From the Department of Immunochemistry, German Cancer Research
Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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INTRODUCTION |
The cytokine TNF-
1
plays a pivotal role in a variety of inflammatory, immunological, and
pathological processes. Two well-studied receptors for TNF-
are
TNF-R55 and TNF-R75. These receptors belong to the rapidly growing
family of TNF receptors, including the recently discovered LT-
receptor, DR3/WSL-1/TRAMP, RANK, and TRAIL (for reviews, see Refs. 1
and 2). Binding of the extracellular ligand leads to trimerization of
the receptors and association of numerous cytoplasmic proteins to the
intracellular receptor domain. This binding is induced by ligation of
the TNF-R55 or clustering of the intracellular C-terminal domains,
which are sufficient for the induction of cell death, which coincides
with the activation of transcription factor NF-
B (3).
NF-
B is normally retained in its inactive state in the cytoplasm of
cells by interaction with an inhibitory I
B molecule (for review, see
Ref. 4). Triggering of cells with a variety of inflammatory cytokines,
including TNF-
, induces phosphorylation, ubiquitinylation, and
degradation of I
B (for reviews, see Refs. 5 and 6). This allows the
DNA-binding dimer to enter the nucleus, to bind to its cognate DNA, and
to induce the transcription of its target genes (for a review, see Ref.
7). The target genes encode a variety of proteins involved in immune
responses, cell growth, and apoptosis. Among those are the
anti-apoptotic proteins Mn-superoxide dismutase, the zinc finger
protein A20, and c-IAP2 (8). Previous studies showed that the
activation of NF-
B can counteract TNF-
-induced cell death (for a
review, see Ref. 9). Fibroblasts from mice lacking the transactivating p65 subunit are more sensitive against the cytopathic effect of TNF-
than fibroblasts from p65+/+ mice (10). The inhibition of
NF-
B in various cell types by stable overexpression of a
transdominant negative form of I
B-
renders those cells more
susceptible to cell killing by TNF-
(11-13). However, NF-
B has
also apoptosis-promoting activities. Expression of high levels of the
NF-
B subunit c-Rel in bone marrow cells leads to apoptosis and
autophagocytic cell death (14). Accordingly, the overexpression of the
viral and cellular anti-apoptotic proteins E1B 19K and Bcl-2
impairs NF-
B-dependent transactivation (15, 16).
In this study, we investigated the impact of NF-
B activation on
TNF-
-mediated cell killing in murine L929 fibrosarcoma cells. This
widely used cell line is of particular interest because TNF-
-induced cell death occurs without co-apoptotic stimuli, such as cycloheximide or actinomycin D. Using several different strategies, we demonstrated that TNF-
-triggered cell killing and activation of transcription factor NF-
B are uncoupled in L929 cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Stable Transfections--
Murine L929sA
fibrosarcoma cells were maintained in Dulbecco's modified Eagle's
medium containing 10% FCS and 1% (v/v) penicillin/streptomycin. All
cells were grown in an incubator at 37 °C and 5% CO2.
The plasmid RSV-I
B-
S32/36A was obtained by inserting the
HindIII fragment from the plasmid CMV-I
B-
S32/36A (17)
into the HindIII site of a eukaryotic RSV expression vector.
The NF-
B-dependent reporter gene construct
p(IL6
B)350hu.IL6 was described previously (18). The
plasmids were purified on CsCl gradients, and the L929 cells were
transfected with CaCl2 as described (16). Stably transfected cells were selected in 200 µg/ml G418 for several weeks.
Electrophoretic Mobility Shift Assays (EMSAs)--
Murine L929sA
cells were stimulated with recombinant TNF-
(Boehringer Mannheim)
for 20 min at the indicated concentrations, washed twice with cold TBS
buffer (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.1 mM MgCl2), and scraped off with a rubber
policeman. After centrifugation for 3 min at 3000 rpm, the total
cellular proteins were extracted from the pellet by resuspension in
TOTEX buffer (20 mM Hepes/KOH, pH 7.9, 0.35 M
NaCl, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM
MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride). The tubes were incubated
on ice for 30 min, and equal amounts of the protein contained in the
supernatant were tested for NF-
B DNA binding activity as described
(19).
