Hydrogen peroxide-induced liver cell necrosis is dependent on
AP-1 activation
Yang
Xu1,
Cynthia
Bradham2,
David A.
Brenner2, and
Mark J.
Czaja1
1 Department of Medicine and
Marion Bessin Liver Research Center, Albert Einstein College of
Medicine, Bronx, New York 10461; and
2 Departments of Medicine and
Biochemistry and Biophysics, Center for Gastrointestinal Biology
and Disease, University of North Carolina, Chapel Hill, North Carolina
27599
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ABSTRACT |
To determine whether intracellular signaling
events involved in apoptosis may also mediate necrosis, the role of the
transcription factor AP-1 was investigated in a hepatoma cell model of
cellular necrosis induced by oxidant stress. Treatment of the human
hepatoma cell line HuH-7 with
H2O2
caused dose-dependent necrosis as determined by light microscopy,
fluorescent staining, and an absence of DNA fragmentation.
H2O2
treatment led to increases in c-fos
and c-jun mRNA levels, Jun nuclear
kinase activity, and AP-1 DNA binding. AP-1 transcriptional activity
measured with an AP-1-driven luciferase reporter gene was also
increased. To determine whether this AP-1 activation contributed to
H2O2-induced
cell necrosis, HuH-7 cells were stably transfected with an antisense
c-jun expression vector. Cells
expressing antisense c-jun had
decreased levels of AP-1 activation and significantly increased
survival after
H2O2
exposure. These data indicate that AP-1 activation occurs during
oxidant-induced cell necrosis and contributes to cell death. Necrosis
is therefore not always a passive process but may involve the
activation of intracellular signaling pathways similar to those that
mediate apoptosis.
c-fos; c-jun; apoptosis; hepatoma cell
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INTRODUCTION |
CELL DEATH is classified as necrosis or apoptosis on
the basis of established morphological criteria (11, 18). Necrosis is
considered a passive event in which the cell is irreversibly damaged by
an environmental insult, leading to cell death (20). In contrast,
apoptosis is an active process in which the cell itself initiates the
molecular machinery to trigger cell death in response to either a
physiological stimulus or an environmental stress (11, 21). Elements of
the genetic machinery that promote or block apoptotic cell death have
begun to be elucidated, but the complete pathway remains unclear and
may in fact vary among different stimuli and cell types (47). Because
necrosis is a passive event, this form of cell death presumably does
not evoke the molecular responses that underlie apoptosis. However, the molecular events that characterize necrosis have not been well studied.
Interestingly, a single stimulus may trigger either apoptotic or
necrotic cell death in vitro, depending on the dose of the stimulus,
the growth state of the cells, and the culture conditions (5, 8).
Similar findings are found in vivo in the liver, where toxin-induced
injury leads to concurrent hepatocyte death by both necrosis and
apoptosis (30). This phenomenon has led to the hypothesis that a high
dose of a death stimulus can cause severe injury and resultant
necrosis, whereas a lower dose may lead to a moderately damaged cell,
which, if incapable of repair, initiates apoptosis (8). Alternatively,
the continuum between apoptosis and necrosis in response to a single
stimulus suggests the possibility that these two forms of cell death in
fact share common mechanisms.
One cellular response that has been identified during apoptosis is
increased expression of c-fos and
c-jun, genes whose proteins form homo-
and heterodimers that constitute the transcription factor AP-1 (4).
Increased levels of c-fos and
c-jun mRNA have been demonstrated in
several in vitro and in vivo models of apoptosis (19, 27, 31, 43). The
functional significance of this response has been shown in neuronal
cells, in which administration of neutralizing antibodies to Fos and
Jun blocked apoptosis resulting from growth factor deprivation (19).
Whether this genetic response also occurs during necrosis is not known.
The exposure of cells to injurious reactive oxygen intermediates (ROI)
also increases c-fos and
c-jun expression (38, 41). Other
investigations have indicated that oxidant-induced cell death occurs by
apoptosis (2, 9, 42). To delineate the mechanism of liver cell death
from oxidative stress, the effects of the ROI
H2O2
were investigated in the human hepatoma cell line HuH-7. Surprisingly,
H2O2-mediated
death in these cells occurred by necrosis and not apoptosis. To
investigate the possibility that common molecular mechanisms such as
AP-1 gene activation regulate the process of necrosis as well as
apoptosis, the expression and function of
c-fos and
c-jun were studied in this model of oxidative stress-mediated necrosis.
