From the Cytokine Research Section, Department of Molecular
Oncology, The University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030 Radiation Research Laboratory,
University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Several recently identified intracellular
proteins associate with the tumor necrosis factor (TNF) receptor and
activate nuclear transcription factor (NF)-B, c-Jun kinase, and
apoptosis. However, the mechanism is not understood. In the present
report, we investigated the role of reactive oxygen intermediates in
TNF-induced signaling. Overexpression of manganese superoxide dismutase
(Mn-SOD) in human breast cancer MCF-7 cells completely abolished
TNF-mediated NF-
B activation, I
B
degradation, p65 nuclear
translocation, and NF-
B-dependent reporter gene
expression. Besides TNF, phorbol ester-, okadaic acid-, ceramide-, and
lipopolysaccharide-induced activation of NF-
B was blocked by Mn-SOD,
indicating a common pathway of activation. H2O2-induced NF-
B activation, however,
was potentiated. In addition, Mn-SOD blocked the TNF-mediated
activation of activated protein-1, stress-activated c-Jun protein
kinase, and mitogen-activated protein kinase kinase. TNF-induced
antiproliferative effects and caspase-3 activation, indicators of
apoptosis, were also completely suppressed by transfection of cells
with Mn-SOD. Suppression of apoptosis induced by okadaic acid,
H2O2, and taxol was also inhibited by Mn-SOD
but not that induced by vincristine, vinblastine, or daunomycin. Overall, these results demonstrate that, in addition to several recently identified signaling molecules, reactive oxygen intermediates play a critical role in activation of NF-
B, activated protein-1, c-Jun kinase, and apoptosis induced by TNF and other agents.
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INTRODUCTION |
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TNF1 plays a major role
in tumorigenesis, inflammation, viral replication, and septic shock
(see Ref. 1 and references within). How a single molecule can mediate
such a wide array of effects is not known, but these effects are known
to be mediated through two different receptors, p60 (type I) expressed
on all cell types and p80 (type II) expressed primarily on immune
cells. Within the last 5 years, over 15 distinct proteins have been
identified that associate with the cytoplasmic domain of the p60
receptor and are involved in activation of apoptosis, NF-B,
N-terminal c-Jun kinase (JNK), and mitogen-activated protein kinase
(see Ref. 2 and references within). Similarly, the cytoplasmic domain of the p80 receptor has been reported to associate with four proteins that play a role in activation of NF-
B and apoptosis. How any of
these proteins mediate apoptosis or NF-
B activation is less clear.
By using metabolic inhibitors, several studies have indicated that
TNF-mediated activation of either NF-
B or apoptosis or activation of
various kinases requires the production of various intermediates including reactive oxygen intermediates (ROI) (3). We conducted a
series of experiments using superoxide dismutase (SOD) to explore the
role of ROI.
SOD is a family of antioxidant enzymes that convert harmful superoxide
radicals into H2O2, which in turn is
metabolized to harmless water and oxygen by catalase and glutathione
peroxidase (4, 5). In mammalian cells, two main types of SOD are found; viz. manganese SOD (Mn-SOD), located in the mitochondria,
and copper-zinc SOD, found in the cytoplasm. Previous studies have shown that overexpression of Mn-SOD blocks TNF-mediated cytotoxicity (6-8). How overexpression of Mn-SOD affects TNF-induced transcription factors including NF-B and AP-1 is not known, however. It is also
not known how overexpression of SOD affects TNF-induced activation of
stress-activated protein kinase, also called JNK, and the growth modulatory kinases of the extracellular signal-regulated kinase family
(MAP kinase kinase kinase/MAP kinase (MEK)/MAP kinase), implicated in
the TNF-induced activation of AP-1 and NF-
B, respectively (9,
10).
In the present report, we demonstrate that overexpression of Mn-SOD in
human breast tumor MCF-7 cells blocks TNF-induced cytotoxicity and
activation of caspase-3 (indicator of apoptosis), NF-B, AP-1, JNK,
and MEK. The effect of SOD was not specific to TNF, since other agents
that activate NF-
B (e.g. PMA, interleukin-1, and okadaic
acid) or induce apoptosis (e.g. okadaic acid and taxol) were
also inhibited, thus suggesting that the ROI has a critical role in
activation of signaling by a wide variety of agents.
