NF-kappa B determines between apoptosis and proliferation in hepatocytes during liver regeneration

Jörg Plümpe, Nisar P. Malek, C.-Thomas Bock, Tim Rakemann, Michael P. Manns, and Christian Trautwein

Department of Gastroenterology and Hepatology, Medizinische Hochschule, 30625 Hannover, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor (TNF)-alpha is a potent inducer of apoptotic cell death in various tissues, whereas the transcription factor nuclear factor (NF)-kappa B is essential to protect against TNF-alpha -induced apoptosis. Human hepatoma cell lines were used to investigate the effectiveness and specificity of the fungal metabolite gliotoxin in inhibiting TNF-alpha -induced NF-kappa B activation in transformed cells. Gliotoxin-TNF-alpha cotreatment induced massive apoptosis in these otherwise TNF-alpha -resistant cell lines. With the use of the mouse partial hepatectomy model, we were also able to demonstrate in vivo the capacity of gliotoxin to act as inhibitor of NF-kappa B activation. Bromodeoxyuridine staining of liver sections showed that the lack of NF-kappa B activation correlated with 80% reduction of DNA synthesis 48 h after hepatectomy compared with untreated controls. Additionally, animals treated with gliotoxin showed nuclear condensation and DNA laddering of hepatocytes indicative of apoptosis 24 h after hepatectomy. In summary, our results demonstrate that NF-kappa B is essential in defining the fate of liver cells in response to TNF-alpha in vivo and furthermore implicate gliotoxin as a potential new response modifier for TNF-alpha -based therapy.

nuclear factor-kappa B inhibitor; liver regeneration; DNA synthesis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PLEIOTROPIC CYTOKINE tumor necrosis factor (TNF)-alpha was originally identified through its capacity to induce hemorrhagic tumors in mice (6). Attempts to use TNF-alpha for systemic anticancer chemotherapy failed due to severe side effects before therapeutic doses could be reached (10, 19). However, recent work provided deeper insights into the molecular mechanisms involved in the regulation of physiological functions of TNF-alpha . These results may help to develop new strategies for modulation of diverse TNF-alpha functions.

Two surface receptors, TNF receptor 1 and 2 (TNF-R1 and 2), are essential in mediation of TNF-dependent signals from cell membrane to nucleus (35, 44). Activation of TNF-R1 results in stimulation of cell death or induction of protective antiapoptotic pathways (14). The proteins associated with the intracellular domain of TNF-R1, which is essential to trigger downstream signals, have recently been identified (32). The TNF receptor-associated protein with death domain (TRADD), which directly interacts with TNF-R1, is involved in mediating the activation of apoptotic and antiapoptotic pathways (15). TNF receptor-associated factor 2 (TRAF 2), which binds to the NH2-terminal region of TRADD, is essential for protective signals (33), whereas Fas-associated protein with death domain (FADD), which interacts with the COOH-terminal region of TRADD, mediates the activation of the proapoptotic caspase cascade (5, 7).

One of the intracellular TNF-dependent pathways that results in the induction of antiapoptotic mechanisms is the activation of nuclear factor (NF)-kappa B. For hepatocytes, this has been shown in mice homozygous for a null mutation in the relA gene that leads to massive apoptosis of hepatocytes during embryogenesis and subsequent death of the animals (1). Another proof of the essential role of NF-kappa B comes from experiments in nontransformed hepatocytes in which NF-kappa B activity is inhibited by antibodies or by an inhibitory protein that triggers apoptosis (3).

Besides its role in the regulation of apoptosis, NF-kappa B has been implicated in direct proliferative processes ranging from B cell and T cell activation to limb development (17, 22). After two-thirds hepatectomy in mice, TNF-alpha serum levels are elevated, which leads to nuclear translocation of NF-kappa B in hepatocytes. In contrast, TNF-R1 knockout mice show impaired NF-kappa B activation and, as a consequence, lack of elevation of interleukin-6 (IL-6) serum levels, resulting in a decrease of DNA synthesis (48).

These results indicate that liver cell proliferation is a process in which NF-kappa B activation is involved in both cell proliferation and the inhibition of apoptosis, thereby offering a unique model for studying strategies for interference with these processes in vivo. One approach could be viral gene transfer by expressing a dominant-negative form of the cellular inhibitor of NF-kappa B, called I-kappa B, which lacks the phosphoacceptor sites essential for degradation of this protein (16). Alternatively, pharmacological compounds able to inhibit NF-kappa B activation specifically might offer another attractive approach to blocking NF-kappa B, thereby enhancing the proapoptotic effects of TNF-alpha in otherwise resistant or less responsive cells (3, 18).