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METHODS |
Materials.
All reagents were obtained from Sigma (St. Louis, MO) unless otherwise
specified.
Cell culture.
The human hepatoma cell line HuH-7 (34) and the transfected cell lines
were cultured in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY)
supplemented with 10% fetal bovine serum (Hyclone, Logan, UT),
antibiotics, and glutamine (GIBCO-BRL). For transient transfections
using the luciferase reporter gene, cells were grown to 80%
confluence. All other experiments were conducted with confluent cultures. For the induction of cell death by tumor necrosis factor-
(TNF-
), cells were pretreated with cycloheximide (20 µg/ml) for 30 min, followed by TNF-
(10 ng/ml; PeproTech, Rocky Hill, NJ).
MTT assays.
Cell number was determined by means of the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay (16) as we have previously described (51). The validity of this
assay as a measure of the number of live cells was established by
comparing results from the MTT assay with the number of trypan
blue-excluding cells recovered from trypsinized dishes.
Microscopic determination of apoptosis and necrosis.
Phase-contrast and fluorescence microscopy were conducted as previously
described (51). The relative numbers of necrotic and apoptotic cells
were determined by fluorescent costaining with ethidium bromide and
acridine orange as described by Duke and Cohen (17) and used previously
(51). Cells were considered necrotic if they exhibited positive
ethidium bromide staining and apoptotic if acridine orange staining
revealed a shrunken cytoplasm and nucleus and a condensed
chromatin, appearing as small spheres or a peripheral
crescent.
DNA fragmentation gels.
DNA was extracted from cells using a modification of the method of Gong
et al. (23) as previously described (51) and analyzed by gel
electrophoresis (51).
RNA extraction and Northern blot hybridizations.
RNA was extracted from cells using a modification of the Chirgwin
procedure (10) as previously described (14). Steady-state mRNA levels
were determined by Northern blot hybridizations using samples of 10 or
20 µg of total RNA (15). The membranes were hybridized with
[32P]dCTP-labeled
(DuPont-New England Nuclear, Boston, MA) cDNA clones for
c-fos (13),
c-jun (39), glutathione peroxidase
(33), catalase (37), and manganese superoxide dismutase (7). Stripped membranes were rehybridized with a radiolabeled cDNA for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (46).
Jun nuclear kinase assays.
Jun nuclear kinase (JNK) activity was measured in nuclear extracts
prepared from HuH-7 cells. In vitro kinase assays were performed as
described previously (49), using purified recombinant glutathione
S-transferase (GST)-c-Jun.
Phosphorylation was assessed by Phosphorimage analysis.
Electrophoretic mobility shift assays.
Nuclear proteins were isolated by the method of Schreiber et al. (40)
with slight modification as previously described (51). Electrophoretic
mobility shift assays (EMSA) were performed with the use of
commercially supplied oligonucleotides for the normal or a mutated
12-O-tetradecanoylphorbol 13-acetate
responsive element (TRE) consensus sequence and the nuclear factor
NF-
B consensus sequence (Santa Cruz Biotechnology, Santa Cruz, CA).
The DNA binding reaction was performed at room temperature for 20 min
in a 20 µl reaction mixture consisting of 5 µg of nuclear extract,
50 µg/ml of polydeoxyinosinic-deoxycytidylic acid, 10 mM
tris(hydroxymethyl)aminomethane, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 mg/ml bovine serum albumin, 10% glycerol, and 25,000 counts/min of 32P-end-labeled
oligonucleotide. After incubation, the samples were resolved on a 4%
polyacrylamide gel, dried, and subjected to autoradiography. For
supershift assays, 8 µg of anti-c-Fos, anti-c-Jun, or anti-p53 antibody (Santa Cruz Biotechnology) were added to the reaction mixture,
and the incubation time was extended for an additional 20 min.
Transfections and luciferase assays.
HuH-7 cells were transfected with a luciferase reporter gene driven by
AP-1 binding sites (2XTRELuc) (26). Cells were transfected in
serum-free medium with 2 µg of DNA and 10 µl of Lipofectamine (GIBCO-BRL) for 2 h. All studies were initiated 48 h after the time of
transfection. To assay luciferase activity, cells were washed in
phosphate-buffered saline, lysed with a buffer containing 1% Triton
X-100 (Promega, Madison, WI), scraped from the dish, and centrifuged,
and the cell extract was assayed for luciferase activity in a
luminometer. All luciferase values were normalized for extract protein
concentration.
