Department of Gastroenterology and Hepatology, Medizinische Hochschule, 30625 Hannover, Germany
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ABSTRACT |
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Tumor necrosis factor (TNF)- is a potent inducer of
apoptotic cell death in various tissues, whereas the transcription
factor nuclear factor (NF)-
B is essential to protect against
TNF-
-induced apoptosis. Human hepatoma cell lines were used to
investigate the effectiveness and specificity of the fungal metabolite
gliotoxin in inhibiting TNF-
-induced NF-
B activation in
transformed cells. Gliotoxin-TNF-
cotreatment induced massive
apoptosis in these otherwise TNF-
-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-
B activation. Bromodeoxyuridine staining of liver sections showed
that the lack of NF-
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-
B is essential in defining the fate of liver cells in response to
TNF-
in vivo and furthermore implicate gliotoxin as a potential new response modifier for TNF-
-based therapy.
nuclear factor-B inhibitor; liver regeneration; DNA
synthesis
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INTRODUCTION |
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THE PLEIOTROPIC CYTOKINE tumor necrosis factor
(TNF)- was originally identified through its capacity to induce
hemorrhagic tumors in mice (6). Attempts to use TNF-
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-
. These results may
help to develop new strategies for modulation of diverse TNF-
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)-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-
B comes from experiments in nontransformed hepatocytes in which
NF-
B activity is inhibited by antibodies or by an inhibitory protein
that triggers apoptosis (3).
Besides its role in the regulation of apoptosis, NF-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-
serum levels are elevated, which leads to
nuclear translocation of NF-
B in hepatocytes. In contrast, TNF-R1
knockout mice show impaired NF-
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-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-
B, called I-
B, which lacks the
phosphoacceptor sites essential for degradation of this protein (16).
Alternatively, pharmacological compounds able to inhibit NF-
B
activation specifically might offer another attractive approach to
blocking NF-
B, thereby enhancing the proapoptotic effects of TNF-
in otherwise resistant or less responsive cells (3, 18).
We addressed the role of NF-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-
B
activation and to trigger TNF-mediated apoptosis. Because gliotoxin was
effective, we administered this compound before liver regeneration. We
demonstrated that NF-
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.
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MATERIALS AND METHODS |
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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 -galactosidase reporter pRSV
Gal as an internal standard.
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-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-
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-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-
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).
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RESULTS |
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Human hepatoma cells can be sensitized against
TNF--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-
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-
treatment. No increase in DNA
fragmentation was found at different time points or even when higher
TNF-
concentrations (200 ng/ml) were used.
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Gliotoxin inhibits TNF--mediated
NF-
B activation and sensitizes human hepatoma cells
against TNF-
-induced apoptosis.
The first results showed that TNF-
and cycloheximide treatment of
hepatoma cells triggers FADD-dependent apoptosis, which can be blocked
by NF-
B activation. TNF-
activates NF-
B in human hepatoma
cells, which was demonstrated by performing gel shift experiments with
nuclear extracts of TNF-
-stimulated Hep G2 cells (Fig.
2B). Because the two pathways,
activation of transcription factor NF-
B and activation of caspases,
trigger antagonistic mechanisms, this system was used to test the
fungal metabolite gliotoxin for its potential to inhibit NF-
B
activation and thus trigger TNF-
-induced apoptosis.
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Gliotoxin specifically interferes with TNF--mediated
NF-
B activity.
Because gliotoxin had a profound effect in inhibiting NF-
B
activation and thus sensitizing hepatoma cells against TNF-
-induced apoptosis, we were interested in assessing the specificity of this regulation.
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Gliotoxin inhibits NF-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-
-dependent activation of NF-
B (8). Because our initial
experiments in hepatoma cells showed that gliotoxin inhibits NF-
B
activation, we treated animals with gliotoxin before hepatectomy to
investigate whether this compound is also an effective NF-
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.
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NF-B activates cell cycle progression and prevents
apoptosis.
Considering the importance of TNF-
-induced signaling pathways for
liver regeneration, as evident from experiments with TNF-R1 knockout
mice (48), NF-
B activation might also be crucial for the reinduction
of the hepatocyte cell cycle after partial hepatectomy.
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DISCUSSION |
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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- 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-
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-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-
B activation through a dominant-negative form of its inhibitor
I-
B (41, 47).
We showed that human hepatoma cells are resistant to TNF--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-
B, as evidenced by cotransfecting increasing amounts of
the p65 subunit of NF-
B (Fig. 1H). Additionally, we used the
fungal metabolite gliotoxin to inactivate NF-
B in liver cells. By injecting gliotoxin before two-thirds hepatectomy in mice, we were able to induce apoptosis in otherwise TNF-
-resistant liver cells by specifically inhibiting the activation of NF-
B (20).
Most NF-B-inhibitors are only effective when cells are treated with
high concentrations (34). In contrast, the specific inhibition of
NF-
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-
B after
stimulation with TNF-
, 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-
B as a protective factor in normal liver
cell physiology (1). Our observation that hepatoma cells are highly
sensitive against TNF-
treatment when NF-
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-B activation in the liver. The immediate TNF-
-dependent activation of NF-
B after partial hepatectomy is essential to trigger
cell proliferation of hepatocytes (16). TNF-R1 knockout mice show
impaired NF-
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-
B activity in vivo.
Gliotoxin significantly diminished early NF-
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-
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-B induction (Fig. 4). The molecular mechanism by which gliotoxin
inhibits NF-
B activation remains unclear. Our experiments in Hep G2
cells showed that degradation of I-
B (Fig. 2D), the
cytoplasmatic inhibitor of NF-
B, is prevented by gliotoxin, although
it does not alter the phosphorylation of I-
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-B activation in vivo is an
adenoviral vector expressing I-kB
(46). In another study, an adenoviral vector expressing dominant-negative I-
B was used to
block NF-
B activation after two-thirds hepatectomy (16). Adenoviral
infection increases the level of proinflammatory cytokines. Among these
cytokines, TNF-
and IL-6 secretion are induced. Although IL-6 is a
proliferative cytokine, this overall immune response might perturb the
effects of NF-
B inhibition (21, 24). This property might also
explain the results observed during liver regeneration in which
infection with the dominant-negative I-
B adenovirus led to an
increase in BrdU staining and apoptosis even before hepatectomy was
performed. After the dominant-negative I-
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-
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-
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-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-
. TNF-
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--dependent activation of
NF-
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-
B activation in vivo,
thereby demonstrating that NF-
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-
.
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ACKNOWLEDGEMENTS |
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We thank D. V. Goeddel (San Francisco, CA) for the FADD expression vector.
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FOOTNOTES |
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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.
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