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(Received for publication, December 13, 1994; and in revised form, January 26, 1995) From the
Tumor necrosis factor (TNF) is a pleiotropic cytokine which has
both cytotoxic and proliferative effects. HuT 78, a T-cell line derived
from a Sezary lymphoma, is resistant to the cytotoxic effects of TNF,
suggesting that TNF may be a growth factor for this cell line. The aim
of this study was to determine whether autocrine TNF production could
function as a growth factor for HuT 78. Resting HuT 78 and K-4 cells, a
protein kinase C- Tumor necrosis factor The regulation of
production of TNF is complex. There are likely to be four levels of
control: the transcriptional level, the mRNA abundance level, the level
of translation and, finally, posttranslationally. Most attention has
been focussed on regulatory elements responsible for transcriptional
activation. Elements responsive to the transcription factor NF Here, we describe for the first time the
constitutive production of TNF and high levels of constitutively active
NF
TNF
Figure 1:
TNF production by T-cell clones. HuT
78 and K-4 cells were incubated for 48 h in medium and TNF levels in
supernatants measured by L929 bioassay (A) or ELISA (B). Mean ± S.E. of three
experiments.
IL2 was not detected in
supernatants of resting HuT 78, K-4, or Jurkat cells (data not shown).
Figure 2:
Inhibition of proliferation of T-cell
clones with a monoclonal anti-TNF
Figure 3:
Time course of activation of NF
All of the complexes detected in each of the cell types
appeared to constitute protein components which interacted specifically
with the NF
Figure 4:
Analysis of NF
Figure 5:
Effect of a neutralizing monoclonal
antibody to TNF
TNF is a pleiotropic cytokine, which is produced mainly by
activated macrophages and lymphocytes in response to many stimuli,
including bacterial toxins and viruses, as well as cytokines, including
TNF itself(1) . It is also produced by a number of transformed
and neoplastic cells(12, 13, 15) . In this
study, we demonstrated that HuT 78, a T-cell lymphoma, and K-4, a PKC
TNF is a growth factor for
many normal lymphoid and non-lymphoid cell types, including thymocytes,
fibroblasts, chondrocytes, and lymphoid cells (T and B lymphocytes) (5, 7, 46, 47) as well as certain
neoplastic cells, including ovarian cancer (10) and B-cell
chronic lymphoid leukemia(9) . HuT 78 and K-4 are resistant
to the cytotoxic effects of TNF, even at concentrations of up to 30
ng/ml TNF, much higher concentrations than that needed to kill L929
cells (data not shown). Jurkat, which do not produce TNF, are sensitive
to TNF-mediated killing(48) . TNF production by other cell
types which are resistant to cytotoxicity by TNF have previously been
reported(14, 43, 49) , suggesting that TNF
may be an autocrine growth factor for certain cell types. A
monoclonal anti-TNF The identification of two TNF receptors on
cells suggested initially that individual receptors might mediate
individual functions of killing and proliferation(51) .
However, both receptors have since been found to mediate both
proliferation and cytotoxicity(52) , therefore both may trigger
proliferation in HuT 78. Once TNF binds to receptors on cells, its
intracellular signaling pathways are complex. TNF has been reported to
activate multiple protein kinases and transcription factors, including
NF Although lymphotoxin is
detected by the L929 bioassay(55) , it is unlikely to be
relevant here as an autocrine growth factor for HuT 78, as the
monoclonal antibody producing virtually complete inhibition of cell
proliferation was specific for TNF Attempts to identify proteins within the
NF An autocrine role for TNF in
NF In conclusion, this study demonstrates that proliferation
of HuT 78 is stimulated by the autocrine production of TNF. This also
results in the activation of NF
Volume 270,
Number 13,
Issue of March 31, 1995 pp. 7399-7404
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
B Are Induced by Autocrine
Production of Tumor Necrosis Factor 
in the Human T Lymphoma Line
HuT 78 (*)
-deficient clone of HuT 78, both produced
significant amounts of TNF compared with Jurkat cells. Thymidine
incorporation by HuT 78 and K-4 cells was inhibited by 90.5 and 73.2%,
respectively, with addition of a neutralizing monoclonal antibody to
TNF
, suggesting that TNF is an autocrine growth factor for these
cells. HuT 78 and K-4 cells also expressed high levels of
constitutively active NF
B, unlike Jurkat cells, which expressed
high levels only upon activation with TNF or phorbol 12-myristate
13-acetate. p50 was the major component in the NFB complexes in
HuT 78 and K-4 cells. Anti-TNF antibody dramatically decreased
levels of NFB in both HuT 78 and K-4 cells. As the TNF gene has an
NFB binding motif, an autocrine loop involving TNF induction of
NFB is therefore likely in these cells. These findings in a
neoplastic T-cell line suggest that therapy directed against TNF could
be effective in a subset of T-cell lymphomas.