Expression vector and stable transfectants.
The full-length c-jun cDNA was excised
from the c-jun clone JAC.1 (39) and
ligated in an antisense orientation in the expression vector pOPRSVICAT
(Stratagene, La Jolla, CA). HuH-7 cells were transfected with vector
alone or vector containing the antisense c-jun construct. Stable transfectants
were selected by culture in 400 µg/ml of G418 (GIBCO-BRL). Individual
clones were isolated by clonal dilution.
Peroxidase activity.
Peroxidase activity was measured in HuH-7 cells using the
tetramethylbenzidine substrate method of Suzuki et al. (44), as modified by Aitken et al. (1). Activity was normalized to the protein
concentrations of the cell extracts.
Glutathione assay.
The 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-glutathione
disulfide recycling assay (3) was used to determine total glutathione (GSH) content as previously described (52). Levels of GSH were normalized to extract protein concentration.
Statistical analysis.
All numerical data are reported as means ± SE. All data represent
the results of three independent experiments unless otherwise specified. Groups were compared by means of Student's
t-test or the Mann-Whitney test.
Statistical significance was defined as P < 0.05. Calculations were made
with the Sigma Stat statistical package (Jandel Scientific, San Rafael,
CA).
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RESULTS |
H2O2 induces
dose-dependent necrotic cell death in HuH-7 cells.
The toxicity of
H2O2
was determined in the well-differentiated human hepatoma cell line
HuH-7 (34). Cells were grown to a high degree of confluence before
H2O2
administration to eliminate effects related to cell proliferation.
Cells were treated with 0.5-5.0 mM
H2O2,
which corresponded to only 0.18-1.8
µmol/106 cells because of the
confluent state of the cells. The effect of
H2O2
on HuH-7 cell survival was determined at 24 h by means of the MTT
assay. The percentage of cell survival at 24 h decreased from 91 ± 0.6 to 47 ± 5.2% over this concentration range of
H2O2 (Fig.
1A). A
study of the time course of cell death revealed that cell death was
minimal for 6 h after
H2O2
administration and then occurred at a relatively constant rate over the
next 18 h (Fig. 1B). Under light
microscopy cells treated with
H2O2
exhibited morphological changes of necrosis that were clearly distinct
from the apoptotic changes that occurred after TNF-
treatment (Fig. 2). Fluorescence microscopy of cells
costained with ethidium bromide and acridine orange substantiated this
finding.
H2O2-treated
cells lacked DNA condensation and fragmentation, which was apparent in
cells undergoing apoptosis from TNF-
(Fig. 2). An examination of
cells dying after 24 h of
H2O2
treatment revealed that 99 ± 0.8% stained with ethidium bromide,
indicating cell death by necrosis, whereas only 1 ± 0.6% had
nuclear changes indicative of apoptosis. Fluorescent staining of cells
6, 12, and 18 h after
H2O2
treatment revealed that at all of these times >98% of the dying
cells were ethidium bromide positive. In contrast, TNF-
-treated
cells were rarely ethidium bromide positive (5 ± 1.9%), whereas
the remaining cells had the characteristic nuclear changes of
apoptosis. In addition, DNA fragmentation gel analysis
revealed no evidence of low molecular weight DNA fragmentation in
H2O2-treated
cells, whereas TNF-
-treated cells displayed such DNA fragments (Fig. 3). These data indicate that
H2O2
induced necrotic cell death in HuH-7 cells under these culture
conditions.

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Fig. 1.
HuH-7 cell survival after
H2O2
treatment. A: confluent HuH-7 cells
were treated with indicated concentrations of
H2O2,
and cell survival was determined 24 h later by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Results are from 4 independent experiments performed in
duplicate. B: HuH-7 cell survival as
determined by MTT assay over the 24-h period after treatment with
indicated concentrations of
H2O2.
Data are from 3 dishes from each of 3 independent experiments.
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Fig. 2.
Morphological evidence that
H2O2
treatment of HuH-7 cells causes necrotic cell death. Phase-contrast
(A-C) and fluorescent
(D-F) micrographs of HuH-7 cells
that were untreated (A,
D), treated with 2.5 mM
H2O2
for 24 h (B,
E), or treated with tumor necrosis
factor- (TNF- ) for 24 h (C,
F).
