University of Southern California Research Center for Liver Diseases, University of Southern California/University of California at Los Angeles Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Staurosporine (STS) induces
apoptosis in various cell lines. We report in this study that
primary cultured mouse hepatocytes are less sensitive to STS compared
with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no
apparent release of cytochrome c or loss of mitochondrial
transmembrane potential was detected in primary hepatocytes undergoing
STS-induced apoptosis. Caspase-3 was activated in primary
hepatocytes by STS treatment, but caspase-9 and -12 were not activated,
and caspase-3 activation is not dependent on caspase-8. These findings
point to a novel pathway for caspase-3 activation by STS in primary
hepatocytes. Pretreatment with caspase inhibitor converted STS-induced
apoptosis of hepatocytes to necrotic cell death without
significantly changing total cell death. Thus STS causes hepatocytes to
commit to death upstream of the activation of caspases. We also
demonstrated that STS dramatically sensitized primary hepatocytes to
tumor necrosis factor--induced apoptosis. STS activated
I
B kinase and nuclear factor-
B (NF-
B) nuclear translocation
and DNA binding but inhibited transactivation of I
B-
, inducible
nitric oxide synthase, and inhibitor of apoptosis protein-1 in
hepatocytes and NF-
B reporter in transfected Huh-7 cells.
caspase; necrosis; tumor necrosis factor-
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INTRODUCTION |
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APOPTOSIS AND NECROSIS ARE two major processes by which cells die, depending on the context and cause of death (16). Necrosis occurs when cells are exposed to extreme variance from physiological conditions. Apoptosis, in contrast, can occur under either physiological or pathological conditions. Apoptosis is characterized by morphological changes that appear with great fidelity in cells of widely different lineages (15, 33). Apoptosis is induced in response to various pathological and physiological stimuli, such as developmental cues, activation of cell surface death receptors, ultraviolet (UV) radiation, serum withdrawal, and cytotoxic drugs (27, 29). Apoptosis plays an important role in liver physiology, i.e., maintenance of liver tissue homeostasis, and pathogenesis of liver diseases, i.e., autoimmune and viral liver diseases, liver cancer, and drug-induced liver injury (1, 8, 14, 26). There are two well-established apoptosis pathways: death receptor-mediated pathway and mitochondria-mediated pathway. These pathways converge on the activation of downstream caspases, the cleavage of key substrates, and morphological features of apoptosis (10). In the first pathway, the ligation of death receptors, such as Fas and tumor necrosis factor receptor (TNFR)-1, leads to the recruitment of adaptor molecule FADD, which then brings two or more pro-caspase-8s in close proximity through the interaction of death effector domains (DEDs). The aggregated caspase-8s engage in auto- or transactivation, producing mature caspase-8. Caspase-8 then activates downstream executioner caspases, such as caspase-3 and caspase-6 (3, 21). In the second pathway, a variety of extra- and intracellular death stimuli trigger the release of cytochrome c from mitochondria (19). Cytosolic cytochrome c binds to apoptotic protease-activating factor 1 (APAF-1), resulting in recruitment of pro-caspase-9 by APAF-1. APAF-1 serves as scaffolding to bring pro-caspase-9 molecules together to promote autoactivation of pro-caspase-9 in the presence of dATP (18). Caspase-9 then activates downstream executioner caspases. In addition, the mitochondrial pathway can act as an amplifying feedback regulator for the death receptor-initiated pathway under some circumstances (17).
Staurosporine (STS) has been viewed as a broad spectrum inhibitor of protein kinases (25). It has been well established and very widely used to promote intracellular stress-induced apoptosis in cell culture models employing malignant cell lines, in which the release of cytochrome c from mitochondria and loss of mitochondrial membrane potential have been reported to be often involved. However, there is very little information about the effect of STS on viability of nonproliferating hepatocytes in primary culture. In recent studies of death receptor-mediated apoptosis of hepatocytes, we used STS as a means of inducing death receptor-independent apoptosis (9). The present work was aimed at examining the mechanism of this effect and has uncovered novel effects of STS that may help to better understand the factors that govern death vs. survival in liver cells.
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MATERIALS AND METHODS |
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Mice and reagents. Male C57BL/6 mice were obtained from Harlan (Indianapolis, IN). All the mice were used at the age of 6-7 wk. STS was purchased from Sigma (St. Louis, MO), dissolved in dimethyl sulfoxide (DMSO) as a stock solution. For each experiment, control and treatments were adjusted to contain equal amount of DMSO vehicle. All other chemicals were purchased from reputable commercial sources.