(TNF) (
)is a potent
cytokine with a wide range of biological activities(1) . It was
initially described in serum of endotoxin-treated mice as the mediator
of the necrosis of some transplantable tumors(2) . It has since
been reported to be cytotoxic to certain transformed cell lines (3, 4, 5) and may act either as a mediator in
beneficial processes of host defense, immunity, and tissue homeostasis
or in the pathogenesis of infection, tissue injury, and
inflammation(6) . In vitro, TNF has been shown to be
involved in mediating a wide range of biological activities, including
differentiation, antiviral responses, and proliferation. TNF stimulates
proliferation of many cell types, both lymphoid and non-lymphoid,
including thymocytes(7) , fibroblasts (5) ,
chondrocytes(8) , and some human cancers, including chronic
lymphoid leukemia and ovarian cancer(9, 10) . TNF is
synthesized by a broad range of cells, including macrophages/monocytes,
T and B lymphocytes, NK cells(1) , mast cells(11) ,
some transformed cell lines (12, 13) , and breast and
epithelial tumor cells(14, 15) . The activated
macrophage is considered to be the main producer of TNF in
vivo(16) . The most potent inducer of TNF production in
these cells is lipopolysaccharide (LPS) derived from the cell wall of
Gram-negative bacteria(1) . Transformed myeloid cell lines such
as U937 and HL60 can also be induced to produce TNF with LPS and such
agents as phorbol esters and the calcium ionophore
A23187(17, 18) . Similarly, T lymphocytes produce TNF
in response to phorbol esters and calcium ionophore or
mitogens(19) . Certain transformed cell lines produce TNF
constitutively(12, 13) , although the basis for such
constitutive production has not been determined.B
are especially important to confer LPS inducibility(20) .
Determining how NFB becomes activated in cells is therefore likely
to be important for our understanding of TNF induction. The NFB
family of proteins currently comprises three main groups of molecules.
The first includes NFKB1 p105/p50 and NFKB2
p100/p52(21, 22) . p105 and p100 represent precursor
proteins which are proteolytically cleaved to form p50 and p52,
respectively, which are transcriptionally
active(23, 24) . The second subclass includes RelA
(formerly p65)(25) , c-Rel (26) , v-Rel(27) ,
and RelB(28) , none of which require processing in order to be
active. The third subclass includes IB (29) ,
IB
(30) , and Bcl-3 (31) which regulate the
cytoplasmic retention, DNA binding, and transcriptional activity of
NFB complexes. The predominant form of NFB in cells appears
to comprise a heterodimer of p50 and RelA complexed to IB,
although other arrangements of NFB occur and are likely to play a
role in NFB-driven gene expression(32) . NFB is
constitutively present and active in the nucleus only in a restricted
subset of cells such as B cells (33) and certain T-cell
lines(34) . Latent NFB is the predominant form in other
cell types where it is maintained in the cytoplasm complexed to an
inhibitory subunit IB(35) . Upon activation with agents
such as LPS, interleukin 1 (IL1), and TNF, IB dissociates from
NFB, which then translocates to the nucleus where it binds its
decameric DNA recognition sequence. Phosphorylation and proteolysis of
IB have been implicated in the dissociation process (36, 37) .B in a human T lymphoma cell line, HuT 78, which is resistant to
cytotoxicity by TNF. In addition, we demonstrate that a monoclonal
antibody to TNF blocks proliferation of the cells and constitutive
NFB. The results suggest that an autocrine loop involving TNF
production and NFB activation may be operating in the cells which
leads to proliferation.
