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J. Biol. Chem., Vol. 278, Issue 32, 29626-29632, August 8, 2003
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(TNF-)-induced Apoptosis in Rat Hepatoma Cells Expressing TNF- Is Linked to Low Antioxidant Enzyme Expression*
From the Institute of Toxicology, Heinrich Heine University of Duesseldorf, P. O. Box 10 10 07, D-40001 Duesseldorf, Germany
Received for publication, August 23, 2002 , and in revised form, April 24, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(TNF-), we have constructed two stable transfectants
producing TNF- (Yv12–2 and Yv13–44) from the rat hepatoma
H4IIE cell, which does not produce TNF-. H4IIE cells were highly
sensitive to apoptosis induced by TNF-, whereas Yv2–12 and
Yv13–44 cells were resistant. Manganous superoxide dismutase was not
up-regulated in Yv2–12 and Yv13–44 cells and was unresponsive to
induction by exogenous TNF- and by H2O2 in H4IIE
cells and in the transfectants. Catalase expression and activity were lower in
Yv2–12 and Yv13–44 cells than in H4IIE cells; furthermore, the
transfectants were more susceptible to H2O2. Treatment
with exogenous TNF- down-regulated catalase in H4IIE cells but not in
Yv2–12 and Yv13–44 cells. Treatment of H4IIE cells with the
catalase inhibitor 3-amino-1,2,4-triazole rendered them resistant to exogenous
TNF-. These data suggest a causal relationship between resistance to
TNF- and low catalase activity. Expression of copper and zinc
containing superoxide dismutase was also decreased, whereas expression of
glutathione peroxidase-1 was unchanged in Yv2–12 and Yv13–44
cells. Data from a microarray point to a down-regulation of genes in the
resistant clones that code for antioxidative proteins and proteins involved in
glutathione synthesis and function. We assume that a prooxidant signal linked
to the down-regulation of antioxidant defense may be associated with
resistance to apoptosis induced by TNF-. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(TNF-)1 is a
pleiotropic cytokine acting in a paracrine or autocrine fashion on a wide
variety of target cells via two members of the TNF- receptor family,
TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Binding of TNF-
trimers to these receptors activates signal transduction pathways that lead to
different and even antagonistic effects on cell function and survival
(reviewed in Refs. 1 and
2): 1) TNF- supports the
formation of proinflammatory molecules that are constituents of the immune
response. 2) TNF- can exert mitogenic actions and supports
proliferation processes. 3) TNF- can induce apoptosis via caspase 8
activation and the mitochondrial pathway. 4) TNF- supports (a) survival
pathway(s), supposedly via the formation of antiapoptotic proteins.
Development of resistance against TNF--induced cytotoxicity has been
demonstrated in a variety of experimental models, including the repeated
administration of exogenous TNF- to cultured cells
(3) and animals
(4,
5) or the overexpression of
endogenous TNF- in cultured cells
(6,
7).
Among the possible mechanisms that might cause TNF- resistance is
the induction of protective proteins that counteract the apoptotic action of
TNF-. Induction of the mitochondrial antioxidant enzyme manganous
superoxide dismutase (MnSOD) by TNF- has been reported in a wide
variety of cell types and has been assumed to function as a central protective
mechanism against TNF--induced apoptosis
(8,
9). In experiments with stable
transfection of the TNF- gene into tumor cells, up-regulation of MnSOD
paralleled TNF- resistance
(10,
11). Overexpression of MnSOD
protected cells against TNF- cytotoxicity, whereas the expression of
antisense MnSOD-RNA increased the susceptibility of cells toward TNF-
(9,
12). It is known that
mitochondrial generation of reactive oxygen species (ROS) is involved in the
cytotoxicity of TNF-
(13,
14) and that inhibitors of
mitochondrial function prevent the induction of MnSOD
(15). It may therefore be
assumed that protection by MnSOD induction against TNF--induced
cytotoxicity is an adaptation reaction to mitochondrial superoxide
production.