We addressed the role of NF-kappa B activation during liver regeneration after two-thirds hepatectomy. We tested the fungal metabolite gliotoxin in hepatoma cells for its capacity to inhibit TNF-dependent NF-kappa B activation and to trigger TNF-mediated apoptosis. Because gliotoxin was effective, we administered this compound before liver regeneration. We demonstrated that NF-kappa B is necessary to trigger cell proliferation and prevent apoptosis after hepatectomy and thus show that gliotoxin could be a useful drug in modifying TNF-mediated mechanisms in vivo.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture, transfection experiments, and luciferase assays. Hep G2 cells and HuH 7 cells were grown in DMEM supplemented with 10% FCS. DNA transfection was performed using a modified calcium phosphate precipitation method as described previously (28). Hep G2 or HuH 7 cells were grown on 60-mm dishes to ~50% confluence when used for transfection experiments. The amounts of reporter and expression vectors are indicated in Fig. 1. By adding pBSK+DNA (Stratagene) to 6 µg of the transfection mix, the total amount of DNA was kept constant in each transfection experiment. All transfections contained 0.2 µg of the beta -galactosidase reporter pRSVbeta Gal as an internal standard.

For stimulation experiments, cells were cultured in DMEM supplemented with 10% FCS immediately after transfection. After 24 h, cells were stimulated with the compounds at time points as indicated.

To measure luciferase activity, cells were washed twice with PBS and lysed by adding 350 µl of extraction buffer [25 mM Tris · H3PO4, pH 7.8, 2 mM EDTA, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, and 2 mM dithiothreitol (DTT)] for 10 min. The lysates were cleared by centrifugation. Fifty microliters of the supernatant were assayed by adding 300 µl of measuring buffer (25 mM glycylglycine, 15 mM MgSO4, and 5 mM ATP). The light emission was measured in duplicate for 10 s in a Lumat LB 9501 (Berthold, Bad Wildbad, Germany) by injecting 100 µl of 250 µM luciferin. Each experiment was performed in duplicate and repeated at least three times. The data show the specific luciferase activity and represent the average of three independent experiments.

Nuclear extracts and gel retardation assays. Hep G2 and HuH 7 nuclear extracts were prepared using the modified Dignam C method (38). For gel retardation assays, nuclear extracts were used as indicated. Binding buffer consisted of 25 mM HEPES, pH 7.6, 5 mM MgCl2, 34 mM KCl, 2 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/µl poly(dI-dC), and 2 µg/µl BSA. A 32P-labeled oligonucleotide representing the NF-kappa B consensus site (5'-TAGTTGAGGGGACTTTCCCAGGCA-3') was used as a probe. The binding reaction was performed for 30 min on ice. Free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel. Super-shift experiments were performed with antibodies directed against the p50 and p65 NF-kappa B proteins (Santa Cruz Biotechnology, Santa Cruz, CA).

SDS-polyacrylamide gel electrophoresis and Western blot analysis. SDS-polyacrylamide gel electrophoresis and Western blot analysis with cytoplasmic or whole cell extracts were performed as described earlier (39). Poly ADP-ribose polymerase (PARP) was detected with an antibody purchased from Boehringer Mannheim, and I-kappa B was detected by using an antibody from Santa Cruz Biotechnology. The antigen-antibody complexes were visualized by using the enhanced chemiluminescence detection system as recommended by the manufacturer (Amersham, Braunschweig, Germany).

Caspase-3 assay. To measure caspase-3 activity, stimulated HuH 7-cells were harvested and washed twice with PBS. The cells were lysed by adding 100 µl of hypotonic lysis buffer (25 mM HEPES, 5 mM MgCl2, 5 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 0.1% Trasylol, 0.4 µM pepstatin A, and 0.4 µM leupeptin) and freezing/thawing the cells four times. The lysate was centrifuged at 16,000 g and 4°C for 20 min. Protein (25 µg) of the resulting supernatant was examined for caspase-3 activity by using the fluorimetric CaspACE Assay System (Promega). The caspace-3 substrate Ac-DEVD is labeled with fluorochrome 7-amino-4-methylcoumarin (AMC). The specificity of caspace-3 cleavage of Ac-DEVD-AMC, which could be monitored by a fluorometer at 460 nm, was proven by using the reversible aldehyde inhibitor Ac-DEVD-CHO.

DNA fragmentation. For semiquantitative determination of DNA fragmentation, one liver lobe was treated in phosphate buffer (50 mM phosphate, 120 mM NaCl, 10 mM EDTA, 10 mM EGTA, pH 7.4) with five strokes of a homogenizer (pestle B). The 20% homogenate was centrifuged at 13,000 g for 20 min. One milliliter of the supernatant was treated with proteinase K for 4 h at 55°C. After phenol/chloroform extraction and precipitation of the DNA with ethanol containing 3 M sodium acetate, RNase digestion was performed. After 1 h of RNase treatment, the DNA was precipitated with ethanol-sodium acetate and resuspended in TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0). DNA (10 µg) was separated on a 1.8% agarose gel and stained with ethidium bromide. The n × 123-bp molecular weight marker used for gel electrophoresis was from GIBCO BRL (Eggenstein, Germany).