Materials
Recombinant human TNF was a
generous gift from Cetus Corp. (Emeryville, CA). The monoclonal
antibody to human TNF (Clone 4H31) was obtained from Sanbio (Uden,
The Netherlands). The anti-IE hybridoma, Y-17, was obtained from ATCC.
T4 polynucleotide kinase, the 22-base pair oligonucleotide containing
the consensus sequence (underlined)(5`-AGTTGAGGGGACTTTCCCAGGC-3`), the
22-base pair oligonucleotide containing the consensus sequence
(underlined) for the immunoglobulin enhancer oligonucleotide factor 1
(OCT1) (5`-TGTCGAATGCAAATCACTAGAA-3`) and poly(dI-dC) were from Promega
(Madison, WI). [-
P]ATP (10 mCi/mmol) was
obtained from Amersham International (Amersham, Buckinghamshire, United
Kingdom). Antibodies to p50, RelA, and c-rel were from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA).Cell Lines
The L929 murine fibrosarcoma and Jurkat
T-cell lymphoma cell lines were obtained from ECACC (Porton Down,
Salisbury, Wiltshire, UK). HuT 78, a T-cell lymphoma derived from
peripheral blood of a Sezary lymphoma patient, was obtained from ATCC
(Rockville, MD). K-4 cells were generated from HuT 78 as described
previously(38) . Cells were cultured in RPMI 1640 containing
10% fetal calf serum, 2 mML-glutamine, penicillin,
and streptomycin and 5 
10
M 2-mercaptoethanol (complete medium) (Life Technologies, Inc.,
Paisley, Scotland).TNF Production
HuT 78, K-4 and Jurkat cells (1
10
cells/ml) were incubated at 37 °C for 48 h
in medium. Supernatants were then removed and stored in 0.5-ml aliquots
at -70 °C. All samples were assayed for TNF by both bioassay
(L929 cytotoxicity assay) and enzyme-linked immunosorbent assay (ELISA)
within 2 months of collection. The L929 cytotoxicity assay was a
modification of that described by Green et al.(39) .
L929 cells between the 5th and 10th passage were incubated in 96-well
flat-bottomed microtiter plates at a concentration of 8
10
cells/well overnight at 37 °C. Titrations of the
recombinant human TNF standard or undiluted test samples were then
diluted (1/2) in medium with actinomycin D (2 µg/ml final
concentration) (Sigma, Poole, Dorset, UK) and incubated for 24 h at 37
°C. 20 µl of
3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Sigma) at a concentration of 6 mg/ml was added to the wells and plates
incubated for 4 h at 37 °C. Dimethyl sulfoxide (British Drug House,
Poole, UK) was used to dissolve the formazan crystals formed in the
wells. Absorbance was measured at 570 nm on an ELISA reader. was also measured with a commercially available ELISA
(Innogenetics NV, Antwerp, Belgium). The detection limit for this assay
is 4 pg/ml.The intraassay and interassay coefficients of variation are
5.5 and 7.9% respectively.Interleukin 2 Production
Interleukin 2 (IL2) was
also measured in the supernatants of resting HuT 78, K-4, and Jurkats
incubated for 48 hr in medium alone, as above. IL2 was measured by an
ELISA obtained from R & D Systems, Inc. (Minneapolis, MN). The
sensitivity of the assay was 6 pg/ml. The intraassay and interassay
coefficients of variation were 5.9 and 7.7%, respectively.Thymidine Incorporation
HuT 78 or K-4 cells were
incubated in 96-well flat-bottomed microtiter plates at a concentration
of 1 10
cells/well for 24 h and pulsed with
tritiated thymidine (0.5 µCi/well) for the last 6 h of incubation.