Up-regulation of MnSOD as a mechanism of TNF- resistance suggests
that TNF--resistant cells might also be resistant against oxidants. It
has been shown that tumor cells overexpressing TNF- and thus rendered
resistant against TNF- were also protected against the redox-cycling
cytostatic drug adriamycin
(10,
16,
11); it may be assumed that
the protection was because of the removal of adriamycin-generated superoxide
anion radicals by the up-regulated MnSOD activity. In the liver, TNF-
has been shown to be involved in viral and immune-mediated hepatitis and
drug-induced liver failure
(17). Consequently, under
pathophysiological conditions related to oxidative stress the liver can be
subject to prolonged exposure to TNF-, and resistance to
TNF--induced liver damage has been shown in animals exposed to
TNF- (4,
5). However, the mechanisms
underlying TNF- resistance in liver cells have not yet been elucidated.
In the present study, two TNF--resistant clones were constructed from a
TNF--sensitive hepatoma cell line in order to investigate whether in
the transfectants the antioxidant enzyme system would be induced.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(R&D, Wiesbaden, Germany),
H2O2 (Sigma), or 3-amino-1,2,4-triazole (ATZ) (Sigma)
was added and remained on the cells for 24 h. Cell viability was tested by the
neutral red assay according to Borenfreund and Puerner
(18).
Stable Transfection of TNF- into H4IIE
Cells—TNF- cDNA was obtained by RT-PCR from total rat
hepatocyte RNA isolated with Trizol (Invitrogen). Hepatocytes were prepared
from male Wistar rats (250–300 g) by a modified method according to
Lindl and Bauer 1989 (19). The
amplified product was purified and cloned into the HindIII site of
the pSPT18 vector. Correct sequence was verified in randomly selected
pSPT18-TNF- clones by the use of the T7 and SP6 sequencing primers. For
stable transfection into H4IIE cells, the pTRE vector (Clontech) was used.
Correct sequence was verified in randomly selected pTRE-TNF- clones
with vector-specific primers. For selection of tetracycline-responsive
transfectants, cells were transfected with 10 µg of pTet-ON or pTet-OFF
using calcium phosphate precipitation. Clones were selected in the presence of
800 µg/ml G418 48 h after transfection for 2 weeks. For establishing
TNF- overexpressing cells, the selected clones were co-transfected with
pTRE-TNF- and pTK-Hyg (Clontech), a plasmid that encodes hygromycin
resistance. Clones were selected in the presence of 500 µg/ml hygromycin B
48 h after transfection, using calcium phosphate precipitation. Isolates of
clones Yv2–12 and Yv13–44 were tested for TNF- expression
by RT-PCR.
TNF- Detection—Total TNF-
concentration in the culture medium was determined by commercially available
rat TNF- ELISA according to the manufacturer's recommendations
(Endogen, Boston, MA). This kit is sensitive to 5 pg/ml TNF- and does
not cross-react with TNF-
, interleukin-1, or interleukin-6. Biologically
active TNF- was quantified by the L929 assay as described previously
(20). One unit of TNF-
was defined as the reciprocal of the dilution at which 50% L929 cytotoxicity
was observed.
Catalase Activity—Catalase activity was determined by measuring the initial rate of decay of H2O2 absorbance at 240 nm, using an extinction coefficient of 43.6 M–1 cm–1. Prior to the catalase assay, homogenates were incubated with ethanol and Triton X-100 according to Cohen et al. (21). Each assay mixture consisted of a cell sample (150 µg of protein/ml) and H2O2 at an initial concentration of 19 mM H2O2 in 50 mM phosphate buffer (pH 7.0).
RNA Isolation and Northern Blot Analysis—Total RNA was
isolated from cells using Trizol (Invitrogen). Total RNA (5–10 µg)
was resolved by electrophoresis in a 1.5% agarose, 2.25 M
formaldehyde gel. RNA was transferred to nylon membrane (Amersham Biosciences)
as described by Sambrook et al.