To determine DNA fragmentation in cell culture experiments, the commercially available cell death detection ELISA (Boehringer Mannheim, Mannheim, Germany) was used. Hepatoma cells (1 × 105) were incubated with 200 µl of lysis buffer. The following steps of the cell death detection ELISA were performed as described by the manufacturer. The level of DNA fragmentation found before treatment was set to 1, and changes were shown as multiples of activation.

To visualize the nuclei of hepatoma cells, DNA was stained with 4'6'-diamidino-2-phenylindole dihydrochloride (DAPI, 1µg/ml) as described previously (4). For staining of cryosections, cells were fixed with methanol-acetone (50:50, vol/vol).

Two-thirds hepatectomy and preparation of nuclear extracts. Six- to nine-week-old mice weighing 20-25 g were obtained from the animal facility of the Medizinische Hochschule Hannover. The animals were maintained on a 12:12-h light-dark cycle. Twelve hours before surgery, food was withdrawn from the animals. Surgery was performed between 8 and 10 A.M. Presurgical treatment was performed by intraperitoneal injection 4 h before surgery. Gliotoxin was chosen according to previous dose-kinetic experiments in mice (37). Gliotoxin, 80 µg per mouse, was injected. Control mice were treated with the saline buffer only.

Animals were anesthetized by intraperitoneal injection of a combination of rompun-ketamine. After a small subxyphoid incision, two-thirds hepatectomy was performed as described by Higgins and Anderson (12). After surgery, the abdominal cavum was closed by a suture. Sham surgery was performed exactly as indicated for two-thirds hepatectomy, except that the liver was only manipulated and not resected.

For each time point indicated, four mice were used in parallel. The remaining livers were removed, pooled, and rinsed in ice-cold PBS, and part of the livers were immediately frozen in liquid nitrogen or Tissue-Tek (Sakura Europe). The remaining liver was used to prepare nuclear extracts as described before (40). All the steps were performed at 4°C.

Bromodeoxyuridine labeling. For in vivo labeling, 30 µg/g bromodeoxyuridine (BrdU; Amersham, Braunschweig, Germany) were injected intraperitoneally 2 h before killing. Liver tissue was frozen immediately in liquid nitrogen. To detect labeled nuclei, cryosections were prepared (5 µm thick). The tissue was fixed in ice-cold acetone/methanol and stained according to the Amersham cell proliferation kit manual.

Immunofluorescence. For immunofluorescence experiments, cryosections (4-5 µm thick) were performed and fixed immediately in ice-cold acetone for 5 min, air dried, and either stored at -80°C or used immediately. Immunofluorescence staining was performed as described before (38). Anti-NF-kappa B (Santa Cruz Biotechnology) was incubated as primary antibody. As secondary antibody, anti-rabbit Cy3-conjugated antibody (Sigma, St. Louis, MO) was used. Sections were analyzed through a 565-nm filter with a fluorescence microscope (Olympus, Hamburg, Germany).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human hepatoma cells can be sensitized against TNF-alpha -induced apoptosis, depending on a functionally active TNF-R1. The human hepatoma cell lines Hep G2 and HuH 7 are known to express TNF-R1 (45). Therefore, we treated human hepatoma cell lines with TNF-alpha and studied DNA fragmentation by cell death detection ELISA (data not shown) and DAPI staining. These experiments showed that HuH 7 and Hep G2 cells are resistant to TNF-alpha treatment. No increase in DNA fragmentation was found at different time points or even when higher TNF-alpha concentrations (200 ng/ml) were used.

Many cell lines, resistant against apoptosis initiated by treatment with TNF-alpha , can be sensitized through costimulation with inhibitors of protein synthesis (14). Therefore, Hep G2 cells were treated with TNF-alpha and 1 µg/ml cycloheximide. DAPI staining (Fig. 1, A-D) indicated that 20 ng/ml TNF-alpha and 1 µg/ml cycloheximide induced massive apoptosis of Hep G2 cells; nearly 100% of the cells underwent nuclear condensation (Fig. 1C). Similar results were found using HuH 7 cells, which were also sensitized against TNF-alpha -induced apoptosis by costimulation with cycloheximide (data not shown).


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Fig. 1.   Cycloheximide sensitizes human hepatoma cells against tumor necrosis factor (TNF)-alpha -induced apoptosis. A-D: 4'6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining of nuclei of Hep G2 cells before (A) and 12 h after treatment with 20 ng/ml TNF-alpha alone (B), 20 ng/ml TNF-alpha and 1 µg/ml cycloheximide (C) or 1 µg/ml cycloheximide alone (D) are shown. DAPI staining was visualized using an emission wavelength of 520 nm. E: HuH 7 cells were treated with 20 ng/ml TNF-alpha or 20 ng/ml TNF-alpha and 1 µg/ml cycloheximide for increasing time intervals as indicated. Western blot analysis was performed to detect poly ADP-ribose polymerase (PARP) and its 89-kDa degradation product. F: HuH 7 cells were treated with 20 ng TNF-alpha and 1 µg/ml cycloheximide for indicated time intervals. Caspase-3 activity was measured with a caspase assay system using 25-µg whole cell extracts. G: HuH 7 cells were transfected with an expression vector for beta -galactosidase (beta Gal) alone or in combination with an expression vector for dominant-negative Fas-associated protein with death domain (Delta FADD). Cells are shown before or 12 h after treatment with 20 ng TNF-alpha and 1 µg/ml cycloheximide. Amount of blue cells is shown as percentage of total amount of cells. H: HuH 7 cells were cotransfected with increasing amounts of an expression vector for nuclear factor (NF)-kappa B p65 (CMV-p65) as indicated and 2 µg of an expression vector for beta -galactosidase. Amount of blue cells is shown as percentage of total cell number.