Titrations of the TNF standard and/or TNF monoclonal antibody
(3.5 mg/ml) or anti-IE antibody (3.5 mg/ml) were added to the wells at
the start of incubation. Samples were harvested on a multiple automated
cell harvester and counted in a Beckman liquid scintillation counter.
In addition, to examine cell viability and numbers, we performed a
non-radioactive cell proliferation assay, using MTS, a tetrazolium salt
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
and an electron coupling reagent, PMS (phenazine methosulfate)
(Promega). Both solutions were stored protected from light at -20
°C, and immediately before use, a working solution containing a
1/20 dilution of PMS in MTS was made. HuT 78 cells were grown as above
with the exception that PMS/MTS was added for the last 4 h of culture.
Absorbance was measured at 490 nm on an ELISA reader.Preparation of Subcellular Fractions
Subcellular
fractions were prepared as described before(40) . HuT 78, K-4,
or Jurkat T lymphomas (1 10/ml, 1 ml/sample) were
treated with IL1, phorbol myristate acetate (PMA), or TNF (all at 10
ng/ml) as indicated for various times or were left untreated. Cells
were then washed with ice-cold phosphate-buffered saline and
resuspended into 1 ml of hypotonic buffer (10 mM Hepes buffer,
pH 7.9, containing 1.5 mM MgCl
, 10 mM KCl, 0.5 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonyl fluoride). Cells were pelleted in hypotonic
buffer by centrifugation at 13,000 g for 10 min at 4
°C and then lysed for 10 min on ice in 20 µl of hypotonic
buffer containing 0.1% (v/v) Nonidet P-40. Lysates were centrifuged
(13,000 g, 10 min, 4 °C), and the supernatant
(nuclear extract) was removed into 75 µl of storage buffer (10
mM Hepes, pH 7.9, containing 50 mM KCl, 0.2 mM EDTA, 10% (v/v) glycerol, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride). Protein concentrations were
determined (41) and the extracts were assayed immediately for
NFB activity or stored at -86 °C until further use. All
of the steps in the above procedure were performed at 4 °C unless
otherwise stated.Electrophoretic Mobility Shift Assay
Nuclear
extracts (2-4 µg of protein) were incubated with 10,000 cpm
of the 22-base pair oligonucleotide containing the NFB binding
motif which had been previously labeled with
[-P]ATP by T4 polynucleotide
kinase(42) . Incubations were performed for 30 min at room
temperature in the presence of 2 µg of poly(dI-dC) as nonspecific
competitor and 100 mM Tris buffer, pH 7.5, containing 100
mM NaCl, 1 mM EDTA, 5 mM DTT, 4% glycerol,
and 100 µg/ml nuclease-free bovine serum albumin. In some
experiments, unlabeled oligonucleotides containing either the NFB
or the OCT1 consensus sequence were added to the extracts before
incubating with labeled oligonucleotide. In experiments involving
antisera to NFB subunits, 0.5 µl of a specific antiserum to
p50, p65, or c-rel was incubated with 4 µg nuclear extract
30 min prior to the binding reaction. All incubations were subjected to
electrophoresis on native 4% (w/v) polyacrylamide gels that were
subsequently dried and autoradiographed in autoradiography cassettes
with intensifying screens for 16-48 h at -86 °C.
