction (RT-PCR).
Cellular mRNA was prepared with RNAzol (Cinna/Biotecx Lab, Houston, TX)
following the protocol suggested by the manufacturer. A commercially
available primer set (TransPrimers, Calbiochem, CA) was used to assess
the presence of BCR/ABL transcripts. RT-PCR for normal c-ABL also was
performed to check RNA quality. A RNA PCR Core kit (GeneAmp; Perkin
Elmer, Branchburg, NJ) was used to perform RT-PCR following the
manufacturer's protocol. A 1.8% agarose gel was used for
electrophoretic analysis of the PCR products. RT-PCR was performed at
least twice to confirm the findings.
Bioassays of stimulatory cytokines.
Factor-independent Dami/HEL and Meg-0l cells were examined for the
capacity to produce autocrine cytokines. Mo7e and TF-1 cell lines, the
survival of which depend on the presence of any of several growth
factors, including GM-CSF, IL-3, IL-6, IL-9, TPO, steel factor/c-kit
ligand, tumor necrosis factor- Dami/HEL and Meg-01 cells grow in a factor-independent manner.
To determine effects of cytokines on DNA synthesis of the proliferating
cells, various concentrations of cytokines were incubated with
Dami/HEL, Meg-01, and Mo7e cells for 3 days, and the cells were labeled
subsequently with [3H]-TdR for the final 4 hours of
culture. When 0 to 200 ng/mL of TPO was incubated with the
cytokine-independent cell lines, Dami/HEL and Meg-01, no significant
stimulation of their basal [3H]-TdR incorporation was
observed (Fig 1, Dami/HEL and Meg-01). The
cells also were tested for their response to other cytokines, including
GM-CSF, IL-3, IL-6 and TNF-
STAT-like DNA-binding factors are activated constitutively in
Dami/HEL and Meg-01 cells.
The fact that the Dami/HEL and Meg-01 cell lines grow in a
factor-independent manner led us to investigate whether an aberrant signal transduction pathway might be involved. Because JAK and STAT
proteins have been reported to have a role in cytokine-induced proliferation of megakaryocytic and leukemic cells,8 we
analyzed STAT DNA-binding factor activation by EMSA with a
[32P]-labeled oligonucleotide containing the IRF-1 GAS
consensus STAT binding site, as the probe. DNA-binding protein(s) were
detected in the nuclear extracts from Dami/HEL and Meg-01 cells in the absence of cytokine exposure (Fig 2).
Addition of as much as 400 ng/mL of TPO or 40 ng/mL IL-3 did not have
any further effect on the constitutive DNA-binding protein activity in
these growth factor-independent cell lines (Fig 2). Exposure of these
cells to GM-CSF, IL-6, erythropoietin (EPO), or TNF-
The constitutive and cytokine-activated STAT-like factor binds
specifically to DNA containing the IRF-1 GAS sequence.
The question remained whether the constitutively activated STAT-like
DNA-binding factor in Dami/HEL or Meg-01 cells is the same one as that
activated by cytokines in Mo7e cells. We performed experiments to
identify the specific STAT protein activated in these leukemic cell
lines. First, we used a set of oligonucleotides to attempt to inhibit
competitively the binding of the STAT-like factor to
[32P]-labeled IRF-1 GAS. When Dami/HEL nuclear extracts
were coincubated with the labeled probe plus a 50-fold molar excess of
unlabeled FIRE, ISRE, or SIE oligonucleotides, which do not contain
TTCNNNGAA sequence, no competitive inhibition of the formation of
DNA-protein complexes was observed (Fig 4,
Dami). However, a 50-fold molar excess of the unlabeled MGFe, which
contains the same TTCCCCGAA STAT-binding sequence as IRF-1 GAS,
completely abolished the formation of the labeled DNA-protein
complexes. Similar results were found with Meg-01 cells (data not
shown).
The GAS-binding factor activated constitutively in Dami/HEL and
Meg-01 cells and induced transiently by cytokine in Mo7e cells is
STAT5.
Because different STAT transcription factors may share the same
binding site to some of the DNA probes, the data from the gel mobility
shift assays with IRF-1 GAS as the probe may not confirm specifically
what STAT protein is activated, although the pattern is most consistent
with activated STAT5. To identify the specific DNA-binding factor(s),
gel mobility supershift assays with antibodies to specific STAT
proteins were used to examine the type of STAT(s) involved in the
DNA-protein complexes. When anti-STAT5 antiserum was used, supershift
of the DNA-protein complexes was observed with nuclear extracts from
Dami/HEL and Meg-01 cells cultured in medium without cytokines or from
TPO-treated Mo7e cells (Fig 5), but not in
untreated Mo7e cells (data not shown). When anti-STAT3 antiserum was
used, no supershift of DNA-protein complexes occurred in TPO-treated
Mo7e or unstimulated Dami/HEL or Meg-01 cells (Fig 5). However,
anti-STAT1, anti-STAT3, and anti-STAT5 antibodies induced supershift of
the DNA-protein complex in nuclear extracts from EGF-treated SK-BR-3
cells (Fig 5), in which STAT1, STAT3, and STAT5 have been shown
previously to be activated.36 Reactions of the DNA-protein
complexes with anti-STAT1, anti-STAT2, anti-STAT4 and anti-STAT6
antibodies also were investigated, and no supershifted DNA-protein
complexes were observed (data not shown). These results indicate that
the predominant or exclusive constitutively activated (in Dami/HEL and
Meg-01 cells) or TPO- or GM-CSF-induced (in Mo7e cells) STAT
DNA-binding factor in these megakaryocytic leukemic cell lines is
STAT5.