H2O2
treatment caused cellular changes characteristic of necrosis, with
confluent areas of swollen cells with disrupted membranes and a loss of
cellular organelles (B). In
contrast, TNF- -treated cells had characteristics of apoptosis, with
cytoplasmic and nuclear condensation and formation of apoptotic bodies
(C). Under fluorescent microscopy
TNF- -treated cells had widespread DNA condensation and fragmentation
(F), which was absent in untreated
(D) or
H2O2-treated
(E) cells.
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Fig. 3.
H2O2-induced
cell death does not result in DNA fragmentation. Ethidium
bromide-stained gel of DNA samples and 100-base pair DNA markers
(lane 1); sizes are shown on
left. DNA samples were prepared from
untreated HuH-7 cells (lane 2),
cells treated for 24 h with 2.5 (lane
3) or 5.0 mM (lane
4)
H2O2,
or cells treated for 24 h with TNF- (lane
5).
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H2O2 treatment
leads to increased c-fos and
c-jun mRNA levels.
To determine whether
H2O2-induced
necrosis of HuH-7 cells was associated with AP-1 gene activation,
c-fos and
c-jun mRNA expression after
H2O2
treatment was determined by Northern blot analysis. Although
c-fos mRNA was barely detectable in
untreated cells, c-fos mRNA content
increased markedly within 0.5 h after the administration of 1 mM
H2O2
and remained elevated for 6-12 h before returning close to
baseline within 24 h (Fig. 4). HuH-7 cells
constitutively expressed c-jun mRNA,
and levels increased from 0.5 to 2 h after H2O2
administration before returning to baseline by 6 h (Fig. 4).
H2O2
treatment did not affect mRNA levels of the constitutive gene GAPDH
(Fig. 4).
H2O2
concentrations as low as 0.5 mM were sufficient to increase
c-fos and
c-jun expression, and higher concentrations of
H2O2
caused dose-dependent increases in the expression of these two genes
(data not shown).

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Fig. 4.
H2O2
treatment increases c-fos and
c-jun mRNA levels. Autoradiograms of
Northern blot hybridizations of 20 µg of total RNA isolated from
untreated HuH-7 cells (control; lane
C) or cells treated with 1 mM
H2O2
for number of hours shown. RNA was hybridized with
c-fos,
c-jun, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNAs as indicated.
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JNK activity is increased by
H2O2.
A mechanism of cellular c-Jun activation alternative to that of
increased gene expression is through activation of JNK, whose phosphorylation of c-Jun increases its transcriptional activity (25).
Treatment of HuH-7 cells with 2.5-5.0 mM
H2O2
led to significant increases in JNK activity of 2.8- to 5.6-fold within
10 min of H2O2
exposure in three independent experiments (Fig.
5A). By
1 h, JNK activity increased 13- to 30-fold over this concentration range (Fig. 5A). Studies of the time
course of JNK activation revealed that 2.5 mM
H2O2
increased JNK activity 8.7-, 10.2-, and 3-fold at 1, 2, and 4 h,
respectively, and that activity returned to baseline by 6 h (Fig.
5B).

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Fig. 5.
H2O2
induces Jun nuclear kinase activity. Nuclear extracts were isolated
from untreated HuH-7 cells (control; lane
C) or cells treated with 0.5-5.0 mM
H2O2
for 10 min or 60 min (A), or with
2.5 mM
H2O2
over a time course of the indicated number of hours
(B). Extracts (25 µg) were
incubated with substrate glutathione
S-transferase (GST)-c-Jun and
[ -32P]ATP.
Phosphorylated substrate was detected by Phosphorimage analysis after
electrophoresis of reaction mixtures in sodium dodecyl
sulfate-polyacrylamide gels.
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H2O2 increases
AP-1 DNA binding and transcriptional activation.