Western Blot Analysis and Luciferase Assays--
For Western
blotting, the proteins were separated by SDS-polyacrylamide gel
electrophoresis and transferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA) using a semidry blot apparatus
(Bio-Rad). The detection of I
B-
proteins was done as described
(19). For the determination of luciferase activity, the cells were
washed with isotonic buffer and lysed in 100 µl of lysis buffer
(Tropix, Bedford, MA). The luciferase assays were performed according
to the manufacturer's instructions (Promega, Mannheim, Germany) and
quantified in a Duo Lumat LB 9507 luminometer (Berthold, Wildbad,
Germany).
Determination of Cell Viability and IL-6 Production--
The
cytotoxic activity of TNF-
was determined by the colorimetric
3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay essentially as described (20). L929sA cells were grown at a
density of 1 × 104 cells per well in 96-well
microtiter plates. Following the addition of TNF-
(2000 units/ml)
and/or the indicated substances, 20 µl of a MTT solution (5 mg/ml
PBS) was added to all wells at the times given in the figure legends.
After another 3-h incubation, supernatants were removed, followed by
the addition of 100 µl of a 24:1 (v/v) isopropyl alcohol-HCl solution
for 15 min. The absorbance of each well was determined with an
automated plate reader (Digiscan, Asys Hitech, Eugendorf, Austria) at
550 nm. The IL-6 concentrations were determined with an anti-human IL-6 enzyme-linked immunosorbent assay kit (Boehringer Mannheim) according to the manufacturer's instructions.
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RESULTS |
BA Reduces TNF-
-induced Cytotoxicity but Not the Activation of
NF-
B--
To study the linkage between TNF-
-induced NF-
B
activation and cell death in L929 cells, we investigated the effect of
BA, which prevents the generation of mitochondrial permeability
transition pores during the apoptotic process (21). The addition of 50 µM BA clearly decreased the cytotoxic effects of various
concentrations of TNF-
on L929 cells (Fig.
1A). In contrast, the
TNF-
-induced DNA binding activity of NF-
B was not influenced by
BA at various concentrations (Fig. 1B). The influence of BA
on TNF-
-stimulated NF-
B-dependent transcription
was tested by adding BA, TNF-
, or a combination of both to a pool of
L929 cells stably transfected with a NF-
B-dependent
luciferase reporter gene. BA was unable to influence the basal or
TNF-
-induced transactivation by NF-
B (Fig. 1C). These
results suggest that it is possible to reduce cytotoxicity without
affecting NF-
B activation in L929 cells.

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Fig. 1.
Influence of BA on TNF- -induced cell
killing and NF- B activation. A, the indicated
concentrations of TNF- were added alone or together with 50 µM BA to L929 cells. After 20 h, cell viability was
determined using the MTT assay. Bars indicate the S. D.
from three experiments. B, L929 cells were incubated for 20 min with the indicated combinations of TNF- and/or BA. Subsequently,
total cell extracts were prepared and tested for DNA binding of
activated NF- B by EMSA. The filled arrowhead indicates
the location of the DNA-NF- B complex, the circle
indicates the position of a constitutively DNA-binding protein, and the
open arrowhead points to the unbound oligonucleotide.
C, TNF- and/or BA were incubated at the indicated
combinations for 8 h with a pool of L929 cells stably transfected
with a B-dependent luciferase reporter gene.
Subsequently, the luciferase activity was determined. Bars
indicate S. D.