(22). Purified cDNAs were
labeled with [32P]dCTP (111 TBq/mmol; Hartmann Analytic,
Braunschweig, Germany) by random hexamer priming (Roche Diagnostics). Blots
were prehybridized and hybridized with cDNAs as described previously
(23) and then exposed to x-ray
films (Kodak XAR) for 1–3 days with an intensifying screen at –80
°C. 18 S rRNA served as internal loading control. Autoradiographs were
analyzed by densitometric scanning using the Quantity One system from
Bio-Rad.
Microarray—Gene expression was analyzed using Atlas Rat
Toxicology Array II (Clontech). Total RNA was extracted from
TNF--transfected and parental H4IIE cells using Trizol (Invitrogen).
The sample was treated with RNase-free DNase I for 60 min, extracted using
phenol-chloroform, and then precipitated using 2.5 volumes ethanol. Integrity
of the RNA was confirmed on an agarose gel. Two µg of total RNA were
reverse-transcribed with nucleotides containing [-32P]dATP;
the labeled cDNAs were purified, denatured, and added to 5 ml of Express-Hyb
hybridization solution (Clontech). The filters were prehybridized, hybridized,
washed according to the manufacturer's recommendations, and exposed to an
x-ray film overnight at –80 °C or to a phosphorimaging screen and
scanned (BAS-MS 2025 phosphorimaging; Raytest, Germany). Signal intensities of
corresponding spots were compared by AIDA software (Raytest).
RT-PCR—cDNA was synthesized from 1 µg of total cellular
RNA using 2.0 µM oligo(dT)16-primer, 0.2
mM of each dNTP, 25 units RNase inhibitor, and 200 units M-MLV
reverse transcriptase (Promega) in a final volume of 25 µl. PCR was
performed using 0.2 mM of each dNTP, 1 unit Taq DNA
polymerase (Promega), and 0.4 µM of each PCR primer. PCR was
then carried out for 1 min at 94 °C, 1 min 60 °C, 1 min 72 °C. The
following primer pairs were used for amplification: rat TNF- (sense,
5'-TACTGAACTTCGGGGTGATTGGTCC-3', antisense,
5'-CAGCCTTGTCCCTTGAAGAGAACC-3'); rat MnSOD (sense,
5'-GGATCCACGTGAACAATCTGAACGTCACC-3', antisense,
5'-AAGCTTAGTAAGCGTGCTCCCACACATC-3'); rat glutathione peroxidase-1
(GPx-1) (sense, 5'-ATGTCTGCTGCTCGGCTCTC-3', antisense,
5'-GTTGCTAGGCTGCTTGGACAG-3); rat glyceraldehyde-3-phosphate
dehydrogenase (sense, 5'-ACCACAGTCCATGCCATCAC-3', antisense,
5'-TTCACCACCCTGTTGCTGTA-3'). The sizes of the amplicons were as
follows: TNF-, 295 bp; MnSOD, 394 bp; GPx-1, 602 bp;
glyceraldehyde-3-phosphate dehydrogenase, 450 bp. The optimal number of PCR
cycles for semiquantitation was previously determined for each gene. PCR
products were then separated on a 1.5% agarose gel, visualized by ethidium
bromide staining, and analyzed by densitometric scanning using the Quantity
One system from Bio-Rad.
Cell Lysis and Western Blot Analysis—Cells were lysed by two rounds of freezing and thawing in lysis buffer (50 mM Tris-HCl, 2.5 mM ethylene diamine tetraacetic acid (EDTA), 150 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, pH 7.4), and then centrifuged at 10 000 x g for 10 min. Protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad). An equal amount of protein for each sample was separated by 10% SDS-PAGE and electrotransferred onto a polyvinylidene fluoride membrane (Roche Diagnostics). Protein loading was assessed by Ponceau S staining of membranes. The membranes were blocked with 3% nonfat dry milk in a buffer containing 20 mM Tris, 500 mM NaCl, and 0.01% Tween 20 (pH 8.0) and incubated with 1:500–1:1000 dilution of antibody against MnSOD (Upstate, Milton Keynes, UK) and catalase (Calbiochem). Blots were then probed with the appropriate horseradish peroxidase-conjugated secondary antibody. Bound antibody was visualized using enhanced chemiluminescence reagent (Roche Diagnostics).