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EXPERIMENTAL PROCEDURES |
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Materials--
Penicillin, streptomycin, RPMI 1640 medium, and
fetal calf serum were obtained from Life Technologies, Inc. Glycine,
NaCl, and bovine serum albumin were obtained from Sigma.
Bacteria-derived recombinant human TNF, purified to homogeneity with a
specific activity of 5 × 107 units/mg, was kindly
provided by Genentech, Inc. (South San Francisco, CA). Antibody against
IB
and double-stranded oligonucleotide having an AP-1 consensus
sequence were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). The rat MDR1bCAT plasmid
243RMICAT containing the
chloramphenicol acetyl transferase (CAT) gene with either wild-type or
mutated NF-
B binding site was kindly supplied by Dr. M. Tien Kuo of
the University of Texas M. D. Anderson Cancer Center (Houston,
TX). The characterization of these plasmids has been described
previously in detail (11).
Cell Lines-- Human breast MCF-7 cells were stably transfected with control (neo) and Mn-SOD genes similarly to what was described in detail previously (8). Two different Mn-SOD cDNAs were examined. Polymorphism in the Mn-SOD gene results in two different amino acids (either Ile or Thr) at position 58 in the human population (12). The replacement of Thr for Ile leads to a protein with about 50% activity and a reduction in protein stability after heat (12). Sense human Mn-SOD cDNA was originally obtained from Dr. Daret St. Clair. After the cDNA was ligated into the pcDNA3 plasmid (Invitrogen) containing a neomycin resistance marker, it was sequenced, which revealed that this cDNA contained an ACA codon for threonine at amino acid 58. Site-directed mutagenesis was performed to change ACA to ATA, the codon for isoleucine.2 The change was confirmed by cDNA sequencing. The MCF-7 cell line was found to express the isoleucine form of Mn-SOD.
These two types of plasmids were transfected into MCF-7 cells (obtained from ATCC at passage number 148) by a protocol similar to that described previously (8). Briefly, at passage 155, these cells were transfected with pcDNA3 plasmids by themselves (neo control) or plasmids containing sense human Ile58 Mn-SOD cDNA or sense human Thr58 Mn-SOD cDNA under the control of a cytomegalovirus promoter. 3 × 105 cells were transfected with 3 µg of plasmids for 20 h using the LipofectAMINE method according to the manufacturer's instructions (Life Technologies, Inc.). Forty-eight hours after the transfection, cells were subcultured and grown in selection media supplemented with 700 µg/ml of G418 for 15 days. G418-resistant clones were selected by a ring colony method. Selected clones were maintained in complete media supplemented with 200 µg/ml of G418. MCF-7 cells were normally maintained in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 1 mM pyruvate, and nonessential amino acids in humidified atmosphere of 5% CO2 at 37 °C.NF-B Activation Assays--
To determine NF-
B activation,
electrophoretic mobility shift assays were carried out essentially as
described (13). Briefly, nuclear extracts prepared from TNF-treated
cells (2 × 106/ml) were incubated with
32P-end-labeled 45-mer double-stranded NF-
B
oligonucleotide (4 µg of protein with 16 fmol of DNA) from the human
immunodeficiency virus long terminal repeat,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (underline indicates NF-
B binding sites) for 15 min at 37 °C, and
the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. 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. The dried gels were visualized, and
radioactive bands were quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
AP-1 Activation Assay-- To determine the activation of AP-1, 4-5 µg of nuclear extract prepared as indicated above were incubated with 16 fmol of the 32P-end-labeled AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (Santa Cruz Biotechnology, Inc.) (underline indicates AP-1 binding sites) for 15 min at 37 °C and analyzed by electrophoretic mobility shift assay. The specificity of binding was examined by competition with unlabeled oligonucleotide. Visualization and quantitation of radioactive bands was carried out by a PhosphorImager (Molecular Dynamics) using ImageQuant software.