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Dexamethasone Impairs TNF-
-induced NF-
B Transactivation and
Cytotoxicity--
A protective role of NF-
B for TNF-
-induced
cell killing would imply that the inhibition of this transcription
factor should result in enhanced cell killing. Therefore, we
investigated the effects of DEX on cell death and
NF-
B-dependent gene expression induced by TNF-
. L929
cells stably transfected with a NF-
B-dependent reporter
gene were treated with different combinations of TNF-
and DEX as
indicated (Fig. 2A). The
TNF-
-induced gene expression was significantly impaired by DEX, a
known inhibitor of NF-
B gene expression (22). Previous reports
attributed the inhibitory effect of glucocorticoids to the increased
production of the inhibitory I
B-
molecule, which then would
dissociate NF-
B from its cognate DNA (23, 24). To test whether this
proposed mechanism also applies to L929 cells, the influence of DEX on
TNF-
-activated NF-
B was tested by EMSAs. As seen in Fig.
2B, DEX had no effect on the DNA binding activity of
NF-
B. The same extracts were assayed for the relative levels of
I
B-
in Western blot experiments. Short-time treatment of cells
with TNF-
resulted in the induced degradation of I
B-
, which
was resynthesized after 2 h (Fig. 2B). Treatment of
cells with DEX had no impact on the relative levels of I
B-
at any
time point, suggesting that DEX interferes in L929 cells with
NF-
B-mediated transactivation rather than detectably influencing the
I
B-
levels. The effect of DEX on TNF-
-induced cell death was
tested by adding DEX, TNF-
, or a combination of both to the cells.
When cell viability was measured after various incubation times (Fig.
2C), it was found that DEX significantly protected the L929
cells from cell death, indicating that NF-
B activation does not
protect this cell line from TNF-
-induced cell death.

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Fig. 2.
Influence of DEX on NF- B
activation and cell death induced by TNF- . A, a pool
of L929 cells stably transfected with a B-dependent
reporter gene was preincubated for 1 h with 1 µM
DEX. The cells were treated for another 8 h with 2000 units/ml
TNF- as shown, and luciferase activity was determined. The error
bars indicate the S. D. obtained from three different experiments.
B, L929 cells were treated with the indicated combinations
of TNF- and DEX for the periods given in the figure. Subsequently,
total extracts were prepared and tested for DNA binding of activated
NF- B by EMSA. The symbols used are as explained in Fig. 1. The same
extracts were tested for the I B- protein in a Western blot
(WB). The arrow points to the I B- protein.
C, L929 cells were treated with the indicated combinations
of TNF- and DEX. Cell viability was determined after 12 and 24 h using the MTT assay. Bars indicate S. D.
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Stable Overexpression of I
B-
Abrogates TNF-
-induced
Transcription of IL-6 without Affecting Cell Killing--
In order to
inhibit NF-
B in a highly specific way, L929 cells were stably
transfected with the plasmid RSV-I
B-
S32A/S36A, an expression
vector encoding a transdominant negative human I
B-
mutant.
Because serines 32 and 36 in this mutant were changed to alanines, the
inducible phosphorylation and subsequent degradation was prevented,
thus conferring an increased half-life to this I
B-
form (17).
Several L929 cell clones were tested for the expression of I
B-
protein in Western blot experiments (Fig. 3A). Three of the cell clones
(2D1, 3A6, and 3B1) were found to constitutively express the
transdominant negative form of human I
B-
, which migrates slightly
slower than the endogenous murine I
B-
protein from L929 cells.
L929 control cells that were stably transfected with the empty
expression vector did not contain this slower migrating I
B-
band
(Fig. 3A). These I
B-
-overexpressing cell clones were
further characterized by testing the TNF-
-induced DNA binding
activity of NF-
B by EMSAs. The induced DNA binding capacity of all
I
B-
-overexpressing clones was strongly impaired when compared
with L929 control cells (Fig. 3B and data not shown). The
effect of I
B-
overexpression on the transcription of the endogenous NF-
B target gene IL-6 was tested by measuring the TNF-
-triggered production of this cytokine. In contrast to the L929
control cells, treatment of the three I
B-
-overexpressing cell
clones with TNF-
did not result in strongly elevated IL-6 levels
(Fig. 4A). In summary, these
results demonstrate that the L929 cell clones 2D1, 3A6, and 3B1 are
unable to significantly activate the transcriptional activity of
NF-
B. However, TNF-
killed these cell clones with the same
efficiency and kinetics as the L929 control cells (Fig. 4B),
indicating that NF-
B activation neither activates nor represses
TNF-
-mediated cell killing, thus establishing that both events are
unrelated and uncoupled in L929 cells.