Analysis of DNA Fragmentation—After incubation with
TNF- or H2O2 for 24 h, 5 x 106
cells were harvested. Cell pellets were resuspended in lysis buffer (20
mM Tris, 5 mM EDTA, 0.1 M NaCl, 0.5% SDS,
RNase A 10 µg/ml, pH 8.0) and incubated for 1 h at 37 °C. DNA was
extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1)
and precipitated with 0.7 volume isopropanol and 0.1 volume 3 M
NaCl. The pellets were resuspended in 50 µl of Tris/EDTA buffer, and DNA
was electrophoresed using a 2% agarose gel at 70 V for 3–4 h. The DNA
bands were visualized by ethidium bromide staining and photographed under UV
light using a transilluminator.
Anti-histone ELISA—Quantitative measurements of apoptosis
were performed using an ELISA that quantifies DNA fragmentation by measuring
cytoplasmic histone-DNA fragments (Cell Death ELISA; Roche Diagnostics). Cells
were incubated with TNF- or H2O2 as described
above; apoptosis was assessed 24 h later according to the manufacturer's
recommendations.
Caspase Assay—Determination of caspase activity was carried out in 96-well plates using 50 µg of protein for each measurement. Colorimetric substrates for caspases were purchased from Calbiochem. Activity of caspase 3, 8, and 9 was detected by measuring the proteolytic cleavage of the substrates in assay buffer (50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 100 mM NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 10 mM dithiothreitol, 100 µM EDTA, 10% glycerol, pH 7.4) using the absorbance of released p-nitroaniline at 405 nm.
Statistical Analyses—All data were analyzed using one-way analysis of variance, followed by least significant difference post hoc analysis to determine statistical significance. p values <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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cDNA resulted in the
emergence of two clones (Yv2–12 and Yv13–44) that constitutively
expressed TNF- to a different extent. These two clones were used for
further experimentation. The concentration of total TNF- protein and
biologically active TNF- in the medium was one order of magnitude
higher in Yv2–12 cells than in Yv13–44 cells. The wild type cells
(wtH4IIE cells) did not produce TNF-. No difference in cell
proliferation and morphology was observed between wtH4IIE cells and
transfectants.
WtH4IIE cells, but not Yv2–12 and Yv13–44 cells, were highly
susceptible to the cytotoxic action of exogenous TNF- at a
concentration of 25 ng/ml. Yv13–44 showed a slight loss of viability at
50 ng/ml (Fig. 1), and
Yv2–12 was completely resistant to TNF- up to 500 ng/ml.
Apoptosis as detected by oligonucleosomal DNA fragmentation
(Fig. 2A) and by an
antihistone ELISA (Fig.
2B) was elicited by treatment with exogenous TNF-
in wtH4IIE cells but not in Yv2–12 and Yv13–44 cells. In wtH4IIE
cells, but not in Yv2–12 and Yv13–44 cells, a slight increase in
p53 and bax expression was found, although bcl-2 expression was not altered.
Activation of caspase 3 (Fig.
3A), caspase 8 (Fig.
3B), and caspase 9
(Fig. 3C) upon
treatment with exogenous TNF- was found in wtH4IIE cells but not in
Yv2–12 cells, whereas Yv13–44 cells displayed intermediate
activation of caspase 3 but not of caspase 8 and 9.
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TNF receptor expression was measured in order to exclude the possibility
that resistance to TNF- in the transfectants is because of a loss of
TNFR1 and/or TNFR2 expression. The wtH4IIE cells as well as the two
TNF--resistant clones expressed the TNFR1, whereas none of them
expressed the TNFR2. TNFR1 functionality was not lost, as shown by the
induction of NF-
B activation by exogenous TNF- in the resistant
clones (data not shown).
Because formation of ROS is assumed to be involved in TNF-
cytotoxicity, TNF- resistance might be caused by the up-regulation of
antioxidant enzymes. An increase of MnSOD expression has been shown to occur
in other TNF--resistant cell types
(10,
11). It might therefore be
assumed that this adaptation process also occurred in Yv2–12 and
Yv13–44 cells. Fig. 4 shows that neither the MnSOD mRNA level nor the MnSOD protein level in
Yv2–12 and Yv13–44 cells differed from those in wtH4IIE cells.