Besides the expression of TNF-R1 on the cell surface, the functional integrity of this receptor is determined by binding of receptor-associated proteins like FADD to the TNF-R1 binding protein TRADD. The interaction of these proteins is required to induce apoptosis in a given cell (15).

To understand whether this pathway is functionally intact in human hepatoma cells, the cleavage activity of caspases downstream of FADD was measured. As shown in Fig. 1E by Western blot analysis, treatment of TNF-alpha and cycloheximide, but not TNF-alpha alone, resulted in a time-dependent cleavage of PARP. The appearance of the 89-kDa degradation product was found 4-8 h after TNF-alpha and cycloheximide treatment. Caspase-3 activity first increased after 4 h and was maximal at the 6-h time point (Fig. 1F), whereas no increase was seen when the cells were treated with TNF-alpha alone (data not shown).

The functional relevance of FADD-dependent signals for TNF-alpha -induced apoptosis was further confirmed by cotransfecting hepatoma cells with a dominant-negative form of FADD (Delta FADD) and beta -galactosidase before stimulation with TNF-alpha and cycloheximide. Twelve hours after stimulation, blue cell analysis was performed. Stimulated or unstimulated cells transfected with beta -galactosidase show the same ratio of blue cells, although total cell number is decreased 12 h after stimulation with TNF-alpha and cycloheximide. In contrast, Delta FADD-expressing cells show reduced apoptosis when stimulated with TNF-alpha and cycloheximide, which results in a significantly higher ratio of transfected blue cells in regard to total cell numbers (Fig. 1G).

Earlier reports have shown that the transcription factor NF-kappa B is able to inhibit apoptosis in response to TNF-alpha stimulation (2). However, the role of NF-kappa B as a protective protein during apoptosis has been challenged by observations implicating NF-kappa B in proapoptotic processes (11, 26). Therefore, the functional relevance of NF-kappa B in the process of TNF-alpha /cycloheximide-induced apoptosis in hepatoma cells was assessed by cotransfecting a vector expressing the p65 (rel A) protein and a vector expressing beta -galactosidase. Thirty-six hours after transfection, the cells were treated with cycloheximide and TNF-alpha . After 12 h of incubation, we performed beta -galactosidase staining of transfected cells. Figure 1H shows that the expression of p65 significantly protected hepatoma cells against TNF-alpha -induced apoptosis, which is indicated by an increased percentage of blue cells in regard to total cell numbers.

Gliotoxin inhibits TNF-alpha -mediated NF-kappa B activation and sensitizes human hepatoma cells against TNF-alpha -induced apoptosis. The first results showed that TNF-alpha and cycloheximide treatment of hepatoma cells triggers FADD-dependent apoptosis, which can be blocked by NF-kappa B activation. TNF-alpha activates NF-kappa B in human hepatoma cells, which was demonstrated by performing gel shift experiments with nuclear extracts of TNF-alpha -stimulated Hep G2 cells (Fig. 2B). Because the two pathways, activation of transcription factor NF-kappa B and activation of caspases, trigger antagonistic mechanisms, this system was used to test the fungal metabolite gliotoxin for its potential to inhibit NF-kappa B activation and thus trigger TNF-alpha -induced apoptosis.


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Fig. 2.   Gliotoxin inhibits TNF-alpha -dependent NF-kappa B activation and renders hepatoma sensitive against TNF-alpha -mediated apoptosis. A: HuH 7 cells were incubated with fungal metabolite gliotoxin. Subsequently, HuH 7 cells were stimulated for 12 h with TNF-alpha . DNA fragmentation was determined by cell death detection ELISA. Rate of DNA fragmentation of untreated cells was set to 1, and other conditions were compared accordingly. Concentrations of gliotoxin are depicted. B: Hep G2 cells were stimulated for 30 min with 20 ng/ml TNF-alpha alone or were pretreated for 1 h with gliotoxin in concentrations indicated. The nuclear extracts of these cells were incubated with a 32P-labeled NF-kappa B consensus oligonucleotide for gel shift experiments. C: super-shift experiments were performed with nuclear extracts of Hep G2 cells treated for 30 min with TNF-alpha and antibodies directed against the p50 and p65 subunits of NF-kappa B (alpha -p50 and alpha -p65). Complexes mainly consisting of p65 (1) or p50 (2) are shown with arrows. D: Hep G2 cells were incubated for indicated periods with 20 ng/ml TNF-alpha , with or without 1-h pretreatment with 1 µg/ml gliotoxin. Western blot analysis was performed with 6.5-µg cytoplasmic extracts to detect I-kappa B levels.