Isolation of mouse hepatocytes and cell culture. Hepatocytes were isolated by in situ retrograde collagenase perfusion. Hepatocytes were dissociated after perfusion from the digested liver by gently scraping with a glass rod suspended in DMEM/F-12 medium (GIBCO-BRL) and filtered through gauze. The cell suspension was then fractionated by Percoll density centrifugation (2,500 rpm for 5 min at 4°C). After Percoll density fractionation, the viability of isolated hepatocyte was assessed by trypan blue dye exclusion, and the cells used for all the experiments had a viability exceeding 86%. Hepatocytes were counted, resuspended in DMEM/F-12 medium containing 10% fetal bovine serum (FBS), 1 nM insulin, 50 nM hydrocortisone, 0.15 mg/ml methionine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, plated at a density of 1.2 × 106 cells/60-mm dish coated with 0.03% rat tail collagen, and incubated under an atmosphere of 95% air-5% CO2. After initial culture for 3 h, the medium was removed and replaced with serum-free medium containing 100 U/ml penicillin and 0.1 mg/ml streptomycin, and incubation continued under an atmosphere of 95% air-5% CO2.
Huh7 cells and Jurkat cells were cultured in DMEM with high glucose and RPMI 1640 with glutamine, respectively, and both media were supplemented with 10% FBS and 100 U/ml penicillin and 0.1 mg/ml streptomycin.DNA fragmentation analysis. After treatment, the hepatocytes were washed with ice-cold PBS. The hepatocytes were lysed in 500 µl lysis buffer [10 mM Tris · HCl, pH 8.0, 10 mM EDTA, 1% Nonidet P-40 (NP-40), and 0.5 mg/ml proteinase K] and incubated at room temperature for at least 1 h. The lysates were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was incubated with 50 µg/ml RNAase at 37°C for 60 min. DNA was extracted with phenol and chloroform (1:1), precipitated by ethanol, and then resuspended in Tris-EDTA buffer. DNA samples (~5 µg for each) were electrophoretically separated on 2% agarose gel containing ethidium bromide (0.5 µg/ml). The gel was destained for 20 min in water before being visualized and photographed.
Measurement of caspase-like activity. At the end of each treatment period, hepatocytes were washed with PBS and then lysed in 500 µl lysis buffer [10 mM Tris · HCl (pH 7.5), 10 mM NaH2PO4/NaHPO4 (pH 7.5), 130 mM NaCl, 1% Triton X-100, 10 mM NaPPi]. Caspase-3-like activity in the lysates was measured using Ac-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-AMC) as caspase-3 substrate. For each sample, 50 µl of the cell lysate, 5 µl of the 1 mg/ml Ac-DEVD-AMC, and 50 µl of reaction buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM 1,4-dithiothreitol (DTT)] were transferred to a 96-well plate, mixed, and incubated at 37°C for 1.5 h. Caspase-3 cleaves a synthetic fluorogenic substrate, Ac-DEVD-AMC, to release the fluorescent AMC. AMC release in the cell lysates was measured by CytoFluor2300 Fluorescence Measurement System (Millipore) using an excitation filter with a wavelength of 360 nm and an emission filter with a wavelength of 460 nm.
Caspase-8- and caspase-9-like activities in the lysates were measured in the same way as caspases-3-like activity except using Ac-IETD-7-amino-4-trifluoromethylcoumarin (Ac-IETD-AFC) as the substrate of FLICE/caspase-8 and Ac-LEHD-AFC as the substrate of caspase-9, a different reaction buffer [20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10% sucrose], and an emission filter with a wavelength of 530 nm.Hoechst dye and Sytox Green staining. After treatment, the cells were stained with 8 µg/ml Hoechst dye 33258 for 10-20 min and 1 µM Sytox Green (Molecular Probes, Eugene, OR) for 5 min. The cells with bright condensed chromatin and fragmented nuclei under Hoechst dye staining were identified as apoptotic cells. The cells with normal-shaped nuclei and stained by Sytox Green were counted as necrotic cells. Hepatocytes in five random fields under the magnitude of ×300 were counted. The results shown are the mean ± SD of three independent experiments.
Fluorescence-activated cell sorter for the mitochondrial potential. The fluorochrome tetramethylrhodamine ethyl ester (TMRE; Molecular Probes) was added to the cells to the final concentration of 100 nM for 20 min before the completion of incubation with STS. The cells were washed twice with PBS to remove excess fluorochrome and then collected by gently scraping. Mitochondrial transmembrane potential was assessed by fluorescence-activated cell sorter (FACS) analysis.