MnSOD induction could not be obtained in either wtH4IIE cells or Yv2–12
and Yv13–44 cells by exogenous TNF-a.
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To provide a possible explanation for the TNF- resistance of the
clones, we examined whether catalase and GPx-1 expression is increased in
Yv2–12 and Yv13–44 cells. Fig.
5 demonstrates that no difference in GPx-1 mRNA expression was
found between wild type cells and TNF-resistant cells and that contrary to
expectations catalase mRNA was even lower in the TNF--resistant clones
than in wtH4IIE cells (Fig.
5B). Catalase protein expression and catalase enzyme
activity were also decreased in the transfectants
(Fig. 5, C and
D). Treatment with exogenous TNF- down-regulated
catalase expression and enzyme activity in wtH4IIE cells but not in the
TNF--resistant clones (Fig.
6). A slight induction of GPx-1 by exogenous TNF- could be
achieved in wtH4IIE cells but not in Yv2–12 and Yv13–44 cells.
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To test the conclusion from Figs.
5 and
6 that low catalase activity is
associated with TNF- resistance, cells were treated with the catalase
inhibitor ATZ. Fig. 7 shows
that in wtH4IIE cells ATZ treatment did, indeed, markedly lower the
sensitivity to TNF-. No influence of ATZ treatment on TNF-
sensitivity was observed in the Yv2–12 and Yv13–44 cells.
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To test the initial hypothesis that TNF--resistant cells also
display resistance toward H2O2, cytotoxicity of
H2O2 was compared in wtH4IIE cells and in Yv2–12
and Yv13–44 cells. Fig.
8A shows that Yv2–12 and Yv13–44 cells are
even more susceptible toward H2O2 than wtH4IIE cells. In
Fig. 8, B and
C, the induction of apoptosis by
H2O2 as detected by oligonucleosomal DNA fragmentation
and by an anti-histone ELISA was compared in wtH4IIE cells versus
Yv2–12 and Yv13–44 cells. The transfectants were considerably more
sensitive to apoptosis induced by H2O2 than the wtH4IIE
cells.
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MnSOD expression as well as catalase expression can be increased by
H2O2 in a number of cells
(22,
24–27).
In our experiments, similar to the results with exogenous TNF-, both in
the wild type cell and the TNF--resistant cells, MnSOD is unresponsive
to H2O2 treatment. Catalase mRNA expression and enzyme
activity could be increased by H2O2 treatment both in
wtH4IIE cells and in Yv2–12 and Yv13–44 cells.
A DNA microarray comparing wtH4IIE cells and Yv2–12 cells was
performed to support the hypothesis that TNF- resistance is paralleled
by a shift toward a more pro-oxidative state and to identify additional genes
differentially regulated in the resistant cells. Of 450 genes, 8 were
up-regulated and 31 were down-regulated in the Yv2–12 cells by a factor
of at least 2. Preliminary evaluation of the microarray data indeed points to
a down-regulation of genes for antioxidant enzymes and of genes involved in
glutathione synthesis and in detoxifying phase II metabolism
(Table I). At present, the
down-regulation of copper and zinc containing superoxide dismutase (CuZn-SOD)
indicated by the microarray has been verified by Northern blotting in the
Yv2–12 and Yv13–44 cells (data not shown). Moreover,
down-regulation of CuZnSOD was obtained in wtH4IIE cells, but not in
Yv2–12 and Yv13–44 cells, upon treatment with exogenous
TNF-.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-induced apoptosis can be associated with a
down-regulation of antioxidant proteins. For this, we have constructed two
TNF--producing stable transfectants from a wild type rat hepatoma cell
that does not express TNF-. The wtH4IIE cell is highly sensitive to
TNF--induced apoptosis, whereas the transfectants are resistant to
TNF--induced apoptosis.