HuH 7 cells were incubated for 12 h with gliotoxin and were subsequently treated for 12 h with 50 ng/ml of TNF-alpha . Apoptosis of hepatoma cells was quantified by using the cell death detection ELISA. The results of the control-treated cells were set to 1, and changes in DNA fragmentation as measured with different gliotoxin concentrations used in these experiments are shown as multiples of activation (Fig. 2A). Gliotoxin sensitized hepatoma cells against TNF-alpha -induced apoptosis, whereas only a moderate increase in DNA fragmentation was found when the cells were treated with the fungal metabolite alone.

To test the direct effect of gliotoxin on inhibiting DNA binding of NF-kappa B, gel shift experiments were performed. Gliotoxin inhibited the TNF-alpha -induced nuclear translocation and DNA binding of NF-kappa B in Hep G2 cells in a dose-dependent manner (Fig. 2B). Super-shift experiments with anti-p50 and anti-p65 antibodies indicated that both proteins are present in the complexes appearing after TNF-alpha treatment (Fig. 2C).

The molecular mechanism by which gliotoxin inhibits the activation of NF-kappa B remains unknown. Earlier reports showed that the phosphorylation of its cytoplasmic inhibitor I-kappa B is unaffected by gliotoxin (30). To examine the effect of gliotoxin on I-kappa B, we performed Western blot analysis with cytoplasmic extracts of TNF-alpha -stimulated Hep G2 cells (Fig. 2D). Without stimulation, I-kappa B is detectable in the cytoplasm of the cells. A 10-min treatment with 20 ng/ml TNF-alpha leads to degradation of the NF-kappa B inhibitor. Pretreatment of Hep G2 cells with 1 µg/ml gliotoxin abolishes the degradation of I-kappa B (Fig. 2D). We speculated that gliotoxin might interact with the ubiquitination of the I-kappa B protein and might act as an nonspecific ubiquitination inhibitor. To investigate this possibility, we overexpressed p21 (waf 1), p27 (kip 1), and cyclin E to determine whether the ubiquitination of these proteins might be affected by gliotoxin. Western blot analysis showed that none of these proteins accumulated after treatment with gliotoxin, nor were ubiquitinated higher molecular forms of these proteins detectable (data not shown).

Gliotoxin specifically interferes with TNF-alpha -mediated NF-kappa B activity. Because gliotoxin had a profound effect in inhibiting NF-kappa B activation and thus sensitizing hepatoma cells against TNF-alpha -induced apoptosis, we were interested in assessing the specificity of this regulation.

Hep G2 cells were transfected with a NF-kappa B-dependent reporter gene construct (3 × kappa B-Luc) and stimulated with 50 ng/ml TNF-alpha for 6 h, a period in which signs of apoptosis were not yet present (Fig. 3A). An incubation with different doses of gliotoxin 30 min before incubation with TNF-alpha leads to strong decrease of luciferase activity. Gliotoxin (1 µg/ml) reduces NF-kappa B-dependent luciferase activity nearly to the level of untreated control cells. In contrast to the decrease in luciferase activity, no changes in the activity of the beta -galactosidase control reporter were found.


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Fig. 3.   Gliotoxin specifically interferes with TNF-alpha -dependent NF-kappa B activation. Hep G2 cells were transfected with a NF-kappa B (A) or Stat3-dependent (B) luciferase construct. Cells were stimulated for 6 h with 50 ng/ml TNF-alpha (A) or interleukin (IL)-6 (B), with or without 30-min preincubation with different concentrations of gliotoxin as depicted. Activity of untreated cells was set to 1, and other activities were compared accordingly. C: HuH 7 cells were transfected with a CREB-dependent luciferase construct (CRE-Luc). Activity of untreated cells was set to 1. As positive control, CRE-Luc was cotransfected with catalytic subunit of protein kinase A. At right, HuH 7 cells transfected with CRE-Luc were incubated with increasing amounts of gliotoxin for 12 h.

To show the specificity of gliotoxin for NF-kappa B, Hep G2 cells were transfected with a Stat3-dependent reporter gene construct. No decrease in reporter gene activity was found when the cells were pretreated with gliotoxin before IL-6 stimulation. Earlier results indicated that gliotoxin activates protein kinase A (PKA), which results in the activation of transcription factor CREB (43). To test this possibility, HuH 7 cells were transfected with a CREB-dependent reporter gene construct (CRE-Luc) and stimulated with gliotoxin. As a positive control, the CRE-Luc plasmid was cotransfected with the catalytic subunit of PKA (Fig. 3C).