TNF Production by HuT 78, K-4, and Jurkat
Cells
We were prompted to measure spontaneous TNF production by
HuT 78 and K-4, as initial experiments demonstrated that they were
resistant to cytotoxicity by TNF (data not shown). Other studies have
suggested that exposure to low concentrations of TNF induce resistance
to subsequent TNF-mediated cytotoxicity and that cells resistant to
TNF-mediated cytotoxicity produce low levels of
TNF(14, 43) . Supernatants of resting HuT 78, K-4, and
Jurkat cells incubated for 48 h were analyzed for TNF production by
L929 bioassay which measures both biologically active TNF and
lymphotoxin and by ELISA, which measures immunoreactive TNF only. Fig. 1A demonstrates that HuT 78 and K-4 produce substantial
amounts of TNF constitutively (HuT 78, 136.6 ± 64.3pg/ml;
K-4, 425.1 ± 122.1 pg/ml, mean ± S.E.), measured by L929
bioassay: Jurkats, however, produce very little TNF (19.8 ± 11.8
pg/ml TNF, mean ± S.E.). TNF levels in supernatants were also
measured by a TNF-specific ELISA. Fig. 1B demonstrates
that the ELISA similarly detected constitutive TNF production by
HuT 78 and K-4 (HuT 78: 56.3 ± 2.3 and K-4: 75.2 ± 16.9
pg/ml TNF, mean ± S.E.): resting Jurkat cells, however, do not
produce TNF levels detectable by ELISA. Differences between the
L929 bioassay and ELISA methods for measuring TNF have been documented
previously(44, 45) .
Inhibition of Thymidine Incorporation of HuT 78 and K-4
by Anti-TNF
HuT 78 and K-4 cells were incubated for 24 h
either alone or with 3 ng/ml TNF or a neutralizing monoclonal antibody
to TNF and proliferation measured by thymidine incorporation (Fig. 2). An irrelevant control antibody (anti-IE) was also
included. 3 ng/ml TNF had no effect on proliferation; higher TNF
concentrations (30 ng/ml) also had no effect (data not shown).
Proliferation of cells treated with the anti-TNF antibody (1/20
dilution) was dramatically inhibited on average by 90.5 ± 10.1%
and 73.2 ± 9.2% for HuT 78 and K-4, respectively (mean ±
S.E., three experiments). A similar concentration of control antibody
had no effect. In addition, we examined cell viability and numbers
using PMS/MTS. Culture of HuT 78 cells alone produced an absorbance of
0.573 ± 0.042 (mean ± S.D., three experiments). Culture
in the presence of anti-TNF antibody resulted in absorbance of
0.333 ± 0.012 (mean ± S.D., three experiments). Hence,
these data confirm that anti-TNF treatment is associated with
significant reduction in number of viable cells.
antibody. HuT 78 and K-4 cells
were incubated for 24 h in medium alone, with 3 ng/ml TNF, with
anti-IE (1/20 dilution of 3.5 mg/ml stock solution), an irrelevant
control antibody, and with anti-TNF (1/20 dilution of 3.5 mg/ml
stock solution). Proliferation was assessed by
[H]thymidine incorporation as described under
``Experimental Procedures.'' Representative of three
experiments, mean ± S.E.
HuT 78 and K-4 Contain Constitutive NF
We
next examined the cells for the presence of NFBB. As can be seen in Fig. 3, A and B, both HuT 78 and K-4 contained
high levels of constitutive NFB which could not be further
enhanced with IL1, PMA, or TNF. This was in contrast to another human
T-lymphoma line, Jurkat, which had no constitutive NFB (Fig. 3C). Upon stimulation with either PMA or TNF,
however, NFB could be activated in Jurkat in a time-dependent
manner.
B in
Hut 78, K-4, and Jurkat T lymphomas. Hut 78 (A), K-4 (B), or Jurkat (C) T lymphomas were incubated with
IL1, PMA, or TNF (all at 10 ng/ml) or left untreated (lanes
C) for the indicated times and then assessed for NFB as
described under ``Experimental Procedures.'' NFB-DNA
complexes are shown.
