From the Cytokine Research Laboratory, Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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How tumor cells develop resistance to apoptosis
induced by cytokines and chemotherapeutic agents is incompletely
understood. In the present report, we investigated apoptosis induction
by tumor necrosis factor (TNF) in two human T cell lines, Jurkat and
HuT-78. While TNF inhibited the growth of Jurkat cells and activated
caspase-3, it had no effect on HuT-78 cells. It was further found that
HuT-78 cells constitutively expressed the nuclear transcription factor
NF-B. TNF activated NF-
B in Jurkat cells but not in HuT-78 cells.
HuT-78 cells were also resistant to NF-
B activation induced by
phorbol ester, H2O2, ceramide, endotoxin, and interleukin-1. Despite the presence of preactivated NF-
B, HuT-78
cells also expressed high levels of I
B-
, the inhibitory subunit
of NF-
B and, unlike Jurkat cells, were resistant to TNF-induced degradation of I
B-
. Its half-life in HuT-78 cells was 12 h
as opposed to 45 min in Jurkat cells. Antibodies against TNF blocked the constitutive activation of NF-
B and proliferation of HuT-78 cells but had no significant effect on Jurkat cells, suggesting an
autocrine role for TNF. The antioxidant pyrrolidine dithiocarbamate also suppressed constitutive NF-
B activation and it reversed the
cell's sensitivity to TNF-induced cytotoxicity and activation of
caspase-3. Overall, these results suggest that constitutive activation
of NF-
B, TNF, and prooxidant pathway in certain T cell lymphomas
causes resistance to apoptosis, and this can be reversed by
antioxidants.
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INTRODUCTION |
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Development of resistance to apoptosis induction by cytokines and
chemotherapeutic agents is one of the major problems in cancer therapy
(1). Overcoming this resistance has been largely unsuccessful, because
the mechanism of development of resistance is not understood. Multiple
drug resistance (MDR)1
P-glycoprotein, Bcl-2, inactivation of p53 and related proteins, glutathione S-transferase, protein kinase C, MDR-related
proteins, transglutaminase, and heat shock proteins (e.g.
hsp 27) all play a role (2-5). Besides these factors, an activated
form of the nuclear transcription factor NF-B has recently been
implicated in development of resistance to tumor necrosis factor (TNF)
(6-8).
Under normal conditions, NF-B is present in the cytoplasm in its
inactive state as a heterotrimer consisting of p50, p65, and I
B-
(9). When activated I
B-
undergoes ubiquitination, phosphorylation, and degradation, and the p50-p65 complex is released to be translocated to the nucleus where it causes gene activation. The
activation of NF-
B is initiated by a wide variety of stress stimuli,
which themselves cause apoptosis. Among these are TNF, IL-1, x-rays,
-radiation, phorbol ester, ceramide, endotoxin, calcium ionophores,
and H2O2 (9, 10). Interestingly, several chemotherapeutic drugs such as the anthracyclines doxorubicin and
daunorubicin (11, 12), taxol, the vinca alkaloids vinblastine and
vincristine (12), camptothecin (13) and etoposide (14) also cause
NF-
B activation. Several genes whose proteins are involved in tumor
promotion and metastasis, such as ICAM-1, VCAM-1, ELAM-1,
cyclooxygenase-2, and matrix metalloprotease-9, are regulated by
NF-
B (15-17).