Although the catalytic subunit of PKA stimulated luciferase activity >10-fold, a small decrease compared with the untreated cells was detected when cells were stimulated with gliotoxin. These results indicate that, at concentrations sufficient to significantly inhibit NF-kappa B activation, the effect on PKA activation was negligible.

Gliotoxin inhibits NF-kappa B activation after two-thirds hepatectomy. Two-thirds hepatectomy in the mouse induces reentry into the cell cycle of the remaining normally resting hepatocytes, ultimately leading to full reconstitution of the organ. An early event during this process is the TNF-alpha -dependent activation of NF-kappa B (8). Because our initial experiments in hepatoma cells showed that gliotoxin inhibits NF-kappa B activation, we treated animals with gliotoxin before hepatectomy to investigate whether this compound is also an effective NF-kappa B inhibitor in vivo. Gliotoxin, 80 µg per mouse, was injected intraperitoneally 4 h before hepatectomy. In control experiments, mice were either not treated before hepatectomy or received gliotoxin only.

To monitor time-dependent DNA binding of NF-kappa B, gel retardation experiments were performed. As shown in Fig. 4A, after hepatectomy we detected a transient increase in DNA binding of NF-kappa B, showing maximal complex formation 1 h after surgery. Importantly, gliotoxin pretreatment significantly diminished the strong increase in DNA binding (Fig. 4B). In contrast, when mice were pretreated with gliotoxin, only weak complex formation was evident 1, 2, and 6 h after hepatectomy. In the gliotoxin control-treated animals, no complex formation was found compared with untreated mice (Fig. 4C). Super-shift experiments with anti-p50 and anti-p65 antibodies indicated that both proteins are present in the complexes appearing 1 h after hepatectomy (Fig. 4D).


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Fig. 4.   Hepatectomy-dependent NF-kappa B activation is inhibited by gliotoxin pretreatment. Gel shift experiments were performed using mouse liver nuclear extracts and a 32P-labeled consensus oligonucleotide for NF-kappa B. Nuclear extracts were prepared from untreated mice and animals that were hepatectomied without (A) and with (B) gliotoxin pretreatment. Additionally, liver nuclear extracts of mice treated with gliotoxin only were used (C). Different time points are indicated. Anti-p50 and anti-p65 antibodies were used for super-shift experiments (alpha -p50 and alpha -p65; D). In these experiments, 2.5-µg nuclear extracts derived 1 h after hepatectomy were incubated. Positions of complexes appearing after hepatectomy and mainly containing either the p50 (1) or p65 (2) subunit are indicated. E-H: Immunofluorescence studies of liver sections with an anti-p65 antibody are shown. Liver sections of untreated animals (E) or mice 1 h after hepatectomy (F), 1 h after hepatectomy with gliotoxin pretreatment (G), or 1 h after gliotoxin treatment (H) were used. Nuclei are shown by arrows.

To further evaluate the gliotoxin-dependent effect on NF-kappa B activation after hepatectomy, immunofluorescence studies were performed using an antibody directed against NF-kappa B (Fig. 4, E-H). As shown in Fig. 4F, an increase in nuclear staining of NF-kappa B was evident 1 h after hepatectomy. In contrast, this effect was blocked in the gliotoxin-pretreated animals (Fig. 4G). No nuclear signal for NF-kappa B was evident before surgery and in the animals treated with gliotoxin only (Fig. 4, E and H).

NF-kappa B activates cell cycle progression and prevents apoptosis. Considering the importance of TNF-alpha -induced signaling pathways for liver regeneration, as evident from experiments with TNF-R1 knockout mice (48), NF-kappa B activation might also be crucial for the reinduction of the hepatocyte cell cycle after partial hepatectomy.

Therefore, we measured DNA synthesis in the regenerating liver using BrdU staining. Only few cells were stained positive in mice before hepatectomy (Fig. 5A). To determine the normal time frame of DNA synthesis after partial hepatectomy, mice were hepatectomized without gliotoxin pretreatment and BrdU staining was performed at different time points. These studies showed that DNA synthesis reaches its maximum 48 h after hepatectomy (data not shown). After a 2-h labeling period, >70% of all hepatocyte nuclei stained positive (Fig. 5B). The number of BrdU-labeled cells 48 h after hepatectomy was set to 100%. Changes in DNA synthesis found in the group treated with gliotoxin were calculated accordingly.


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Fig. 5.   Gliotoxin pretreatment before hepatectomy inhibits DNA synthesis. Bromodeoxyuridine (BrdU) staining was performed to monitor DNA synthesis in untreated mice (A) and in animals 48 h after hepatectomy without (B) and with (C) gliotoxin pretreatment. Mice treated with gliotoxin only for 48 h are shown in D. Quantitative analysis of BrdU experiments are depicted in E. The absolute amount of BrdU-positive cells 48 h after hepatectomy was set to 100%, and changes were calculated accordingly.