Western Blot for IB
, p50, and p65--
To determine the
levels of I
B
, postnuclear (cytoplasmic) extracts were prepared
(14) from TNF-treated cells and resolved on 10% SDS-polyacrylamide
gels. To determine the levels of NF-
B proteins, p50, and p65,
nuclear and postnuclear extracts prepared from TNF-treated cells were
resolved on 8% SDS-PAGE. After electrophoresis, the proteins were
electrotransferred to nitrocellulose filters, probed with rabbit
polyclonal antibodies against I
B
, p50, or p65 and detected by
chemiluminescence (ECL, Amersham Pharmacia Biotech) (14). The bands
obtained were quantitated using Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Molecular Dynamics).
Cytotoxicity Assays-- The cytotoxic effects of TNF on MCF-7 cells were determined by the amount of [3H]thymidine incorporated by the cells as described previously (15). Briefly, cells were plated at 5000/well in 0.1 ml of medium in 96-well flat-bottomed Falcon plates. Different concentrations of TNF were added in an additional 0.1 ml of medium and incubated at 37 °C for 48 h. During the last 6 h before harvesting, [3H]thymidine (5 mCi/mmol; Amersham Pharmacia Biotech) was added to each well (0.5 µCi/well), and then cells were harvested with the aid of a Filtermate 196 harvester (Packard Instruments Co., Meriden, CT). Radioactivity bound to the filter was measured in a liquid scintillation counter (model 1600 TR; Packard Instrument Co.).
The cytotoxicity was also measured by the modified tetrazolium salt 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay (16). Briefly, cells (5000 cells/well) were incubated in the presence or absence of the indicated test sample in a final volume of 0.1 ml for 72 h at 37 °C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in phosphate-buffered saline) was added to each well. After a 2-h incubation at 37 °C, 0.1 ml of the extraction buffer (20% SDS, 50% dimethyl formamide) was added. After an overnight incubation at 37 °C, the optical densities at 590 nm were measured using a 96-well multiscanner autoreader (Dynatech MR 5000), with the extraction buffer as a blank. Percentage of cytotoxicity was determined as follows: percentage of cytotoxicity = (1Immunoblot Analysis of PARP Degradation-- TNF-induced apoptosis was examined by proteolytic cleavage of PARP (17). Briefly, cells (2 × 106/ml) were treated with TNF for different times at 37 °C and then extracted by incubation 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, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml benzamidine, and 1 mM DTT. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 µg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP antibody, and then detected by chemiluminescence (ECL; Amersham Pharmacia Biotech). Apoptosis was represented by the cleavage of 116-kDa PARP into 85- and 41-kDa peptide products (18).
MAP Kinase Kinase Assay-- MCF-7 cells, stimulated with different concentrations of TNF for 30 min at 37 °C were washed with Dulbecco's phosphate-buffered saline and then lysed with buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml benzamidine, 1 mM DTT, and 1 mM sodium orthovanadate. A 50-µg aliquot of protein was resolved on 10% SDS-PAGE, electrotransferred to nitrocellulose filters, and probed with the phosphospecific anti-p44/42 MAP kinase (Thr202/Tyr204) antibody (New England Biolabs, Inc.) raised in rabbit (1:3000 dilution). Then the membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1:3000 dilution), and the bands were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech).
c-Jun Kinase Assay--
The c-Jun kinase assay was performed by
a modified method as described earlier (18). Briefly, after treatment
of cells (3 × 106/ml) with TNF for 10 min, cell
extracts were prepared by lysing cells in buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM
NaCl, 1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml benzamidine,
and 1 mM DTT. Cell extracts (150-250 µg/sample) were
immunoprecipitated with 0.3 µg of anti-JNK antibody for 60 min at
4 °C. Immune complexes were collected by incubation with protein
A/G-Sepharose beads for 45 min at 4 °C. The beads were extensively
washed with lysis buffer (4 × 400 µl) and kinase buffer (2 × 400 µl; 20 mM HEPES, pH 7.4, 1 mM DTT, 25 mM NaCl). Kinase assays were performed for 15 min at
30 °C with glutathione S-transferase-Jun-(1-79) as a substrate in 20 mM HEPES, pH 7.4, 10 mM
MgCl2, 1 mM DTT, and 10 µCi of
[32P]ATP. Reactions were stopped with the addition of
15 ml of 2 × SDS sample buffer, boiled for 5 min, and subjected
to SDS-PAGE (9%). Glutathione S-transferase-Jun-(1-79) was
visualized by staining with Coomassie Blue, and the dried gel was
analyzed by a PhosphorImager (Molecular Dynamics).