To determine whether increases in AP-1 gene mRNA levels resulted in
changes in DNA binding, AP-1 DNA binding activity was determined by
EMSA in nuclear extracts isolated from untreated and
H2O2-treated
HuH-7 cells. Within 2 h after treatment,
H2O2 concentrations
2.5 mM increased AP-1 DNA binding 2-fold (data not
shown). By 4 h,
H2O2
concentrations >0.5 mM increased AP-1 DNA binding activity 2- to
10-fold (Fig. 6). DNA binding remained markedly elevated at 8 and 20 h after the addition of
H2O2
(Fig. 6). The shifted complex was specific for AP-1, because it could be abolished by competition with cold oligonucleotide with the consensus sequence for AP-1 but not that of NF-
B, and no binding occurred when a mutated TRE oligonucleotide was used (data not shown).

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Fig. 6.
H2O2
induces AP-1 binding activity in HuH-7 cells. Nuclear extracts were
isolated from untreated HuH-7 cells or cells treated with various
concentrations of
H2O2
for indicated lengths of time. These extracts were then used for
electrophoretic mobility shift assays (EMSA) as described in
METHODS. Solid arrow, AP-1 binding
complex; open arrow, free probe.
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The AP-1 DNA binding proteins induced by
H2O2
were predominantly composed of c-Fos and c-Jun, as determined in
supershifts performed with c-Fos and c-Jun antibodies (Fig.
7). The prolonged induction of AP-1 was
specific for this factor and was not a generalized response to cellular
stress and death, as dissimilar effects were found for the
transcription factor NF-
B.
H2O2
increased NF-
B DNA binding activity at 2 h, but, in contrast to the
prolonged expression of AP-1, NF-
B binding returned to baseline
levels within 4 h (data not shown).

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Fig. 7.
H2O2-induced
AP-1 complex is composed of Jun and Fos proteins. Nuclear extract was
isolated from HuH-7 cells treated with 2.5 mM
H2O2
for 8 h. After incubation with radiolabeled probe, extract was further
incubated in absence or presence of anti-Jun, anti-Fos, or anti-p53
antibodies as indicated. Large solid arrow, AP-1-shifted complexes;
small solid arrows, supershifted complexes; open arrow, free probe.
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To evaluate whether this increased DNA binding corresponded to changes
in AP-1 transcriptional activity, HuH-7 cells were transfected with a
luciferase reporter gene driven by AP-1 binding sites (2XTRELuc).
Treatment of transfected cells with 1.5 or 2.5 mM
H2O2
led to slight increases of 1.3- and 2-fold at 4 h and marked increases
of 3- and 6-fold at 8 h and 11- and 21-fold by 20 h in AP-1-driven
luciferase activity (Fig. 8).
H2O2
treatment therefore resulted in sustained increases in both AP-1 DNA
binding and transcriptional activation.

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Fig. 8.
H2O2
stimulates AP-1 transcriptional activation. HuH-7 cells were
transfected with a luciferase reporter gene containing two AP-1 binding
sites, cultured to confluency, and treated with 1.5 (hatched bars) or
2.5 (solid bars) mM
H2O2.
Luciferase activity was assayed at indicated time after
H2O2
treatment and expressed as degree of increase in activity over
untreated HuH-7 cells. Data are from 3 independent experiments
performed in duplicate.
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AP-1 activation mediates
H2O2-induced
HuH-7 cell necrosis.
To assess the functional role of AP-1 transcriptional activation during
H2O2-induced
necrosis, HuH-7 cell AP-1 activation was inhibited by the use of an
antisense c-jun expression vector. Cells were transfected with either the expression vector pOPRSVICAT without insert, or vector containing a full-length
c-jun cDNA in an antisense
orientation. Stably transfected clones were selected containing vector
alone (CAT-16 H-7 cells) or the antisense
c-jun construct (AS-Jun-11 H-7 cells).
Differences in the levels of AP-1 binding between the two cell lines
after
H2O2
treatment were determined by EMSA. After 8 h of stimulation with 2.5 mM
H2O2, AS-Jun-11 H-7 cells had a level of AP-1 activation that was only 20%
of that found in CAT-16 H-7 cells (Fig.
9A).
When survival of the two cell lines was assessed by MTT assay after 24 h in various concentrations of
H2O2,
AS-Jun-11 H-7 cells had a survival rate that was 1.7- to 2.4-fold
greater than that of CAT-16 H-7 cells (Fig.