Northern blot analysis.
Total hepatic RNA was extracted from cultured primary hepatocytes by
Trizol reagent (GIBCO-BRL) according to the manufacturer's instruction. RNA was denatured at 65°C for 5 min in loading buffer, electrophoresed through 1.2% agarose gel containing 6.8%
formaldehyde, transferred to Zeta-Probe blotting membrane (Bio-Rad) and
cross-linked with a UV cross-linker (Stratagene, La Jolla, CA). The
probes for IB-
, IAP-1, and inducible nitric oxide synthase (iNOS)
mRNAs were prepared by RT-PCR and labeled with
32P-deoxycytidine triphosphate using Megaprimer labeling
kit (Amersham). PCR primer pairs were 1) mouse I
B-
:
upstream primer, 5'-tcg ttc ctg cac ttg gca atc and downstream primer,
5'-gcc tcc aaa cac aca gtc atc; 2) mouse IAP-1: upstream
primer, 5'-tca gac cct gtg aac ttc cga g and downstream primer, 5'-acg
aca tct tcc gaa ctt tct cc; 3) mouse iNOS: upstream primer,
5'-gac aag ctg cat gtg aca tcg ac and downstream primer, 5'-cga cct gat
gtt gcc att gtt g. Hybridization was performed in Rapid-hyb buffer
(Amersham) at 65°C. Membranes were washed twice in 2× sodium
chloride-sodium citrate (SSC), 0.1% SDS for 20 min at room temperature
and subsequently washed twice in 0.1× SSC, 0.1% SSD for 10 min at
65°C. Membranes were then exposed to film at
80°C.
Subcellular fractionation for assessment of cytochrome c release. Subcellular fractionation was conducted according to the method of Gross et al. (11), with some modification. Cells were washed once in PBS, resuspended in HEPES isotonic mitochondrial (HIM) buffer [(in mM) 200 mannitol, 70 sucrose, 1 EGTA, 10 HEPES (pH 7.5)] supplemented with 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, and 2 µg/ml leupeptin and homogenized in HIM buffer in a 7-ml Wheaten Dounce glass homogenizer using 15 complete up and down cycles of a glass tight-type pestle. Nuclei and unbroken cells were separated at 120 g for 5 min as the low-speed pellet (P1). The supernatant was centrifuged at 10,000 g for 10 min to collect the mitochondria-enriched heavy-membrane pellet. This supernatant was centrifuged at 100,000 g for 30 min to yield the light-membrane pellet and final soluble fraction.
Western blot analysis.
Cells were lysed with ice-cold RIPA buffer (1× PBS, 1% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS) containing 100 µg/ml PMSF, 2 µg/ml
aprotinin, 2 µg/ml leupeptin, and 1 mM sodium orthovanadate. After
centrifugation at 14,000 g for 30 min, protein in the
supernatants were quantitated by Bradford method (Bio-Rad). Forty
micrograms of protein per lane were run in 10% denatured
polyacrylamide gel. After protein was transferred from the gel to
nitrocellulose membrane or polyvinylidene difluoride (PVDF) membrane,
the membranes were blocked at 4°C overnight in PBS containing 5%
fat-free powdered milk or at room temperature for 1 h in PBS + 0.05% Tween 20 (PBS-T) containing 5% fat-free powdered milk. After
the membrane was briefly rinsed with PBS-T, the membrane was incubated
with primary antibody for 1 h at room temperature. Rabbit
polyclonal antibody against IB-
(mouse, rat, and human specific)
and rabbit polyclonal antibody against Rel A (mouse, rat, and human
specific) were purchased from Santa Cruz Biotechnology, and rabbit
polyclonal antibody against caspase-3 (mouse, rat, and human specific),
rabbit polyclonal antibody against caspases-9 (mouse specific), and
rabbit polyclonal antibody against caspases-12 (mouse specific) were
purchased from Cell Signaling Technology (Beverly, MA). All the
antibodies described above were used at 1:1,000 dilution. Mouse
anticytochrome c monoclonal antibody (PharMingen) was used
at 1:500 dilution. After the membranes were washed and incubated with
corresponding horseradish peroxidase-labeled secondary antibody (Santa
Cruz Biotechnology) for 45 min, membranes were washed with PBS-T four
times for 10 min. Proteins were visualized using Luminol enhanced
chemiluminescence reagent (Santa Cruz Biotechnology).