B recognition sequence, as their formation was
inhibited by addition of unlabeled oligonucleotide, containing the
NFB binding site, to nuclear extracts before incubation with
labeled probe (Fig. 4A). An unlabeled oligonucleotide
containing the OCT1 sequence motif failed to exhibit any such
inhibitory effect. We also examined the extracts for the presence of
p50, RelA, and c-Rel, as shown in Fig. 4B. Samples
prepared from TNF-treated Jurkats were shown to contain p50 and RelA
but no c-Rel, as indicated by the ability of p50 and RelA antisera to
cause a further retardation in probe mobility (Fig. 4B), lanes 2 and 3). In HuT 78
and K-4, a similar result was obtained, although much larger amounts of
p50 were detected than RelA (Fig. 4B, lanes 6 and 7 for HuT 78 and lanes 10 and 11 for K-4). Again,
no c-Rel was detected in either cell type.
B in HuT 78, K-4, and
Jurkat T lymphomas. A, 2 µg of nuclear extract protein
prepared from Jurkat cells stimulated with TNF (10 ng/ml) for 1 h
or unstimulated Hut 78 and K-4 were incubated in the absence (lanes
0) or presence of unlabeled oligonucleotides (1.75 pmol)
containing the NFB or OCT1 consensus sequences prior to assaying
for NFB binding activity as described under ``Experimental
Procedures.'' NFB-DNA complexes are shown. B, 2
µg of nuclear extract protein prepared from Jurkat cells stimulated
with TNF (10 ng/ml) for 1 h or unstimulated Hut 78 and K-4 were
incubated with the indicated antisera on ice prior to assaying for
NFB binding activity as described under ``Experimental
Procedures.'' NFB-DNA complexes are shown. Supershifted
complexes corresponding to p50 and RelA are
indicated.
A Monoclonal Antibody to TNF Inhibits NF
Because both HuT 78 and K-4 were shown to produce TNF
constitutively, we next determined whether the monoclonal antibody to
TNF, which we had found to inhibit proliferation in the cells, would
also interfere with the constitutive NFB in HuT 78
and K-4B, which we found in both
cell types. Fig. 5A demonstrates that incubating both HuT 78
and K-4 for 48 and 72 h with anti-TNF dramatically reduced the levels
of NFB in both HuT 78 and K-4. Similar concentrations of an
irrelevant antiserum had no effect, as seen in Fig. 5B.
This indicates that the constitutively active NFB is a result of
constitutive TNF production by the cells. As the TNF gene is NFB
regulated, an autocrine loop is therefore likely to be operational in
the cells.
on NFB in HuT 78 and K-4. A, Hut 78
cells and K-4 were washed twice in RPMI and were then incubated in
medium alone(-) or 125 µl TNF antibody (3.5 mg/ml stock
solution) (+) for 24 h, 48 h, or 72 h as indicated. NFB
binding activity was measured as described under ``Experimental
Procedures.'' NFB-DNA complexes are shown. B, Hut 78
cells and K-4 were washed twice in RPMI and were then incubated in
medium alone(-) or 125 µl of control antibody anti-IE IgG1
(3.5 mg/ml stock solution) (+) for 24 h, 48 h, or 72 h as
indicated. NFB binding activity was measured as described under
``Experimental Procedures.'' NFB-DNA complexes are
shown.
-deficient clone of HuT 78, produce TNF spontaneously but Jurkat,
another T-cell line, however, does not. antibody significantly inhibited proliferation
of HuT 78 and K-4, suggesting that TNF was a growth factor for these
cells. Recently, Pimentel-Muinos et al.(50) suggested
that PMA-induced TNF was an autocrine growth factor for T-cells,
stimulating IL 2 secretion, which then stimulated proliferation.