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Fig. 3.
Characterization of I B- -overexpressing
cell clones. A, equal amounts of total cellular
proteins contained in the indicated cell clones were separated by
SDS-polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride membrane. The positions of the human
(h) and murine (m) I B- proteins, which were
detected with a polyclonal -I B- antibody, are indicated.
B, the indicated cell clones were treated for 20 min with
TNF- , and the DNA binding of activated NF- B contained in
total extracts was tested by EMSA. The symbols used are as explained in
Fig. 1.
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Fig. 4.
TNF- -induced IL-6 production and cell
killing of I B- -overexpressing cell clones. A, the
indicated cell clones were stimulated for 10 h with 2000 units/ml
TNF- , and subsequently, IL-6 production was measured by
enzyme-linked immunosorbent assay. Bars indicate the S. D.
obtained from four independent experiments. B, the different
cell clones were treated with TNF- for the indicated periods. Cell
viability was determined with the MTT assay. The viability of untreated
cells was set as 100%. A representative experiment is shown.
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DISCUSSION |
The role of NF-
B in the regulation of apoptosis does not yield
a homogeneous picture yet. There are numerous examples showing apoptosis-promoting as well as apoptosis-antagonizing effects of
NF-
B activation (for a review, see Ref. 9). Some inducers of cell
death, such as TNF-
, directly activate NF-
B; others may influence
B-dependent gene expression by alternative pathways. In
this respect, it was shown that an activated form of Caspase-3 can
cleave the unphosphorylated I
B-
protein, which leads to the
generation of a constitutive inhibitor of
B-dependent
gene expression (25). The events leading to TNF-
-triggered cell death and NF-
B activation are initiated by the trimerization of the
TNF receptor. This leads to the induced association of the
intracellular TRADD protein and subsequent recruitment of further
proteins, including TRAF-2 and MORT1/FADD. The molecular events leading
to NF-
B activation and cell killing diverge relatively early in this
signal cascade. The expression of a transdominant negative form of FADD
inhibits TNF-R55-mediated cell death but not
B-dependent
transcription (26). TRAF-2-deficient mice display a functional NF-
B
activation and an increased sensitivity to TNF-
, suggesting an
additional NF-
B-independent pathway of cell protection that is
mediated by TRAF-2 (27).
In this study, we show that the TNF-
-triggered activation of NF-
B
and the induction of cell death are uncoupled events in L929 cells. A
protective role of NF-
B would imply that the inhibition of this
transcription factor would lead to enhanced cell death. However, even
the partial inhibition of NF-
B transactivation by DEX was
accompanied by a decrease in cell killing. In the case of L929 cells,
the DEX-mediated repression of NF-
B seems to be independent from
up-regulation of I
B-
and can probably be attributed to the
recently proposed repression of transactivation (28). Conversely, the
activation of NF-
B does not promote killing of L929 cells, because
BA allowed the full activation of NF-
B while reducing the cytotoxic
effects of TNF-
. Evidence for the uncoupling of NF-
B activation
and cytotoxicity in L929 cells was obtained by the unchanged
susceptibilities to cell death in cell lines stably overexpressing a
transdominant negative variant of I
B-
. The role of NF-
B as a
promoter or attenuator of cell killing may therefore depend on the
nature of the apoptosis-inducing stimulus as well as on the cell type.
Accordingly, the inhibition of NF-
B activation by I
B-
overexpression did not alter the sensitivity of the human breast cancer
cell line MCF7 toward TNF-
(29), whereas the HT1080 fibrosarcoma
cells and Jurkat T-cells were more susceptible to the detrimental
effects of TNF-
(12, 13).
We thank Dr. J. A. Duine (Delft, Netherlands)
for kindly providing bongkrekic acid.