9B). Similar survival figures were
seen in two additional clones (data not shown). In contrast to the
prior experiments with
H2O2,
which were conducted in serum-exhausted medium, transfected cells were
refed with fresh medium 24 h before the addition of H2O2
to maximize transgene expression. The addition of fresh medium decreased cell survival after
H2O2
treatment relative to the studies performed with wild-type cells in
serum-depleted medium. Cell death in the transfected cells occurred by
necrosis in both cell lines, as indicated by fluorescent staining
studies. The percentage of dying cells undergoing apoptosis at 12, 18, and 24 h as determined by acridine orange-ethidium bromide costaining
averaged only 1.5 ± 0.5% for CAT-16 H-7 cells and 1.2 ± 0.7% for AS-Jun-11 H-7 cells.

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Fig. 9.
Inhibition of AP-1 DNA binding activity prevents
H2O2-induced
necrosis. A: effect of
H2O2
on CAT-16 and AS-Jun-11 H-7 cell AP-1 DNA binding activity. Nuclear
extracts were obtained from untreated cells or cells treated with 2.5 mM
H2O2
for 8 h. Solid arrow, AP-1-shifted complexes; open arrow, free probe.
B: survival of CAT-16 H-7 cells
(hatched bars) and AS-Jun-11 H-7 cells (solid bars) 24 h after
H2O2
treatment. Cells were treated with indicated concentrations of
H2O2,
and survival was determined 24 h later by MTT assay. Results are from 3 independent experiments performed in duplicate. Differences in survival
between the 2 cell lines are statistically significant at all
concentrations tested (1 mM, P < 0.01; all others P < 0.001).
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AP-1 inhibition does not affect peroxidase activity or levels of
antioxidants.
The ability of inhibition of AP-1 activation to prevent
H2O2-induced
cell death could have resulted from effects unrelated to a role of AP-1
in promoting cell necrosis. Specifically, AS-Jun-11 H-7 cells may have
had increased survival because of accelerated metabolism or antioxidant
neutralization of
H2O2.
To exclude these possibilities, peroxidase activity and antioxidant
levels were determined in CAT-16 and AS-Jun-11 H-7 cells. Relative
peroxidase activity normalized to total protein in CAT-16 H-7 cells was
99% of the level found in AS-Jun-11 H-7 cells (results of 3 independent experiments performed in triplicate). Northern blot
analysis also revealed no difference in mRNA levels for the
H2O2-metabolizing enzymes glutathione peroxidase and catalase between the two clones, either untreated or after 6 h of treatment with 2.5 mM
H2O2
(Fig. 10). Levels of mRNA for the
superoxide metabolizing enzyme manganese superoxide dismutase and GAPDH
were also equivalent between the two clones (Fig. 10). To ensure that
AP-1 inhibition did not alter levels of GSH, the principal nonenzymatic
cellular antioxidant, total GSH content was determined in the two cell
clones. There was no significant difference in intracellular GSH levels
between untreated CAT-16 and AS-Jun-11 H-7 cells (Table
1). GSH levels were also decreased by
H2O2
treatment to a similar extent in both clones (Table 1). These results
indicate that the enhancement in cell survival provided by inhibition
of AP-1 activation did not result from effects on cellular metabolism
or neutralization of
H2O2.

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Fig. 10.
CAT-16 and AS-Jun-11 H-7 cells have similar antioxidant enzyme mRNA
levels. Autoradiograms of Northern blot hybridizations of 10 µg of
total RNA isolated from CAT-16 and AS-Jun-11 H-7 cells that were
untreated or treated with 2.5 mM
H2O2
for 6 h. RNA was hybridized with cDNAs for glutathione peroxidase
(GPx), catalase (Cat), manganese superoxide dismutase (MnSOD), and
GAPDH.
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DISCUSSION |
The production of ROI by an oxidant stress has been implicated in a
variety of forms of cell injury (25), including that in hepatocytes
(28, 35, 53). Sufficient production of these compounds presumably
overwhelms cellular antioxidant defenses, leading to toxic levels of
oxidants and injury from their effects on cellular macromolecules.
Prior studies of
H2O2
administration in nonhepatic cell types have indicated that injury from
this ROI leads to death from apoptosis (2, 9, 42). However, H2O2-induced
cell death in HuH-7 cells occurred by a process of necrosis, as
determined by morphology under light microscopy, fluorescent staining,
and a lack of DNA fragmentation. These findings indicate that
H2O2-mediated
oxidant stress may induce cell death by necrosis as well as apoptosis.
Similarly, in a human lung adenocarcinoma line it was recently
demonstrated that hyperoxia, another form of cellular oxidant injury
(22), also led to cellular necrosis rather than apoptosis (29).