A progressive activation of constitutive NF-B has recently been
correlated with progression of breast cancer, melanoma, and juvenile
myelomonocytic leukemia (18-21). How NF-
B is constitutively activated in some tumor cells and what role it plays in induction of
resistance to apoptosis is not clear. In the present study, we show
that in contrast to acute T cell leukemic Jurkat cells, cutaneous T
cell lymphoma HuT-78 cells were resistant to the apoptotic effects of
TNF and constitutively expressed high levels of activated form of
NF-
B and of I
B-
simultaneously. The latter was resistant to
TNF-induced I
B-
degradation and had a very long half-life. Neutralizing anti-TNF antibodies down-regulated the constitutive NF-
B activation and induced apoptosis in HuT-78 but not in Jurkat cells. Pyrrolidine dithiocarbamate (PDTC), a quencher of reactive oxygen intermediates, also inhibited constitutively activated NF-
B
and rendered HuT-78 cells susceptible to TNF-induced killing.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Reagents--
Bacteria-derived human rTNF,
purified to homogeneity, was kindly provided by Genentech (San
Francisco, CA). PMA, lipopolysaccharide, hydrogen peroxide, PDTC,
ceramide, and cycloheximide were purchased from Sigma. Okadaic acid (LC
Laboratories, Woburn, MA), [-32P]ATP (ICN
Pharmaceutical, Inc., Costa Mesa, CA), polynucleotide kinase (New
England Biolabs, Beverley, MA) poly(dI-dC) (Amersham Pharmacia Biotech)
were purchased. The plasmids (wild-type and mutant)
243RMICAT with
rat MDR1b promoter possessing either wild-type or mutated NF-
B
binding site linked to chloramphenicol acetyltransferase (CAT) reporter
gene were kindly supplied by Dr. M. Tien Kuo of the University of Texas
M. D. Anderson Cancer Center (Houston, TX).
Cell Lines and Culture--
An acute T cell leukemia cell line,
Jurkat, was purchased from American Type Culture Collection (Rockville,
MD). HuT-78, a cutaneous T cell lymphoma derived from peripheral blood
of a Caucasian patient with Sezary syndrome with the properties of a
mature T cell line of inducer/helper phenotype, was a generous gift
from National Institutes of Health AIDS Research and Reference Reagent Program of National Institutes of Health. These cells were grown in
RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (Life Technologies, Inc.), 2 mM glutamine, and
antibiotics at 37 °C in an atmosphere of 5% CO2 in air.
GST-IB-
was prepared as described previously (31).
Antibodies--
The polyclonal antibodies used were as follows:
anti-p65, against the epitope corresponding to amino acids mapping
within the amino-terminal domain of human NF-B p65; anti-p50,
against a peptide 15 amino acids long mapping at the NLS region of
NF-
B p50; anti-I
B-
, against amino acids 297-317 mapping at
the carboxyl terminus of I
B-
/MAD-3 and anti-I
B-
(amino acid
339-358) and anti-cyclin D1 against amino acids 1-295, which
represents full-length cyclin D1 of human origin. All these antibodies
were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA.).
Anti-PARP monoclonal antibody that recognizes PARP and its degradation
product was purchased from PharMingen (San Diego, CA). Anti-TNF
antibodies were raised in rabbits using recombinant TNF as described
(22).
Electrophoretic Mobility Shift Assays (EMSA)--
The details of
the preparation of nuclear extracts and the assay procedure have been
described elsewhere (23, 24). Nuclear extracts were either used
immediately or stored at 70 °C. Typically, 4-6 µg of protein
was used per assay. The protein content of the extract was measured by
the method of Bradford (25). EMSAs were performed by incubating nuclear
extract with 32P-end-labeled 45-mer double-stranded NF-
B
oligonucleotide from the human immunodeficiency virus terminal repeat,
5'TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3'. A double-stranded mutated oligonucleotide,
5'TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-
B to the DNA.
The specificity of binding was also examined by competition with the
unlabeled oligonucleotide. For supershift assays, antibodies against
p50 or p65 subunits of NF-
B were used as described (26). Visualization and quantitation of radioactive bands were carried out
using a PhosphorImager (Molecular Dynamics) using Image Quant software
(National Institutes of Health, Bethesda, MD).
Determination of IB by Western Blot--
I
B-
and -
Western blot assays were carried out with 25-30 µg of cytoplasmic
extracts. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE), the proteins were electrophoretically
transferred to nitrocellulose membranes. The membranes were blocked
with phosphate-buffered saline with 0.5% Tween 20 (PBST) containing
5% fat-free milk and then exposed to I
B-
antibodies at 1-3000
dilution. The membranes were washed with PBST and treated with
secondary antibody conjugated to horseradish peroxidase. The
antigen-antibody reaction was visualized by an enhanced
chemiluminescence (ECL) assay using Amersham ECL reagents and exposure
to film.