Transient Transfection and CAT Assay--
To determine the
TNF-induced NF-B-mediated reporter gene transcription, MCF-7
(neo and Mn-SOD) cells were transiently transfected by the
calcium phosphate method with the plasmids 243RMICAT (contains wild-type NF-
B binding site) and
243RMICAT-
m (mutated binding site), according to the instructions supplied by the manufacturer (Life
Technologies). After 6 h of transfection, the cells were plated
for 24 h at 37 °C, stimulated with 100 pM of TNF
for 1 h, washed, and examined for CAT activity as described
(19).
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RESULTS |
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In the present study, we investigated the effect of transfection
of the Mn-SOD gene on the transcription factors NF-B and AP-1 and
the associated kinases of the MAP kinase family induced by TNF. We also
examined the effect of this gene on apoptosis induced by TNF and a wide
variety of other agents. For all studies, untransfected cells and those
transfected with the neo gene were used as a control. Two
different types of clones were studied. Clone SOD18 was transfected
with Thr58 Mn-SOD cDNA and was used for the majority of
the experiments. SOD18 had an approximately 6-fold increase in Mn-SOD
immunoreactive protein as determined by Western blotting compared with
wild type or neo lines, but only a 1.8-fold increase in
Mn-SOD enzymatic activity). Clones 11, 40, and 59 were obtained from
cells transfected with Ile58 Mn-SOD cDNA. The increases
in immunoreactive protein over wild type were 13-, 14-, and 8-fold for
clones 11, 40, and 59, respectively. The increases in enzymatic
activity over wild type and neo controls were 24-, 40-, and
8-fold, for clones 11, 40, and 59, respectively. Thus, both types of
clones have similar increases in immunoreactive protein, but the
Ile58 clones have far more specific activity than the
Thr58 clone. The actual enzymatic activities in units per
mg of protein for wild type, neo, SOD18, Mn11, Mn40, and
Mn59 were 7 ± 5, 7 ± 3, 12 ± 1, 142 ± 20,
238 ± 56, and 46 ± 13, respectively. Mn-SOD-transfected cells grow more slowly than wild type or neo controls,
perhaps because of H2O2 production in these
cells. All other clones grow at similar rates to each other, however.
To ascertain the stability of the gene, cells were routinely checked
for the expression of SOD enzyme activity. When examined for cell
surface expression of TNF receptors by radioreceptor assay, both
neo- and Mn-SOD-transfected cells were found to show similar
specific binding of labeled TNF (3896 ± 100 versus
3975 ± 21 cpm).
Mn-SOD Inhibits TNF-induced NF-B Activation--
Nontransfected
and neo- and Mn-SOD-transfected MCF-7 cells were stimulated
with different concentrations of TNF, and nuclear extracts were
prepared and assayed for NF-
B by electrophoretic mobility shift
assay. As little as 10 pM TNF activated NF-
B in both
nontransfected and neo gene-transfected cells, whereas in Mn-SOD gene-transfected cells, no significant NF-
B activation was
noted, even at 1000 pM TNF concentration (Fig.
1A). Since antibodies against
the p50 and p65 subunits of NF-
B supershifted the band on the gel
shift (Fig. 1B), NF-
B suppressed by Mn-SOD consisted
of p50 and p65 subunits. This supershift was specific, since antibodies
to c-Rel or cyclin D1 or preimmune serum (PIS) had no
effect. Specificity is also indicated from the observations that this
band competed with unlabeled probe and that it did not bind to a
oligonucleotide with mutated NF-
B site (Fig. 1B). Since no difference was noted between nontransfected and
neo-transfected MCF-7 cells, for all following experiments
only neo-transfected cells were used as a control.