JAK2 is activated constitutively in Dami/HEL and Meg-01 cells.
To examine protein phosphorylation patterns and to identify the
mechanism for constitutive STAT5 activation, Dami/HEL and Meg-01 cells
were incubated in medium without serum, and Mo7e cells were deprived of
growth factors in medium with 10% FBS for 18 hours, and cells were
then exposed to TPO or GM-CSF for 10 minutes. Two growth factor-induced
tyrosine-phosphorylated protein bands of approximately 130 kD and 116 kD were observed in Mo7e cells (Fig 6,
lanes 2 and 3, Mo7e). When Dami/HEL cells were examined, there was no
detectable difference in the protein phosphorylation patterns in
control Dami/HEL cells or cells treated with cytokines (Fig 6, lanes 1 through 3, Dami). Interestingly, the pattern of tyrosine
phosphorylation in untreated Dami/HEL cells was very similar to that of
the cytokine-treated Mo7e cells (compare Fig 6, lane 1, Dami and lanes
2 and 3, Mo7e). The results show that phosphorylated protein bands,
which are undetectable or barely detectable in control Mo7e cells, but
are induced by TPO or GM-CSF, are phosphorylated constitutively in
untreated control Meg-01 (data not shown) and Dami/HEL cells. Because
it has been reported that JAK2 is activated by TPO or other cytokines
in megakaryocytic cells,14,15 we examined the tyrosine
phosphorylation of JAK2 in Dami/HEL, Meg-01, and Mo7e cells. Lysates
from cells treated with or without cytokines were immunoprecipitated
with antiphosphotyrosine agarose. The results show that JAK2 is
phosphorylated constitutively in control Dami/HEL
(Fig 7, lane 1, Dami) and Meg-01 cells
(data not shown). However, JAK2 was unphosphorylated in control Mo7e cells (Fig 7, lane 1, Mo7e) and phosphorylated in cytokine-treated Mo7e
cells (Fig 7, lanes 2 and 3, Mo7e). No further effect on tyrosine-phosphorylation of JAK2 was observed when Dami/HEL cells were
exposed to TPO or IL-3 (Fig 7, lanes 2 and 3, Dami). These results show
that JAK2 is phosphorylated constitutively in the two growth
factor-independent megakaryocytic leukemic cell lines and that its
phosphorylation is induced by TPO and IL-3 in factor-dependent Mo7e
cells.
Proto-oncogene Raf-1 is constitutively phosphorylated in Dami/HEL
cells.
To examine the phosphorylation of Raf-1, Western blot with a specific
anti-Raf-1 antibody was used to examine the band shift of Raf-1. The
band shift of Raf-1 in Western blot has been identified to be induced
by the phosphorylation of Raf-1.33,37 When cell lysates
were prepared from cells treated with or without GM-CSF, and equal
amounts of cellular proteins were subjected to SDS-PAGE, band shift of
Raf-1 was detected by Western blot in Mo7e cells in response to GM-CSF
treatment (Fig 8). In the cell lysates
prepared from Dami/HEL and U937 cells without cytokine exposure,
phosphorylation (band shift) of Raf-1 was detected (Fig 8, Dami and
U937, CTL). Treating Dami/HEL cells with GM-CSF had no further effects
on the phosphorylation of Raf-1 (Fig 8, Dami, GM). In Meg-01 cells without GM-CSF exposure, no significant band shift was observed (Fig 8,
Meg-01, CTL). GM-CSF treatment also failed to induce any significant
phosphorylation of Raf-1 in Meg-01 cells (Fig 8, Meg-01, GM). U937
cells, which have been shown previously to have increased phosphorylation of Raf-1 in cells without cytokine
exposure,37 were used as the control to locate the
phosphorylated Raf-1.
No increased activity of MAP kinase kinase (MEK) is detected in
Meg-01 or Dami/HEL cells.