Reporter transfection and luciferase assay.
Huh-7 cells (1 × 106) were plated in six-well plates.
After 1 day of culture, cell transfection was performed according to the Superfect transfection protocol (QIAGEN) with some modifications. In brief, 0.5 µg of pNFB-Luc vectors (Clontech) together with 0.5 µg of pSV-
-galactosidase vectors (Promega) were diluted with cell
growth medium containing no serum or antibiotics to a total volume of
50 µl and then mixed with 10 µl SuperFect transfection reagent and
incubated at room temperature for 10 min to allow transfection-complex
formation. Cell growth medium (1 ml) containing serum and antibiotics
was added into the reaction tube containing the transfection complexes
and mixed and then transferred to Huh-7 cells in the plates. After a
3-h incubation, the medium was replaced with normal culture medium and
further incubated for 12 h. The transfected cells were first
pretreated with 25 µM z-VAD for 30 min to avoid the interference of
caspases, which could be activated by subsequent treatment, and then
treated with STS for 30 min before the treatment with 20 ng/ml TNF-
for 6 h. Cell lysates were prepared using Promega Reporter lysis
buffer. Luciferase and
-galactosidase activity were measured using
Luciferase assay system and and
-galactosidase assay system
(Promega), respectively. Luciferase activity was normalized with
-galactosidase activity.
Electrophoretic mobility shift assay.
After treatments, ~1.2 × 106 hepatocytes in 60-mm
dishes were washed twice with ice-cold PBS and then were gently scraped
in 0.4 ml of hypotonic lysis buffer [10 mM HEPES (pH 7.6), 60 mM KCl,
1 mM EDTA, 0.05% NP-40, 1 mM PMSF, 2.5 µg/ml each of aprotinin and
leupeptin], transferred to microcentrifuge tubes, and lysed on ice for
30 min with occasional vortexing. Nuclei were pelleted (200 g for 5 min
at 4°C). The nuclear pellet was resuspended in two pellet volumes of
nuclear extract buffer [(in mM) 20 Tris (pH 7.9), 420 NaCl, 1.5 MgCl2, 0.2 EDTA, and 0.5 PMSF with 25% glycerol, 2.5 µg/ml of aprotinin, and 2.5 µg/ml leupeptin].The tubes were kept
on ice for 15 min with occasional vortexing. Nuclear extracts were
obtained by centrifugation at 16,000 g, 4°C for 10 min.
Protein concentration of extracts was measured using the Bradford assay
with the Bio-Rad protein assay reagent. Nuclear extracts (10 µg) were
mixed with 1.5 µg poly dI-dC (Sigma) and 0.5-ng end-labeled nuclear
factor B (NF-
B) oligonucleotide probe (5'-agt tga ggg gac ttt ccc
agg c-3') in the binding buffer [(in mM) 10 Tris (pH 7.9), 50 NaCl,
0.5 EDTA, and 1 DTT with 10% glycerol] and were incubated for 20 min
at room temperature. DNA-protein complexes were resolved by
electrophoresis through a 4% polyacrylamide gel containing 25 mM Tris,
0.19 M glycine, and 1 mM ETDA. The gel was dried and visualized by autoradiography.
Immunoprecipitation and IB kinase assay.
IP-I
B kinase (IKK) assay was performed as described in Bhullar et
al. (5). Briefly, cells were treated with STS for various times. Antibody against IKK-
(Santa Cruz Biotechnology) was used to
immunoprecipate IKK complex from whole cell lysates, and the immunoprecipitates were subjected to an IKK assay using GST-I
B-
fusion protein (2 µg) as the IKK substrate. After the reaction cocktail was incubated at 30°C for 30 min, the reaction products were
separated by electrophoresis through 10% SDS-polyacrylmide gel and
then transferred to PVDF membranes. IKK activity was analyzed by
Phosphoimager (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
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STS treatment induces apoptosis of primary hepatocytes.
STS induces apoptosis in a wide variety of proliferating
transformed cells. Normal primary cultured hepatocytes are
nonproliferating cells, and very little is known about the effect of
STS on these cells. To compare the effect of STS treatment on primary
murine hepatocytes with malignant cell lines, dose-dependent (Fig.
1A) and time-dependent (Fig.
1B) effects of STS on primary hepatocytes were examined.