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Mn-SOD Inhibits NF-B Activation Induced by a Wide Variety of
Agents--
Different agents may activate NF-
B by different
mechanisms (20). For instance, TNF-induced NF-
B activation is
protein kinase C-independent, whereas that activated by PMA is protein kinase C-dependent. Therefore, we examined the effect of
Mn-SOD on the activation of NF-
B by PMA, LPS,
H2O2, okadaic acid, and ceramide (Fig.
2). As we have shown previously, all five
agents activated NF-
B in human histiocytic lymphoma U-937 cells
(upper panel). Although the overall activation was less
pronounced than in U-937 cells, all five activated NF-
B in
neo-transfected MCF-7 cells (middle panel).
Mn-SOD transfection blocked NF-
B activation by all agents except
H2O2 (lower panel). The NF-
B
activation by the latter was actually potentiated by Mn-SOD, thus
suggesting that H2O2 activates NF-
B by a
mechanism that is quite different from that of other agents.
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Mn-SOD Inhibits TNF-dependent Degradation of
IB
--
The translocation of NF-
B to the nucleus is preceded
by the phosphorylation and proteolytic degradation of I
B
. To
determine whether Mn-SOD also inhibits TNF-induced I
B
degradation, the cytoplasmic levels of I
B
proteins after
treatment of cells with TNF for different times were examined by
Western blot analysis. I
B
was degraded within 10 min of TNF
treatment and reappeared by 60 min in MCF-7 (neo) cells
(Fig. 3A). In
Mn-SOD-transfected cells, however, no degradation of I
B
was
observed after TNF treatment even up to 60 min. In contrast to
neo-transfected cells, levels of p65 remained high in
cytoplasmic extracts of Mn-SOD-transfected cells (Fig. 3B).
The nuclear levels of p65 protein increased in neo cells but
not in Mn-SOD-transfected cells, thus suggesting that in
Mn-SOD-transfected cells TNF is unable to dissociate p65 from
I
B
.
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Mn-SOD Blocks NF-B-dependent Reporter Gene
Expression Induced by TNF--
Our results up to now show that Mn-SOD
blocks the binding of NF-
B protein to DNA. Whether it also blocks
NF-
B-dependent gene expression was also examined. The
rat multiple drug resistance gene promoter contains an NF-
B binding
site; the promoter was ligated with CAT reporter gene. This plasmid was
transfected into neo and Mn-SOD-transfected cells, and then
the cells were stimulated with 10 and 100 pM TNF. TNF
activated CAT activity by 3-4-fold in neo cells, whereas a
minimal increase in CAT activity over the basal level was observed in
Mn-SOD-transfected cells (Fig. 4). These
effects were specific, since the plasmid with mutated NF-
B binding
site was not activated on treatment of cells with TNF. These results
thus demonstrate that Mn-SOD also blocks TNF-induced transcriptional
activity.
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Mn-SOD Inhibits TNF-induced AP-1 Activation--
TNF is a potent
activator of another transcriptional factor, AP-1 (9). Several reports
indicate that the requirements for AP-1 activation differ from that of
NF-B activation (21). Whether activation of AP-1 by TNF is through
ROI is not known. Therefore, we investigated the effect of Mn-SOD on
TNF-induced AP-1 activation. As shown in Fig.
5, TNF activated AP-1 in a
dose-dependent manner with 6-fold activation at 100 pM in neo cells. Mn-SOD-transfection, however,
resulted in only 2-fold activation of AP-1 even with 10,000 pM TNF. This activation was specific, since it was
completely inhibited by the unlabeled probe.
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Mn-SOD Inhibits TNF-induced c-Jun Kinase Activation-- TNF is also a potent activator of JNK. This kinase is activated in response to different kinds of stress and is needed for activation of AP-1. As shown in Fig. 6A, TNF activated JNK in neo cells in a dose-dependent manner, with 27-fold activation noted with 10,000 pM TNF. Mn-SOD transfection, in contrast, completely suppressed TNF-induced JNK activation. These results thus indicate that ROI generated by TNF is also essential for JNK activation.