The possibility that intracellular signaling pathways are activated
during necrosis was investigated in this model of cell death mediated
by oxidative stress. Necrotic cell death is thought to occur by passive
biochemical events rather than the initiation of active gene expression
and/or protein synthesis (18, 20, 21), but gene expression
during necrosis has not been well investigated. To determine whether a
molecular response was triggered during H2O2-induced
necrosis, the state of AP-1 activation was investigated, because
increased expression of components of this transcription factor has
been linked to apoptosis (12, 19, 27, 31). Necrosis in HuH-7 cells was
associated with AP-1 activation as indicated by the fact that
H2O2
treatment 1) induced increased mRNA
expression for the AP-1 genes c-fos
and c-jun,
2) resulted in increased JNK
activity, and 3) increased AP-1 DNA
binding and transcriptional activity. The findings of increased
c-fos and c-jun gene expression and JNK
activation rule out the possibility that changes in AP-1 activation
were simply the result of
H2O2-induced cellular redox shifts, which can directly modify AP-1 DNA binding activity (36). Rather, the process of
H2O2-induced
necrosis involved active changes in gene expression and kinase activity that led to AP-1 activation. The marked stimulation of JNK activity in
HuH-7 cells by
H2O2
differs from reported findings in NIH 3T3 cells in which only a minor
activation of JNK occurred after
H2O2 treatment (24), despite the use of an
H2O2
concentration (1 µmol/106 cells)
comparable to that used in our studies. Cell type-specific responses to
oxidant stress may explain these divergent findings. Interestingly the
response to
H2O2
in HuH-7 cells was one of prolonged activation of AP-1.
H2O2
readily diffuses across the cell membrane and is metabolized within a
few minutes by liver cells (6). Despite the brevity of this
pharmacological effect, HuH-7 cells responded with an activation of
AP-1 that was sustained for at least 20 h.
Inhibiting
H2O2-induced
AP-1 activation through the introduction of a
c-jun antisense expression vector led
to a significant decrease in cell necrosis. This effect of AP-1
inhibition did not result from alterations in cellular metabolism or
neutralization of
H2O2.
These findings demonstrate that AP-1 activation directly mediates
oxidant-induced cell death. Although recent studies in U-937 cells and
bovine endothelial cells demonstrated that JNK activation mediated
apoptotic cell death induced by a variety of agents, including
H2O2
(48), JNK phosphorylates other known (ATF-2, p53) (32, 45) and perhaps
unknown transcription factors in addition to c-Jun that may have
accounted for this effect. Our data suggest that the effect of JNK
activation on
H2O2-induced cell death is mediated ultimately through c-Jun. These data also demonstrate that activation of the transcription factor AP-1 induces cell death by necrosis as well as apoptosis.
To our knowledge this is the first demonstration of cellular necrosis
mediated by a specific genetic response. Prior investigations implicating JNK or AP-1 activation in apoptosis (19, 43, 50), together
with the present studies, demonstrate that AP-1 gene expression, JNK
activation, and AP-1 transcriptional activation are central molecular
mechanisms of the cellular response to an injurious environmental
insult, whether the terminal result is cell death from apoptosis or
necrosis. Our findings contradict the concept that the absence or
presence of an active cellular response is always a critical
distinguishing feature between necrosis and apoptosis. These two forms
of cell death instead share elements of a common pathway, which may
explain why both necrosis and apoptosis can occur from a single
stimulus. The existence of shared signaling mechanisms between these
two forms of cell death implies that it may be possible to prevent both
necrosis and apoptosis with a single therapeutic strategy aimed at this
common element.
 |
ACKNOWLEDGEMENTS |
We thank Emily Bobe and Anna Caponigro for secretarial assistance,
K. Chien for the 2XTRELuc construct, Marc-Edouard Mirault for the
glutathione peroxidase cDNA, and Yaffa Beck for the manganese superoxide dismutase cDNA. The c-fos,
c-jun, GAPDH and catalase cDNAs were
obtained from American Type Culture Collection.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-44234 (M. J. Czaja), DK-34987, and GM-41804 (D. A. Brenner).
Address for reprint requests: M. J. Czaja, Liver Research Center,
Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY
10461.
Received 2 December 1996; accepted in final form 30 May 1997.
 |
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