Jurkat cells and Huh-7 cells were treated with 1 µM STS. STS (1 µM) rapidly induced massive apoptosis (~90%) of Jurkat in
4 h and moderate apoptosis (~20%) of Huh-7
cells within 16 h (Fig. 1C). However, 1 µM STS
did not have a significant apoptotic effect on normal hepatocytes
up to 16 h. STS at a higher concentration (5 µM), however, did
induce significant amounts of apoptosis of primary cultured
hepatocytes in 16 h. DNA laddering confirmed apoptosis
(Fig. 1E). We also found that 10 µM STS dramatically sensitized hepatocytes to TNF-induced apoptosis (90.3 ± 4.5%), whereas 1 µM did not significantly sensitize hepatocytes to
TNF-induced apoptosis (Fig. 1D). Because STS is
believed to be a broad-spectrum inhibitor of protein kinases, we
examined various protein kinase inhibitors to see if they have a
similar apoptotic effect on hepatocytes or a sensitizing effect on
TNF-induced apoptosis of primary hepatocytes. None of the PKC
inhibitors (GO6983, GO6976, calphostin C, chelerythrine), PKA inhibitor
(H-89), tyrosine kinase inhibitor (genistein), or PI 3-kinase inhibitor
(wortmannin) had apparent apoptotic effects on primary hepatocytes,
and none of these protein kinase inhibitors significantly sensitized
primary hepatocytes to TNF-
(data not shown).
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STS did not induce cytochrome c release from mitochondria in
primary hepatocyte in contrast in Jurkat and Huh7 cells.
Cytochrome c release from mitochondria is essential to
apoptosis in many systems. We examined whether STS induces
release of cytochrome c into cytosol from mitochondria in
primary hepatocytes. As expected, 1 µM STS induced rapid release of
cytochrome c from mitochondria in Jurkat cells in 4 h
and also an appreciable amount of cytochrome c from
mitochondria in Huh7 within 16 h. However, 10 µM STS did not
induce apparent cytochrome c release to cytosol from
mitochondria in primary hepatocytes after up to 16 h treatment, whereas cytochrome c was released from TNF-/ActD-treated
primary hepatocytes (Fig. 2A).
Nearly 100% primary hepatocytes treated with TNF-
plus ActD
underwent apoptosis, whereas only ~20% of primary
hepatocytes treated with STS did so. Therefore, to ensure our method is
sensitive enough to measure the release of cytochrome c from
20% of cells, we did a titration of loading of the TNF plus
ActD-treated sample. Cytochrome c was readily detected from one-fifth (8 µg) the amount of cytosol fraction from the TNF plus ActD-treated sample (data not shown). Furthermore, the cytochrome c release in Huh7 cells treated with 1 uM STS, which undergo
~20% apoptosis, was also readily detected.
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Effect of STS on activation of caspases.
Caspase-3 in primary hepatocytes was activated by STS treatment, as
shown by both Western analysis and assaying for caspase-3-like activity (Fig. 3A). However,
no caspase-9 activation in primary hepatocytes was detected after
STS treatment by either Western blot analysis and assay for
caspase-9-like activity, which supports the finding that no cytochrome
c release occurred in primary hepatocytes after STS
treatment. As shown in Fig. 3B, both caspase-3 and caspase-8 were activated in STS-treated hepatocytes. As expected in a death receptor-independent apoptosis, caspase-8 activation lagged
behind caspase-3 activation, suggesting that caspase-8 was not
responsible for caspase-3 activation. To further exclude the
possibility that caspase-3 activation is dependent on caspase-8
activation, hepatocytes were pretreated with a low concentration (1 µM) of a selective caspase-8 inhibitor z-IETD-fmk. z-IETD-fmk
completely blocked caspase-8-like activity induced by either
death receptor-dependent TNF or death receptor-independent STS (Fig.
3C). However, z-IETD was unable to block STS-induced
caspase-3 activation and only slightly inhibited STS-induced
apoptosis, whereas z-IETD-fmk completely blocked caspase-3
activation and apoptosis induced by TNF- plus ActD (3D),
indicating that caspase-8 was not responsible for caspase-3 activation
in STS-treated hepatocytes and the delayed increase in caspase 8 activity is secondary to caspase-3 feedback. Slight inhibition of
STS-induced apoptosis by z-IETD-fmk might be due to
interruption of caspase-3 activation self-amplification loop through
caspase-8. Furthermore, caspase-12 was not activated in hepatocytes by
STS treatment up to 16 h (Fig. 3E), suggesting that
endoplasmic reticulum (ER) stress-initiated apoptotic pathway could
not account for STS-induced activation of caspase-3. Thus a
novel mechanism for STS-induced activation of caspase-3 exists that is
distinct from the death receptor-DISC-caspase-8 and
apoptosome-caspase-9 pathways and the ER stress pathway.