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Mn-SOD Inhibits TNF-induced Mitogen-activated Protein Kinase Kinase
Activation--
It has been reported that TNF activates a
serine/threonine kinase, MAP kinase kinase kinase (also called MEKK),
which in turn activates a dual specificity kinase, MAP kinase kinase
(also called MEK) and that activates another serine/threonine MAP
kinase, mitogen-activated protein kinase/extracellular signal-regulated
kinase. There are reports that MAP kinase kinase kinase is required for
TNF-induced NF-B activation, so the question of whether Mn-SOD
affects TNF-induced activation of MEK was investigated. MEK was
activated by TNF in a dose-dependent manner in
neo cells but not in Mn-SOD-transfected MCF-7 cells (Fig.
6B). These results indicate that activation of MEK by TNF
also occurs through the generation of ROI.
Mn-SOD Blocks TNF-mediated Cytotoxicity and Activation of Caspase-3-- By cell counting and by clonogenic assay, we have previously shown that Mn-SOD blocks the antiproliferative effects of TNF (8). We now show by thymidine incorporation assay (DNA synthesis) as well as by MTT assay (mitochondrial activity) that Mn-SOD completely protected MCF-7 cells from TNF-induced cytotoxicity (Fig. 7, upper panel). TNF exhibited its cytotoxic effects either by inducing necrosis or apoptosis. One hallmark of apoptosis is the activation of caspase-3 activity, which leads to cleavage of PARP substrate from 116 to 85 kDa. TNF induced cleavage of PARP within 1 h in neo cells (Fig. 7, lower panel), whereas Mn-SOD cells were resistant to PARP cleavage even after 2 h of TNF treatment. These results thus suggest that Mn-SOD also blocks TNF-induced activation of caspase-3.
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Mn-SOD Protects Cells from Taxol-, Okadaic Acid-, and
H2O2-induced Cytotoxicity and NF-B
Activation--
Besides TNF, MCF-7 cells are sensitive to cytotoxic
effects of a wide variety of other agents including chemotherapeutic
agents such as vinblastine, vincristine, taxol, and daunomycin and to H2O2 and okadaic acid. How Mn-SOD affects the
cytotoxicity induced by these agents was also investigated. Mn-SOD
completely protected the cells from okadaic acid- and taxol-induced
cytotoxicity (Fig. 8A). Unlike
these agents, however, Mn-SOD had no effect on cytotoxicity induced by
vincristine, vinblastine, and daunomycin. Cells treated with
H2O2 were only partially protected. These
results thus indicate that different agents induce cytotoxicity by
different mechanisms. Most chemotherapeutic agents that induce
apoptosis also appear to activate NF-
B. Whether this activation is
through ROI is not known. Therefore, we also investigated the effect of
Mn-SOD on NF-
B activation by various chemotherapeutic agents. The
results shown in Fig. 8B indicate that all of the agents
that induced cytotoxicity also activated NF-
B, but only
taxol-induced activation was suppressed significantly by Mn-SOD. These
results suggest that taxol induces cytotoxicity and NF-
B activation
through the same free radical species, whereas other agents transduce
their signal through a different mechanism.
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DISCUSSION |
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The aim of the present report was to investigate the role of ROI
in TNF-induced activation of NF-B, AP-1, JNK, MEK, and caspase-3. To
determine this, human breast cancer MCF-7 cells were transfected with
the Mn-SOD gene; neo-transfected cells were used as
controls. Overexpression of Mn-SOD suppressed TNF-induced I
B
degradation, NF-
B activation, and NF-
B-dependent
reporter gene expression. It also blocked TNF-induced activation of
AP-1, JNK, MEK, caspase-3, and cytotoxicity (see Fig.
9). The effects of Mn-SOD were not specific to TNF, since NF-
B activation by okadaic acid, PMA, LPS,
and ceramide and apoptosis induced by okadaic acid, taxol, and
H2O2 were also blocked. All of these results
suggest that ROI are intermediates for transmission of a wide variety
of TNF effects. These effects are not cell type-specific. Moreover,
both of the polymorphic forms of Mn-SOD led to similar effects.