However, this does not seem to occur in HuT 78 or K-4, as IL2 was not
detected in the supernatants of resting cells after 48 h, suggesting
that IL2 is not involved here (data not shown). Also, K-4 produce
significantly less IL2 than HuT 78 upon stimulation with
PMA(38) , yet the anti-TNF antibody inhibited
proliferation of K-4.B(1) . We detected high levels of constitutive NFB
in HuT 78 and K-4 cells. In most cell types, NFB exists in a
latent form in the cytosol complexed to an inhibitory subunit
IB(35) . Only a limited number of lymphoid cells have been
found to contain constitutively active nuclear NFB. These include
mature B cells (33) and a few T lymphomas(34) . In
these cells, however, the constitutive activation of NFB is only
partial as stimulation of the cells results in a further activation.
This does not appear to be the case in HuT 78 and K-4 as stimulation of
the cells with agents which potently activate NFB in other cells
failed to cause increased activation. The mechanism here appears to
involve the constitutive production of TNF by the cells as a
neutralizing monoclonal antibody to TNF completely blocked the
appearance of NFB in the nucleus of the cells. As NFB is
known to play a key role in the control of TNF gene
transcription(20) , an interesting autocrine loop may therefore
be operating in the cells. The role of activated NFB in the
proliferation of the cells through the induction of other genes is
unresolved. For example, NFB is known to regulate the c-myc gene(53) , which has a well established role in growth
control. Kitajima et al.(54) have previously
demonstrated that inhibiting NFB causes an inhibition of
tumorigenesis, in experiments using antisense technology. Hence,
NFB could have indirect effects on growth control regulation
through interaction with either proto-oncogenes or growth factor genes
involved in tumor growth and proliferation.. Also, Ware et al.(56) reported that lymphotoxin is not produced by HuT 78
and recently, it has been reported that lymphotoxin does not activate
NFB(57) .B complex in the cells revealed the presence of both p50 and
RelA. Two recent studies have shown that HuT 78 nuclei contain high
levels of both p52 (58) and p50 (59) . Two additional
proteins of p84 and p85 were also detected and represented aberrantly
processed p100. The basis for these observations was found to be a
rearrangement of the NFB2 gene in the cells, resulting in a
protein which had lost an ankyrin repeat domain in its C terminus.
Because ankyrin domains on p100 and p105 are important for cytoplasmic
retention, the authors suggest that the lack of the C-terminal ankyrin
domain results in a protein which is not processed in the normal way
and escapes cytoplasmic retention, thereby localizing to the nucleus.
p85 and p52 were also shown to be transcriptionally
active(58) . The studies did not explain why normal p52 is
found in the nucleus of the cells as this would be expected to be
retained in the cytoplasm complexed to IB. Our results indicate
that constitutive TNF production by the cells is critical for the
nuclear localization of both p52 and p85, along with p50 and RelA, as a
neutralizing antibody to TNF dramatically decreased nuclear NFB
levels. This suggests that the missing ankyrin repeat in p85 would not
be sufficient to cause nuclear translocation, the five remaining
repeats being sufficient to retain the protein in the cytoplasm. It is
likely, however, that once the aberrant NFB has translocated to
the nucleus in response to TNF, a dysregulation in TNF gene expression
must occur which results in a lack of feedback inhibition. In normal
cells, NFB activation leading to TNF production is self-limiting
through mechanisms likely to involve, at least in part, IB
induction which acts to neutralize transactivating NFB. Such
mechanisms appear to be absent in HuT 78. This is most likely due to
the presence of abnormal NFB2 proteins altering the functioning of
the NFB system in the cells.B activation has also been demonstrated in a study involving
PMA-stimulated T lymphocytes, where it was shown that activation of
NFB by PMA required the induction of TNF as neutralizing
antibodies to TNF blocked the response(48) . Our results with
HuT 78 are analagous except that no stimulus was needed to trigger TNF
production. Both studies confirm the importance of autocrine regulation
in NFB activation in cells and suggest that once TNF is induced,
it will feed back on the cells and activate NFB. However, the
current study differs significantly from studies on peripheral blood
T-cells. HuT 78 is a malignant T-cell clone derived from a cutaneous
T-cell lymphoma. Inhibition of TNF blocked proliferation of this
malignant cell line, suggesting the possibility that pharmacological
interventions involving inhibition of TNF production may have potential
effects.B in the cells, which is likely to
lead to further TNF production. The HuT 78 T-cell line is derived from
a cutaneous Sezary type T-cell lymphoma. The finding that proliferation
of this cell line responds to inhibition of TNF production suggests the
intriguing possibility that anti-TNF strategies could be employed in
the treatment of these lymphomas in the clinical environment.