TNF- induces apoptosis via the death receptor pathway, and
down-regulation of TNF- receptors could theoretically be the mechanism
of TNF- resistance. This is, however, not the main mechanism in our
transfectants because (a) both the wild type cell and the
transfectants express the TNFR1 to a similar extent, (b) both the
wild type cell and the transfectants do not express the TNFR2 at all, and
(c) NF-B activation by TNF-, assumed to require TNF-R1
functionality, can be induced in the transfectants. In vivo, Sass
et al. (28) also did
not find a down-regulation of TNFR1 in TNF--resistant mice.
A mitochondrial component is involved in TNF--induced cytotoxicity
(13,
14). This has supported the
assumption that fortification of mitochondrial defense against oxidative
stress by up-regulating MnSOD could be a mechanism of TNF- resistance.
In contrast to our initial expectations, up-regulation of MnSOD expression did
not occur in our TNF--producing transfectants and thus could be
excluded as a mechanism of TNF- resistance in this cell type. MnSOD is
expressed to the same extent in the wtH4IIE cell and in the
TNF--resistant clones derived from it and is unresponsive to induction
by exogenous TNF-. Induction of the enzyme in wtH4IIE cells and in the
TNF--resistant clones could not be achieved with
H2O2 either. In contrast, MnSOD can be induced by
TNF- (29,
30) and
H2O2
(23) in primary rat
hepatocytes. The reason for the intrinsic inability of the H4IIE cell and its
descendents to up-regulate MnSOD has not yet been elucidated.
It might have been expected that the expression of other antioxidant
enzymes in the TNF--resistant cells was increased as a substitute for
the lack of up-regulation of MnSOD. However, gene expression analysis revealed
that in the transfectants none of the antioxidant enzymes examined is
up-regulated, whereas two antioxidant enzymes, catalase and CuZn-SOD, are even
down-regulated. This expression pattern is mimicked by TNF- treatment
of the TNF--sensitive wtH4IIE cell: Exogenous TNF- leads to a
decrease of catalase and CuZnSOD expression in these cells. As a consequence
of low catalase activity, the sensitivity of the transfectants to
H2O2 was increased. A decrease in catalase activity
(31) and catalase mRNA
(32) upon TNF-
treatment has also been observed in vivo. The finding that exposure
to endogenous TNF- in the TNF--resistant clones and to exogenous
TNF- in the TNF--sensitive wild type cell leads to a decrease in
catalase expression and activity suggests that the down-regulation of this
antioxidant enzyme is not merely an epiphenomenon of the transfection process.
Additionally, we performed a pharmacological experiment that strongly
indicates a causal relationship between low catalase activity and TNF-
resistance: Exposure of TNF--sensitive wtH4IIE cells to the catalase
inhibitor ATZ renders these cells partially resistant to exogenous
TNF-.
We conclude from these findings that a pro-oxidant signal linked to
down-regulation of antioxidant defense contributes to the
TNF--resistant state. This signal may transfer information via mediator
concentrations of ROS too low to exert cytotoxic effects. The conclusion that
a shift of the pro-oxidantantioxidant balance of the cell has occurred in the
TNF--resistant clones is further supported by the preliminary results
from a cDNA microarray analysis that suggest the down-regulation of a number
of proteins with antioxidative properties in the Yv2–12 cell, including
thioredoxin-2 and metallothionein-1 as well as a number of enzymes involved in
glutathione supply and function.
The conclusion that low activity of antioxidant enzymes may be involved in
TNF- resistance is at a first glance not consistent with two
interpretations brought forward in the literature. First, it is commonly
assumed that the antioxidant enzyme MnSOD, induced via the NF-B
survival pathway, is the main protective principle against TNF--induced
cytotoxicity (8). In our cells,
MnSOD cannot contribute to protection against TNF- because the enzyme
is intrinsically unresponsive to induction. Even in cells in which MnSOD is
induced, protection may not be linked to the antioxidant character of the
enzyme. The reaction product is H2O2, and the
antioxidant potential of the MnSOD reaction can only be realized by subsequent
reduction of H2O2 to H2O by glutathione
peroxidases or catalase. Siemankowski et al.