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Human breast carcinoma MCF-7 cells are known to express only the p60
form of the TNF receptor (22). Recently, over 15 different proteins
have been identified that physically associate with the cytoplasmic
domain of the TNF receptor and are responsible for mediation of
apoptosis and activation of NF-B, JNK, MEK, and caspase-3 as
investigated here. How any of these receptor-associated proteins
generate ROI is not clear. By using pyrrolidine dithiocarbamate (a
metal chelator) and N-acetyl-L-cysteine (a
precursor of glutathione), ROI has been implicated in TNF-induced
NF-
B activation (23-26). There are other reports, however, that
show that both pyrrolidine dithiocarbamate and
N-acetyl-L-cysteine not only fail to inhibit activation of NF-
B but by themselves activate NF-
B (27, 28). Our
results, however, support the notion that ROI are critical for
TNF-induced NF-
B activation. It is not possible to ascertain the
reactive oxygen species from studies with SOD-transfected cells alone.
This is because increased SOD levels certainly lead to decreased
superoxide radical levels but may also lead to increased concentrations
of hydrogen peroxide and other hydroperoxides (4, 5). Moreover, since
the nitric oxide radical effectively competes with SOD for superoxide,
increasing SOD will lead to increased levels of NO. Thus, SOD
transfection can lead to changed levels of superoxide, hydrogen
peroxide, and nitric oxide, as well as any species derived from them.
It is not possible with a single transfection to determine the effector
species. Double transfection with peroxide-removing enzymes may,
however, allow identification of the reactive species involved.
How TNF activates NF-B is not fully understood. Recently, several
kinases have been identified that play a critical role in TNF-induced
NF-
B activation. This includes MAP kinase kinase kinase, Ikk-
,
Ikk-
, and NIK (for a review, see Ref. 29). We found that the
overexpression of Mn-SOD blocked the activation of TNF-induced MEK,
suggesting that superoxide radical is needed for activation of this
kinase. It is possible that the suppression of NF-
B is due to
down-regulation of MEK.
Mn-SOD converts superoxide radicals to H2O2 and
molecular oxygen (30). Interestingly, we found that
H2O2-activated NF-B was potentiated by
Mn-SOD. These results indicate that ROI species involved in activation
of NF-
B by TNF, and most other agents must differ from that of
H2O2. It is possible that TNF-induced NF-
B
activation is through a superoxide radical, whereas that induced by
H2O2 is through a hydroxyl radical.
N-acetyl-L-cysteine, however, has been shown to
inhibit both H2O2 (24) and TNF-induced NF-
B
activation, perhaps because it quenches both the superoxide radical and
hydroxyl radical species. A redox-independent mechanism of activation
of NF-
B has also been described. For instance, interleukin-1,
okadaic acid, and vanadium analogues induce NF-
B activation without
involvement of ROI (27, 31-33). Our results based on Mn-SOD
overexpression, however, indicate that NF-
B activation by okadaic
acid requires superoxide radical. Some of these differences may be due
to different cell types as demonstrated by others (26, 27) and also may
be due to the lack of specificity of ROI quenchers used as indicated
above.
Our results show that various chemotherapeutic agents, including taxol,
doxorubicin, daunomycin, vincristine, and vinblastine activate NF-B.
These results confirm recent reports (34, 35). How any of these agents
activate NF-
B is not known. Our results show that agents that act by
different mechanisms induced both cytotoxicity and NF-
B activation
in MCF-7 cells. Mn-SOD, however, blocked only taxol-induced
cytotoxicity and NF-
B activation. These results suggest that taxol
transduces its signal both for cytotoxicity and NF-
B activation
through the superoxide radical, whereas anthracyclins (doxorubicin and
daunomycin) and vinca alkaloids (vincristine and vinblastine) act
through an ROI-independent mechanism.