Figure 5E shows the dramatic decrease in DNA synthesis caused by gliotoxin treatment before partial hepatectomy. Compared with 100% maximum DNA synthesis 48 h after hepatectomy, only 20.8 ± 10% of the cells were marker positive (Fig. 5, C and E). We observed no difference in mice treated with gliotoxin alone or control mice (Fig. 5, D and E). These experiments revealed that gliotoxin treatment inhibited cell cycle progression after hepatectomy before the G1/S-phase checkpoint.

The gel shift and immunofluorescence studies demonstrated that gliotoxin treatment diminishes normal NF-kappa B activation after two-thirds hepatectomy. Because NF-kappa B is involved in controlling antiapoptotic mechanisms, we were interested in studying whether this function might also be relevant during liver regeneration. Therefore, fragmentation of genomic DNA isolated 24 h after treatment was measured using agarose gel electrophoresis. No DNA fragmentation was found 24 h after manipulation in gliotoxin control-treated animals and in mice not treated with gliotoxin before hepatectomy. In contrast, genomic DNA isolated from mice treated with gliotoxin before partial hepatectomy showed DNA laddering specific for DNA fragmentation induced by apoptosis (Fig. 6A).


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Fig. 6.   Gliotoxin treatment before two-thirds hepatectomy triggers apoptosis of hepatocytes. A: Genomic DNA prepared from total liver was separated on an agarose gel to monitor DNA fragmentation. Samples are depicted of untreated mice (M; lane 1), animals treated with gliotoxin alone for 36 h (lane 3) or animals 24 h after partial hepatectomy either without or with gliotoxin pretreatment (lanes 4 and 5). Co, controls. B-E: DAPI staining of liver nuclei before hepatectomy (B), 24 h after hepatectomy (C), 24 h after hepatectomy with gliotoxin pretreatment (D), or after gliotoxin treatment alone (E). DAPI staining was visualized using an emission wavelength of 456 nm.

To observe the characteristic nuclear condensation pattern of cells undergoing apoptosis, we stained frozen liver sections from mice 24 h after treatment with the nuclear dye DAPI (Fig. 6, B-E). When mice were treated with gliotoxin before hepatectomy, condensed DNA was found in hepatocyte nuclei, indicating DNA fragmentation and thus apoptosis (Fig. 6D). This observation was not evident in mice undergoing partial hepatectomy without gliotoxin pretreatment or mice treated only with gliotoxin for 24 h (Fig. 6, C and E).

Besides the increase in apoptosis of hepatocytes in the group that underwent hepatectomy and gliotoxin pretreatment, there was a significant increase in serum transaminases compared with the animals with hepatectomy alone. Additionally, histological examination revealed areas with profound hemorrhages in the livers of these animals (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatocellular carcinoma is one of the main complications of liver cirrhosis and a serious health problem worldwide. Because most hepatocellular carcinomas are diagnosed in advanced stages, therapeutic options are limited and prognosis is rather poor. Clinical trials using the cytokine TNF-alpha as a potent antiproliferative drug have been hampered by severe side effects elicited by this cytokine if used in effective concentrations (10, 19). Meanwhile, the intracellular pathways that are activated after binding of TNF-alpha to its cellular receptors (TNF-R1 and TNF-R2) have been characterized in detail (23, 33).

It is known that several proteins bind to the intracellular domain of the TNF-R1, thereby enabling induction of its different biological functions (15, 31, 32). Besides the FADD-mediated induction of apoptosis, TNF-R1 activates NF-kappa B via TRAF 2 and thereby induces a potent antiapoptotic pathway (27). The significance of this antiapoptotic mechanism has recently been demonstrated by blocking NF-kappa B activation through a dominant-negative form of its inhibitor I-kappa B (41, 47).

We showed that human hepatoma cells are resistant to TNF-alpha -induced apoptosis. Costimulation with the protein synthesis inhibitor cycloheximide renders hepatoma cells sensitive to apoptosis, which is mediated by TNF-R1 via the FADD/caspase cascade. In contrast, in these hepatoma cells, antiapoptotic pathways are efficiently activated through NF-kappa B, as evidenced by cotransfecting increasing amounts of the p65 subunit of NF-kappa B (Fig. 1H). Additionally, we used the fungal metabolite gliotoxin to inactivate NF-kappa B in liver cells. By injecting gliotoxin before two-thirds hepatectomy in mice, we were able to induce apoptosis in otherwise TNF-alpha -resistant liver cells by specifically inhibiting the activation of NF-kappa B (20).

Most NF-kappa B-inhibitors are only effective when cells are treated with high concentrations (34). In contrast, the specific inhibition of NF-kappa B by gliotoxin can already be detected at low concentrations and it is most likely responsible for its potent immunosuppressive activity (30). Initially, we tested the effect of gliotoxin in human hepatoma cells. It strongly suppresses the activation of NF-kappa B after stimulation with TNF-alpha , thereby shifting the normal balance between pro- and antiapoptosis towards cell death (Fig. 2). The relevance of this balance for the developing liver was recently shown in rel A knockout mice, which die during embryogenesis from massive apoptosis of liver cells, implicating NF-kappa B as a protective factor in normal liver cell physiology (1). Our observation that hepatoma cells are highly sensitive against TNF-alpha treatment when NF-kappa B is blocked is an example of the high level of conservation of the TNF-inducible system in normal liver and cells derived from hepatocellular carcinomas (43).