Transient Transfection and CAT Assay--
HuT-78 or Jurkat cells
were transiently transfected with 243RMICAT (wild-type) or
243RMICAT-
m (mutant) gene for 6 h by the calcium phosphate
method, according to the instructions supplied by the manufacturer
(Life Technologies, Inc.). After transfection, the cells were examined
for CAT activity as described (44).
Analysis of PARP Cleavage-- Apoptosis induced by TNF was studied by proteolytic cleavage of the death substrate, PARP (27). Briefly, 2 × 106 cells/ml were treated with TNF (1 nM) for 2 h in the presence of cycloheximide (10 µg/ml). Following incubation, cell extracts were prepared by incubating the cells for 30 min on ice in 0.05 ml of buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 250 mM NaCl, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.5 mg/ml benzamidine, and 1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged and the supernatant collected. A 50-µg protein sample from the supernatant was resolved by SDS-PAGE using a 7.5% gel and electrotransferred onto a nitrocellulose membrane. The transferred proteins were probed with anti-PARP antibody and detected by anti-mouse IgG conjugated to horseradish peroxidase and visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech). PARP degradation was represented by detection of both cleaved (86 kDa) and uncleaved (116 kDa) proteins recognized by the antibody.
Determination of Cell Viability by MTT Assay-- Aliquotes of 5 × 103 cells/well were distributed in 96-well tissue culture plates in 0.1 ml of medium and incubated at 37 °C in the presence or absence of the test materials. Following incubation for the indicated time periods, MTT solution was added to the culture to achieve a final concentration of 1 mg/ml. The culture was incubated at 37 °C for 2 h, and the cells were lysed in a lysis buffer containing 20% SDS in 50% N,N-dimethyl formamide solution. The absorbance was read at 570 nm and the percent cell viability calculated.
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RESULTS |
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HuT-78 Cells Are Resistant to Apoptosis by TNF-- The aim of the present study is to understand the molecular basis for induction of sensitivity or resistance of tumor cells to the apoptotic effects of TNF. Two human T cell lines, Jurkat and HuT-78, were examined. The proliferation of Jurkat cells was completely inhibited by TNF, whereas it had no significant effect on the proliferation of HuT-78 cells (Fig. 1A). Activation of caspase-3 leading to cleavage of PARP is one of the major hallmarks for apoptosis. When examined for the ability of TNF to induce PARP cleavage, it was found that PARP was cleaved by TNF in Jurkat cells but not in HuT-78 cells (Fig. 1B). Cycloheximide enhanced the TNF-induced PARP cleavage only in Jurkat cells, not in HuT-78 cells. These results indicate that HuT-78 cells are resistant to growth inhibition and apoptosis induction by TNF.
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HuT-78 Cells Constitutively Express Activated NF-B--
As
NF-
B activation has been implicated in induction of resistance, we
examined the constitutive and inducible NF-
B activation in the two
cell types. Gel shift assay showed that under normal conditions, Jurkat
cells did not express activated NF-
B, but on treatment with TNF,
activated NF-
B was noted within 5 min, reached maximum at 30 min,
and declined thereafter (Fig.
2A). In contrast, HuT-78 cells
constitutively expressed high levels of activated NF-
B and could not
be further activated by TNF (Fig. 2A). Supershift assays
using antibodies to either the p50 or p65 subunits of NF-
B retarded
the mobility of NF-
B proteins in both Jurkat and HuT-78 cells (Fig.
2B). This shift was specific, as the irrelevant antibodies
anti-cyclin D1 and preimmune serum had no effect. Thus it is possible
that HuT-78 cells were resistant to TNF-induced apoptosis, because they
constitutively expressed activated NF-
B.
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HuT-78 Cells Constitutively Express High Levels of
IB-
--
We used Western blot analysis to further
investigate why HuT-78 cells constitutively express activated NF-
B.
NF-
B is present in its inactive state in the cytoplasm where it is
bound to I
B-
. The degradation of I
B-
is critical for
NF-
B activation and translocation to the nucleus. Western blot
analysis of the levels of I
B-
in the cytoplasm before and after
TNF treatment showed that in Jurkat cells, TNF caused the degradation
of I
B-
within 15 min; it was resynthesized by 60 min (Fig.