We found that Mn-SOD also blocked TNF-induced AP-1 activation. While we
examined TNF-inducible activation, other investigators recently showed
that overexpression of Mn-SOD abolished the constitutive AP-1 activity
in growing fibrosarcoma cells (36). Our finding on the suppression of
TNF-induced AP-1 by Mn-SOD was surprising in view of studies that
showed that the antioxidants pyrrolidine dithiocarbamate and
N-acetyl-L-cysteine activate AP-1 by themselves (21). Since activation of AP-1 requires activation of JNK, we found
that Mn-SOD also suppressed TNF-induced JNK activity. There is a recent
report (37), however, that indicates that the TNF-receptor-associated kinase NIK is required for activation of NF-B and AP-1 but not JNK,
indicating that TNF-induced AP-1 activation is JNK-independent.
We also demonstrated that Mn-SOD suppresses the TNF-induced activation
of caspase-3, an enzyme needed for TNF-induced apoptosis. While there
are reports that Mn-SOD can block TNF-induced cytotoxicity (7, 8), ours
is the first to show that it is through inhibition of caspase-3
activation. Mn-SOD blocked the cytotoxic effects not only of TNF, but
also of taxol and okadaic acid, suggesting that all of these agents
kill cells through a superoxide radical-dependent mechanism. Similar to these agents,
H2O2-induced cytotoxicity was also blocked by
Mn-SOD, suggesting that H2O2 activates
cytotoxicity and NF-B by different mechanisms. The lack of effect of
Mn-SOD on cytotoxicity induced by vincristine, vinblastine, and
daunomycin suggests that these agents kill the cells by a mechanism
independent of the superoxide radical.
Mn-SOD also blocked TNF-induced NF-B-dependent gene
expression. Previously, it has been shown that TNF-induced
interleukin-1
expression in human fibrosarcoma cells is blocked by
overexpression of Mn-SOD (38). Since NF-
B activation is needed for
interleukin-1 expression, it is possible that suppression of NF-
B as
shown in our studies is involved in interleukin-1 inhibition. Mn-SOD has also been reported to inhibit the malignant phenotype of human tumor cells (39). Since several genes involved in tumorigenesis contain
NF-
B in their promoter (20), it is possible that suppression of
NF-
B by Mn-SOD is linked with its effects on tumorigenesis.
Recent studies showed that TNF-activated NF-B plays an antiapoptotic
role in TNF-induced apoptosis (40-42). How is not clear, but Mn-SOD is
one of the genes that is regulated through NF-
B (43), thus
suggesting a negative feedback loop. While results from our laboratory
and others show that various effects of TNF are mediated through the
generation of superoxide radicals, TNF is also known to induce Mn-SOD
gene expression in almost all cell types (6). This shows the complexity
in the mechanism of action of TNF. The susceptibility of cells to
TNF-mediated killing might be influenced (besides by Mn-SOD) by
catalase and glutathione peroxidase, which degrades
H2O2 to form water. Overexpression of catalase
has been shown to attenuate TNF and okadaic acid-induced NF-
B
activation (44). Overall, it is quite likely that the total cellular
balance of prooxidants and antioxidant intermediates determines the
signaling mechanism of various responses to TNF and other agents. In
the future, we will examine the other antioxidants in the
Mn-SOD-overexpressing cells to determine if they are also involved in
the mode of action of TNF.
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FOOTNOTES |
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* This work was supported in part by the Clayton Foundation for Research.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.
§ Supported by National Institutes of Health Grant CA 66081.
¶ 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: TNF, tumor necrosis
factor; NF-B, nuclear transcription factor-
B; I
B, inhibitory
subunit of NF-
B; SOD, superoxide dismutase; Mn-SOD, manganese
superoxide dismutase; ROI, reactive oxygen intermediate(s); DTT,
dithiothreitol; CAT, chloramphenicol acetyltransferase; MTT,
3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzoil)-1-piperazinyl)-2(1H)-quinolinone; PARP, poly(ADP-ribose) polymerase; PMA, phorbol 12-myristate
13-acetate; JNK, c-Jun N-terminal kinase; AP-1, activated protein-1;
PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered
saline; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; LPS, lipopolysaccharide.
2 H. J. Zhang, T. Yan, and L. W. Oberley, manuscript in preparation.
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