)B,
nuclear factor B; PKC, protein kinase C; PMA, phorbol
12-myristate 13-acetate; PMS, phenazine methosulfate; MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Long, D. Kelleher, S. Lynch, and Y. Volkov Cutting Edge: Protein Kinase C{{beta}} Expression Is Critical for Export of IL-2 from T Cells J. Immunol., July 15, 2001; 167(2): 636 - 640. [Abstract] [Full Text] [PDF]
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 D. W. Kim, M. A. Sovak, G. Zanieski, G. Nonet, R. Romieu-Mourez, A. W. Lau, L. J. Hafer, P. Yaswen, M. Stampfer, A. E. Rogers, et al. Activation of NF-{kappa}B/Rel occurs early during neoplastic transformation of mammary cells Carcinogenesis, May 1, 2000; 21(5): 871 - 879. [Abstract] [Full Text] [PDF]
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 C. H. Selzman, B. D. Shames, L. L. Reznikov, S. A. Miller, X. Meng, H. A. Barton, A. Werman, A. H. Harken, C. A. Dinarello, and A. Banerjee Liposomal Delivery of Purified Inhibitory-{kappa}B{alpha} Inhibits Tumor Necrosis Factor-{alpha}–Induced Human Vascular Smooth Muscle Proliferation Circ. Res., April 30, 1999; 84(8): 867 - 875. [Abstract] [Full Text] [PDF]
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U. Dobbeling, R. Dummer, E. Laine, N. Potoczna, J.-Z. Qin, and G. Burg Interleukin-15 Is an Autocrine/Paracrine Viability Factor for Cutaneous T-Cell Lymphoma Cells Blood, July 1, 1998; 92(1): 252 - 258. [Abstract] [Full Text] [PDF]
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 D. K. Giri and B. B. Aggarwal Constitutive Activation of NF-kappa B Causes Resistance to Apoptosis in Human Cutaneous T Cell Lymphoma HuT-78 Cells. AUTOCRINE ROLE OF TUMOR NECROSIS FACTOR AND REACTIVE OXYGEN INTERMEDIATES J. Biol. Chem., May 29, 1998; 273(22): 14008 - 14014. [Abstract] [Full Text] [PDF]
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A. Bellocq, S. Suberville, C. Philippe, F. Bertrand, J. Perez, B. Fouqueray, G. Cherqui, and L. Baud Low Environmental pH Is Responsible for the Induction of Nitric-oxide Synthase in Macrophages. EVIDENCE FOR INVOLVEMENT OF NUCLEAR FACTOR-kappa B ACTIVATION J. Biol. Chem., February 27, 1998; 273(9): 5086 - 5092. [Abstract] [Full Text] [PDF]
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M. R. Hassuneh, P. S. Nagarkatti, and M. Nagarkatti Evidence for the Participation of Interleukin-2 (IL-2) and IL-4 in the Regulation of Autonomous Growth and Tumorigenesis of Transformed Cells of Lymphoid Origin Blood, January 15, 1997; 89(2): 610 - 620. [Abstract] [Full Text] [PDF]
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R. Y. Liu, C. Fan, G. Liu, N. E. Olashaw, and K. S. Zuckerman Activation of p38 Mitogen-activated Protein Kinase Is Required for Tumor Necrosis Factor-alpha -supported Proliferation of Leukemia and Lymphoma Cell Lines J. Biol. Chem., July 7, 2000; 275(28): 21086 - 21093. [Abstract] [Full Text] [PDF]
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