3A). In contrast, HuT-78
expressed large amounts of I
B-
, and these were not affected by
TNF treatment. Thus HuT-78 cells constitutively express high levels of
I
B-
along with activated NF-
B.
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IB-
Turnover Is Slow in HuT-78 Cells--
Why I
B-
does
not inhibit NF-
B activation in HuT-78 cells was investigated. The
synthesis of I
B-
was blocked by treatment of cells with
cycloheximide, which allowed us to determine the constitutive rate of
degradation. The half-life of I
B-
was found to be less than 60 min in Jurkat cells but greater than 12 h in HuT-78 cells (Fig.
3B). These results suggest that I
B-
from HuT-78 cells
is either resistant to proteolysis or its protease is not as active in
HuT-78 cells.
HuT-78 Cells Express Various Rel Proteins and IB-
and
-
--
To determine why NF-
B is constitutively activated in
HuT-78, but not in Jurkat cells, we examined the levels of p50, p65, p52, c-Rel, I
B-
, and I
B-
in the cytoplasm and nucleus of
these cell types by Western blot analysis. As shown in Fig.
4A, all these NF-
B proteins
are expressed in the cytoplasm of both cell types, but levels may be
somewhat higher in HuT-78 cells. In addition, HuT-78 cells expressed
two different isoforms of p52, whereas Jurkat cells expressed only one
form. Besides p50 and p65, the nucleus of HuT-78 cells also expressed
high level of c-Rel. It is possible that either c-Rel or an additional
isoform of p52 may prevent the degradation of I
B-
in HuT-78
cells.
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IB-
Interacts with p50/p65 in HuT-78 Cells in Vitro and in
Vivo--
It is possible that p50/p65 interacts with I
B-
differently in Jurkat cells than in HuT-78 cells. To investigate this
possibility, the p50-p65 complex prepared from TNF activated cells was
incubated in vitro with recombinant GST-I
B-
and then
examined for binding to DNA by EMSA. As shown in Fig. 4B,
I
B-
inhibited the DNA binding activity of p50/65-derived from
either cell type in a concentration dependent manner. Heat-denatured
GST-I
B-
was inactive in blocking DNA binding activity of p50-p65
complex (data not shown). To demonstrate the interaction of p50/p65
with I
B-
in vivo, cell extracts were first
immunoprecipitated with anti-p65 antibody and then the pellet was
analyzed for I
B-
by Western blot. As shown in Fig. 4C,
p65 was found to interact with I
B-
in both cell types to same
extent (lanes 1 and 2). This interaction was
specific as no I
B-
band was observed with cyclin D1 as a control.
Thus these results indicate that I
B-
and p50/65 interact in
HuT-78 cells.
NF-B in HuT-78 Cells Constitutively Activates Gene
Transcription--
To further ascertain that constitutive NF-
B
activation observed by DNA binding assay in HuT-78 cells is functional,
both Jurkat and HuT-78 cells were transiently transfected with
NF-
B-CAT gene reporter construct and then assayed for CAT
expression. As shown in Fig. 5, the
NF-
B-dependent CAT gene was not constitutively expressed
in Jurkat cells, but it was active in HuT-78 cells. The plasmid with a
mutated NF-
B site was inactive in both cell types. These results
further confirm that NF-
B is functional in HuT-78 cells.
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HuT-78 Cells Are Also Resistant to Other Inflammatory
Stimuli--
Increasing evidence shows that different stimuli activate
NF-B through pathways consisting of both overlapping and
nonoverlapping steps. PMA, lipopolysaccharide,
H2O2, ceramide, okadaic acid, and IL-1
activated NF-
B in Jurkat cells, but none had any effect on NF-
B
in HuT-78 cells (Fig. 6). It is possible
that NF-
B is constitutively maximally activated in HuT-78 cells.