(33) have reported that in
MCF-7 cells TNF- induces MnSOD without any change in the expression of
other antioxidant enzymes, including GPx-1 and catalase. This may indicate
that the mechanism by which MnSOD protects against TNF- could be
pro-oxidative in nature because the steady state level of
H2O2 increases. It has been concluded from experiments
in cRel overexpressing HeLa cells that H2O2 produced via
MnSOD induction creates an oxidative injury leading to mitochondrial
degeneration, proliferation arrest, and resistance to TNF--induced
cytotoxicity (34). On the
other hand, the down-regulation of the cytoplasmic CuZnSOD observed in the
present work, not only in the TNF--resistant clones but also in the
TNF--treated wild type H4IIE cells, is not consistent with this view
because the resulting pattern of SOD activities would decrease
H2O2 delivery to GPx and catalase. We assume that a
coordinated response of H2O2-delivering and
H2O2-consuming enzymes to TNF- exposure is
required for the maintenance of a well dosed pro-oxidant signal to the
TNF--dependent signaling cascade. Secondly, in the mouse,
transcriptional and/or translational inhibitors sensitize cells and animals to
the toxic action of TNF-
(35). This suggests that the
de novo synthesis of protective proteins is needed for TNF-
resistance and down-regulation (e.g. of catalase) as a protective
mechanism does not seem to fit in this picture. However, catalase is known to
be in part regulated post-transcriptionally via the degradation of the
catalase mRNA by a protein
(36). If in the presence of a
transcriptional or translational inhibitor (a situation linked to high
TNF- sensitivity) this protein is no longer formed, this would result
in an apparent up-regulation of catalase while uninhibited de novo
synthesis of this regulatory protein would be required to achieve a state of
TNF- resistance.
In the literature, a number of findings support the view that a pro-oxidant
environment confers resistance to death receptor-dependent apoptosis.
Overexpression of catalase increases the sensitivity of HepG2 cells to
TNF- (37). From
experiments with radiation-exposed leukemia cells, it has been concluded that
the primary role of catalase in multicellular organisms could be the
regulation of apoptosis rather than the detoxification of
H2O2
(38). In lymphocytic cell
lines, glutathione depletion prevents TNF--induced apoptosis
(39). NF-B activation,
a process shown to be a survival pathway in TNF--treated cells
(40), is a candidate target
for a pro-oxidative anti-apoptotic action because a co-stimulatory role of
reactive oxygen species in NF-B activation has been postulated
(41).
In the experiments on TNF- resistance reported here, we have focused
on the antioxidant enzyme system and on the existence of a pro-oxidant signal
maintaining the resistant state. This, of course, does not exclude further
mechanisms, either related or unrelated to the redox state of the cell, that
may also be involved in the process of TNF- resistance in hepatoma
cells as well as in other cell types. In the mouse in vivo, it has
been excluded that interleukin-1, inducible nitric oxide synthase, and
heme oxygenase-1 confer to resistance TNF-, although they are induced
in the resistant animals (28).
By introduction of a randomized hybrid ribozyme library into MCF-7 cells, a
number of genes functional in TNF--induced apoptosis were recently
identified (42). It may be
worthwhile testing whether these genes are down-regulated in a
TNF--resistant cell.
In conclusion, we have shown that in TNF--resistant transfectants
derived from a TNF--sensitive rat hepatoma cell line a down-regulation
of certain antioxidant proteins occurs in the absence of any increase in the
expression of the other antioxidant proteins examined. We suggest that a
pro-oxidant signal linked to the down-regulation of antioxidant defense could
be associated with the TNF--resistant state.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 49-211-8113022; Fax:
49-211-8113013; E-mail:
kahl{at}uni-duesseldorf.de.
1 The abbreviations used are: TNF-, tumor necrosis factor-;
TNFR1, TNF receptor 1; TNFR2, TNF receptor 2; MnSOD, manganous superoxide
dismutase; ROS, reactive oxygen species; ATZ, 3-amino-1,2,4-triazole; GPx-1,
glutathione peroxidase-1; CuZnSOD, copper and zinc-containing superoxide
dismutase; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, p < 0.05
versus Yv2–12 control.
