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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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-alpha (TNF-alpha ), cells were pretreated with cycloheximide (20 µg/ml) for 30 min, followed by TNF-alpha (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-kappa 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).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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-alpha 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-alpha (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-alpha -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-alpha -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-alpha (TNF-alpha ) 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-alpha -treated cells had characteristics of apoptosis, with cytoplasmic and nuclear condensation and formation of apoptotic bodies (C). Under fluorescent microscopy TNF-alpha -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-alpha (lane 5).

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.

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 [gamma -32P]ATP. Phosphorylated substrate was detected by Phosphorimage analysis after electrophoresis of reaction mixtures in sodium dodecyl sulfate-polyacrylamide gels.

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-kappa 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.

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-kappa B. H2O2 increased NF-kappa B DNA binding activity at 2 h, but, in contrast to the prolonged expression of AP-1, NF-kappa 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.

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.

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).

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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Table 1.   GSH levels in CAT-16 and AS-Jun 11 H-7 cells

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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