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PDTC Inhibits NF-B Activation and Renders HuT-78 Cells
Susceptible to TNF-induced Apoptosis--
The mechanism of NF-
B
activation is not fully understood. Activation by most stimuli,
however, requires the generation of reactive oxygen species (ROS). PDTC
is known to inhibit the formation of ROS and NF-
B activation by
certain stimuli in a cell type-specific manner (28). Whether PDTC can
also inhibit constitutive NF-
B activation is not known. We showed
that PDTC suppressed the TNF-induced NF-
B in Jurkat cells (Fig.
7A). PDTC also suppressed
NF-
B activation in HuT-78 cells (Fig. 7B). Thus, like
inducible activation of NF-
B in Jurkat cells, ROS was involved in
constitutive activation of NF-
B in HuT-78.
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Anti-TNF Antibodies Inhibit NF-B Activation and Proliferation of
HuT-78--
TNF is known to induce ROS (28). In HuT-78 cells
constitutive synthesis of TNF may be responsible for production of ROS and thus for constitutive expression of activated NF-
B. To determine this, we treated HuT-78 cells for 24, 48, and 72 h with anti-TNF antibodies and then assayed them for NF-
B. Anti-TNF treatment down-regulated the nuclear levels of activated NF-
B in a
time-dependent manner (Fig.
9A). This decrease correlated
with a decrease in the constitutive levels of I
B-
in the
cytoplasmic pool (Fig. 9B). Anti-TNF antibodies also
decreased HuT-78 cell proliferation, but not Jurkat cell proliferation
in a dose-dependent manner (Fig. 9C). Consistent
with these results, anti-TNF antibodies induced PARP cleavage in HuT-78
cells but not in Jurkat cells (Fig. 9D). In addition, when
measured for TNF production, HuT-78 cells were found to express TNF but
not Jurkat cells (data not shown). These results indicate that HuT-78
cells constitutively express TNF, which causes NF-
B activation
through ROS and that in turn induces resistance to apoptosis, also
consistent with the effect of PDTC, indicating that NF-
B activation
leads to resistance to apoptosis.
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DISCUSSION |
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In the present study we investigated the mechanism by which tumor
cells develop resistance to apoptosis induced by TNF. Two human T cell
lines, Jurkat and HuT-78, were examined. While TNF inhibited the growth
of Jurkat cells and activated caspase-3, it had no effect on HuT-78
cells; constitutive activation of NF-B was demonstrated in HuT-78
cells and may account for this difference in growth and apoptosis
induction. TNF and various other stimuli activated NF-
B in Jurkat
cells but not in HuT-78 cells. Despite preactivated NF-
B, HuT-78
cells also expressed high levels of I
B-
, which unlike Jurkat
cells, were resistant to TNF-induced degradation. The half-life of the
I
B-
in HuT-78 cells is much longer than Jurkat cells. Antibodies
against TNF down-regulated the constitutive activation of NF-
B and
proliferation of HuT-78 cells, suggesting an autocrine role for TNF.
The antioxidant PDTC also suppressed constitutive NF-
B activation
and reversed sensitivity to TNF for cytotoxicity and caspase-3
activation. These results suggest that constitutive NF-
B activation
causes resistance to apoptosis through generation of ROS and TNF.
Our results show that HuT-78 cells express activated NF-B and
I
B-
simultaneously. This is not too surprising as the synthesis of I
B-
requires NF-
B activation (29), but why I
B-
fails to inhibit constitutively activated N
-kB, however, is not clear. Our
results show that I
B-
in HuT-78 is degraded very slowly compared
with Jurkat cells. In WEHI-3 cells, which were also shown to express
constitutively activated NF-
B, the rate of degradation of I
B-
is faster than normal (30). Previously, we have shown that I
B-
phosphorylated at Tyr-42 is refractory to TNF-induced degradation (31).
Thus, it is possible that I
B-
from HuT-78 cells is phosphorylated
at Tyr-42. Phosphotyrosine blots, however, did not reveal any tyrosine
phosphorylation (data not shown). A lack of retarded mobility of
I
B-
from HuT-78 cells on SDS-PAGE gels also indicated a lack of
phosphorylation. It is possible that the p50-p65 heterocomplex is
mutated so that it can no longer bind to endogenous I
B-
. However,
it is unlikely because we found that the p50-p65 heterocomplex can bind
both exogenously added I
B-
, can bind to the NF-
B binding site
in the DNA, and is supershifted by the antibodies.