It has been reported that TPO and other cytokines stimulate both the
MAPK and JAK/STAT signaling pathways in megakaryocytic cells. Because
we observed the constitutive activation of the JAK2/STAT5 signaling
pathway in Dami/HEL and Meg-01 cells and the increased Raf-1
phosphorylation in Dami/HEL cells, it was of interest to see whether
the MEK also was activated constitutively in these cells. Our data show
that all of the cells had a low level of MEK functional activity and no
difference of the MEK activity could be distinguished among Mo7e,
Meg-01, and Dami cells without any cytokine exposure (Fig 8B, lanes 1, 3, and 5). However, a significant increased level of MEK activity was
detected transiently in all three cell lines after treatment with
GM-CSF (lanes 2, 4, and 6), IL-3, TPO, or IL-6 (data not shown).
Furthermore, when phosphorylation of p44/p42 MAPK was investigated in
control and cytokine-treated cells to confirm the cytokine-induced MEK
activation, the data showed that there was minimal phosphorylated MAPK
detectable in the absence of any cytokine, and strong cytokine-induced
phosphorylation of MAPK was observed in all three cell lines (data not shown).
No BCR/ABL oncogene product is detected in Mo7e or Dami/HEL cells.
There are several reports showing that the BCR/ABL chimeric oncoprotein
activates STATs and usually JAKs and induces cytokine-independent growth in cells transfected with the BCR/ABL oncogene.38-40
Our data show no BCR/ABL transcripts detectable in factor-dependent Mo7e cells with or without cytokine exposure
(Fig 9A, lanes 3 and 4). Although
constitutive activation of STAT5 and phosphorylation of JAK2 are
observed in Dami/HEL cells without cytokine exposure, no BCR/ABL
transcript was detected in this cell line, either without (Fig 9, lane
5) or with (Fig 9, lane 6) TPO treatment. BCR/ABL transcripts were
observed in Meg-01 and K562 cell lines (Fig 9, lanes 1, 2, 7, and 8),
both of which are cell lines that were derived from patients with
chronic myelogenous leukemia (CML). In Meg-01 cells, a smaller BCR/ABL
PCR product was detected (consistent with p185 BCR/ABL), compared with
that of K562 (consistent with p210 BCR/ABL) (compare Fig 9, lanes 7, 8 and 1, 2).
No detectable stimulatory factors are secreted by Dami/HEL or Meg-01
cells.
To investigate further the mechanism for the constitutive activation of
JAK2 and STAT5 in Dami/HEL and Meg-01 cells, we performed experiments
to examine whether the cytokine-independent Dami/HEL and Meg-01 cell
lines acquired the capacity to produce cytokines that may result in
autocrine stimulation of cell proliferation and JAK2/STAT5 activation.
The Mo7e cell line, the survival of which is dependent on the presence
of at least one of several growth factors, including GM-CSF, IL-3,
IL-6, IL-9, steel factor/C-kit ligand, TNF-
Our studies show that the JAK2/STAT5 signal transduction pathway is
activated constitutively in the growth factor-independent leukemic cell
lines, Dami/HEL and Meg-01. In contrast, the activation of this
signaling pathway is strictly cytokine-induced transiently in the
growth factor-dependent leukemic cell line, Mo7e. Others also have
reported transient cytokine-dependent activation of the JAK2/STAT
signaling pathway in several growth factor-dependent cell
lines.8-12,15 Although our present study is focused on two growth factor-independent megakaryocytic leukemic cell lines (Dami/HEL cells also have erythroid characteristics), the aberrant signaling pathway could also exist in other factor-independent leukemic cell
lines. Based on the published observation that STAT-related transcription factors are activated constitutively in primary cells
from acute myeloid and lymphoid and chronic myeloid leukemia patients41-44 and given the close relationship between cell
proliferation and the activation of JAK2/STAT5, constitutive activation
of the JAK2/STAT5 pathway may be one of the important mechanisms of
leukemogenesis and of maintaining the leukemic phenotype.
Submitted March 23, 1998; accepted November 23, 1998.
Supported by Grants No. CA56072, CA55652, and P30 CA76292 from the
National Institutes of Health/National Cancer Institute, Bethesda, MD.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Richard Y. Liu, PhD, H. Lee Moffitt Cancer
Center, MRC 3007, 12902 Magnolia Dr, Tampa, FL 33612; e-mail:
liur{at}moffitt.usf.edu.
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TOP
ABSTRACT
INTRODUCTION
(TNF-
-casein promoter, but also in a number of other genes,
such as those encoding interferon regulatory factor-1 (IRF-1), interferon consensus sequence binding protein (ICSBP), rat serine protease inhibitor, human interstitial cell adhesion molecule, high
affinity immunoglobulin G receptor, Oncostatin M, and cytokine-induced SH2 protein (CIS).
activation site (GAS) that binds to interferon
regulatory factor (IRF-1).
70°C with Kodak X-OMAT film (Eastman Kodak, Rochester, NY) and a DuPont Cranex lightning-plus intensifying screen (DuPont, Wilmington, DE).
(TNF-