Because this observation may have implications for the treatment of hepatocellular carcinoma, we studied whether gliotoxin may block NF-kappa B activation in the liver. The immediate TNF-alpha -dependent activation of NF-kappa B after partial hepatectomy is essential to trigger cell proliferation of hepatocytes (16). TNF-R1 knockout mice show impaired NF-kappa B activation and, as a consequence, impaired elevation of IL-6 serum levels, which results in decrease of DNA synthesis in hepatocytes (48). Therefore, we used the liver regeneration model to study the ability of gliotoxin to block NF-kappa B activity in vivo. Gliotoxin significantly diminished early NF-kappa B activation after hepatectomy, which was followed by induction of apoptosis and blockage of liver cell proliferation as assessed by DNA fragmentation assay and BrdU in vivo labeling. These results demonstrate the essential role of NF-kappa B in protecting liver cells against apoptosis and triggering cell cycle progression of hepatocytes during liver regeneration.

Although earlier studies implicated gliotoxin in a variety of different biological functions, we performed control experiments (9, 43). In the concentrations used in our mouse model, gliotoxin selectively inhibited NF-kappa B induction (Fig. 4). The molecular mechanism by which gliotoxin inhibits NF-kappa B activation remains unclear. Our experiments in Hep G2 cells showed that degradation of I-kappa B (Fig. 2D), the cytoplasmatic inhibitor of NF-kappa B, is prevented by gliotoxin, although it does not alter the phosphorylation of I-kappa B (30). The obvious lack in hepatocyte proliferation caused by this compound in our experiments might therefore reflect a general inhibition of ubiquitination. This could in turn lead to accumulation of short-lived cell cycle inhibitors like p21 or p27, known to be degraded through the ubiquitin/proteasome system (25, 29). However, no changes in the degradation of these proteins were detected when cells expressing p21 or p27 were treated with various amounts of gliotoxin (N. P. Malek and J. M. Roberts, unpublished results).

An alternative approach to blocking NF-kappa B activation in vivo is an adenoviral vector expressing I-kBalpha (46). In another study, an adenoviral vector expressing dominant-negative I-kappa B was used to block NF-kappa B activation after two-thirds hepatectomy (16). Adenoviral infection increases the level of proinflammatory cytokines. Among these cytokines, TNF-alpha and IL-6 secretion are induced. Although IL-6 is a proliferative cytokine, this overall immune response might perturb the effects of NF-kappa B inhibition (21, 24). This property might also explain the results observed during liver regeneration in which infection with the dominant-negative I-kappa B adenovirus led to an increase in BrdU staining and apoptosis even before hepatectomy was performed. After the dominant-negative I-kappa B construct was used, cell cycle progression after hepatectomy was impaired at late S or G2 (16). In contrast, our data and the results in the TNF-R1 knockout mice suggested that the lack in NF-kappa B activation results in blocking cell cycle progression before the G1/S-phase check point (48). Because this block can be prevented by injection of IL-6, infection by adenoviruses may mask the relevance of NF-kappa B to entering S phase. Therefore, the adenovirus-induced proinflammatory response might also account for the different effects on cell cycle progression found during liver regeneration.

Our in vitro and in vivo results show that gliotoxin blocks NF-kappa B activation in cells of hepatic origin. A future therapeutic approach could thus integrate the ability of gliotoxin to sensitize hepatocellular carcinoma cells against TNF-alpha . TNF-alpha could either be applied alone or in combination with chemotherapy. The concept of regional chemotherapy as already used to treat hepatocellular carcinoma would selectively target tumor cells.

In summary, our results show that TNF-alpha -dependent activation of NF-kappa B is essential for survival of human hepatoma cells in vitro. Furthermore, our experiments using the hepatectomy model in mice show that gliotoxin is an effective inhibitor of NF-kappa B activation in vivo, thereby demonstrating that NF-kappa B is a crucial factor during liver regeneration able to prevent apoptosis and trigger cell cycle progression of hepatocytes. From this we conclude that gliotoxin might be a valuable tool in treating hepatocellular carcinomas and other conditions rendering otherwise resistant cells sensitive to TNF-alpha .


    ACKNOWLEDGEMENTS

We thank D. V. Goeddel (San Francisco, CA) for the FADD expression vector.


    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinaschaft Grant Tr 285 3-4. The first and second authors contributed equally to this work.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. Trautwein, Dept. of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany (E-mail: trautwein.christian{at}mh-hannover.de).

Received 12 April 1999; accepted in final form 8 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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