NF-B activation in HuT-78 cells was not only refractile to TNF, the
constitutive DNA binding activity in these cells remained unchanged by
a host of other NF-
B activators, including lipopolysaccharide, H2O2, ceramide, IL-1, and phorbol 12-myristate
13-acetate. Western blot analysis indicated that besides a nuclear pool
there is a cytoplasmic pool of p50-p65 in HuT-78 cells. Coprecipitation
followed by Western blot analysis results indicated that the
cytoplasmic pool of p50-p65 subunits in HuT-78 cells exists in complex
with I
B-
. The absence of further activation by various stimuli
suggest the lack of nuclear translocation of p50-p65 from the
cytoplasm, perhaps because of decreased proteolytic activity of the
enzyme required to degrade I
B-
, as noted earlier.
Our results indicate that NF-B activation and apoptosis are linked,
but how constitutive activation of NF-
B prevents apoptosis of
HuT-78 cells is not clear. Several genes that are known to down-regulate apoptosis are regulated by NF-
B activation, including the zinc finger protein A20 (32), manganese superoxide dismutase (33),
and cIAP2 (cellular inhibitor for apoptosis) (34). It is possible that
these genes are constitutively expressed in HuT-78 cells, thus leading
to inhibition of apoptosis. Our results are consistent with reports
that mice that lack the NF-
B p65 gene die early in embryonic
development from massive cellular death of hepatic parenchyma (35). The
antiapoptotic role of NF-
B was also demonstrated from the
observation that embryonic fibroblast from I
B knockout mice are
resistance to TNF (6). Similarly, transfection of a dominant negative
form of I
B-
cDNA prevented the TNF-induced apoptosis of cells
(7, 8).
Our results show that pretreatment of HuT-78 cells with PDTC inhibited
constitutive NF-B activation and sensitized the cells to TNF-induced
apoptosis, thus suggesting a critical role of ROS. PDTC is a potent
inhibitor of inducible NF-
B activation (28, 36) in most cells. It
displays antioxidant property both by metal chelating and by acting as
a radical scavenger (37, 38). Generation of ROS has been proposed as an
important mechanism to mediate the apoptotic and gene regulatory
effects of TNF (39, 40). Our results, however, indicate ROS is also
involved in protection of cells from apoptosis by activating
NF-
B.
Besides PDTC, treatment of cells with anti-TNF antibodies also
down-regulated NF-B and inhibited cell growth. These results, which
are consistent with previous report (41), suggest an autocrine role for
TNF in induction of resistance. In addition, studies employing a TNF
expression vector and an antisense TNF mRNA expression vector
transfected into TNF-sensitive and -insensitive cells clearly demonstrated that endogenously made TNF is protective against TNF-induced cytotoxicity (42). These observations further confirm the
inhibitory role of NF-
B in apoptosis. Additionally, our study demonstrates the autocrine growth-promoting role of TNF through the
generation of reactive oxygen intermediates. Inhibitors of both nuclear
factor-
B and activator protein-1 activation have been shown to block
the neoplastic transformation response (43). In summary, our study
demonstrates the role of constitutively expressed NF-
B in the
induction of resistance in HuT-78 cells through generation of TNF and
ROS.
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ACKNOWLEDGEMENT |
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We thank Dr. Sunil Manna for performing
NF-B-CAT reporter assay.
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FOOTNOTES |
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* This work was conducted by the Clayton Foundation for Research and had financial support in the form of an associateship (to D. G.) from the Department of Biotechnology, Government of India.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. Tel.:
713-792-3503/6459; Fax: 713-794-1613; E-mail:
aggarwal{at}utmdacc.mda.uth.tmc.edu.
1 The abbreviations used are: MDR, multiple drug resistance; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13 acetate; EMSA, electrophoretic mobility shift assay; ROS, reactive oxygen species; PDTC, pyrrolidine dithiocarbamate; PAGE, polyacrylamide gel electrophoresis; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; IL, interleukin; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase.
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REFERENCES |
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