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Inhibition of caspases converts STS-induced apoptosis to
necrotic death in primary hepatocytes.
To further address the role of caspases in the STS-induced death of
primary hepatocytes, the cells were pretreated with broad-spectrum caspase inhibitor z-VAD-fmk (50 µM). The pretreatment with the caspase inhibitor converted STS-induced apoptosis to necrosis without significantly changing the viability of the cells. In contrast,
z-VAD-fmk pretreatment protected the cells from TNF- plus
ActD-induced apoptosis without shifting to necrosis (Fig. 4). Similar results were obtained with
pretreatment with DEVD-fmk, a relatively selective inhibitor of
caspase-3 (data not shown). Thus the commitment to STS-induced death of
primary hepatocytes was independent of caspases, although apoptotic
cell death required caspases.
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STS sensitizes primary hepatocytes to TNF- by inhibition of
transcription of NF-
B-dependent survival genes.
TNF-
treatment initiates simultaneously both death and survival
pathways (4, 28). In most systems, including primary hepatocytes, TNF-
alone does not kill cells. One of the most established pathways to sensitize cells to TNF-
is to block the expression of genes of the survival pathway (30).
Therefore, we examined whether STS inhibits TNF-
-induced NF-
B
nuclear translocation and DNA-binding activity. We hypothesed that the
broad-spectrum kinase inhibitory effects of STS might encompass
inhibition of phosphorylation of I
B-
by IKK. Unexpectedly, we
demonstrated that STS alone activated IKK and caused the degradation of
I
B-
in both Huh-7 cells and primary hepatocytes (Fig.
5, A and B). Furthermore, STS did not inhibit the TNF-induced nuclear translocation and DNA binding activity of NF-
B, and STS alone induced the
translocation of Rel A to nuclei and increased NF-
B binding
activity in primary hepatocytes (Fig. 5C).
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DISCUSSION |
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STS is widely employed as an intracellular stress inducer of apoptosis. In most studies that employ cancer cell lines, STS appears to induce cytochrome c release and mitochondrial depolarization (6, 22, 32, 34). We confirmed this mechanism in Huh-7 and Jurkat cells but observed that STS-induced apoptosis in primary hepatocytes by a mechanism independent of caspase-8, cytochrome c, caspase-9, and caspase-12. The precise mechanism for caspase-3 activation is uncertain, and its elucidation will be of considerable interest in defining a novel pathway to apoptosis independent of caspase-8 activation, cytochrome c-mediated caspase-9 activation, and caspase-12. Although inhibition of protein kinases might be involved, a wide variety of protein kinase inhibitors could not reproduce the effect of STS.
STS appears to cause a commitment to cell death. In the presence of caspase-3 activation, hepatocytes undergo typical apoptosis. However, when caspases are inhibited, the mode of cell death changes to necrosis. It is unclear what determines this phenomenon, because the expected alteration of mitochondria integrity was not observed. Increasing evidence appears to support the concept that profound intracellular stress could initiate both apoptotic and necrotic pathways in many cell types. On the inhibition of caspases, a relatively latent necrotic pathway may become evident and necrosis may emerge as a prevalent mode of cell death under certain circumstances.
In addition, STS exerts unique effects on the survival gene pathway.
STS itself appears to activate IKK, cause IB-
degradation and
NF-
B translocation, and does not alter the response TNF-
of these
initial steps in the survival pathway. Thus this is opposite to our
initial speculation that the kinase inhibitory effect of STS might
inhibit IKK and subsequent events. The precise mechanism for
STS-mediated intracellular stress-induced IKK activation is uncertain.
STS however, interfered with NF-
B-mediated gene expression. Thus
despite increasing NF-
B DNA binding, and not interfering with
TNF-induced NF-
B DNA binding, the expected increased gene expression
was inhibited as reflected in I
B-
, IAP-1, and iNOS. The
mechanism for this effect is uncertain but might be due to selective
inhibition of protein kinases, which have been implicated in the
regulation of NF-
B transcriptional activity (20, 35, 36). This effect of STS most likely contributes to the ability of STS alone to cause apoptosis of hepatocytes and to STS
sensitization to TNF-
-induced apoptosis.
In summary, we have investigated the effect of STS on the survival of
primary hepatocytes and have discovered several novel actions of this
substance that may lead to a better understanding of the factors that
control survival and death of liver cells. STS exhibits two major
effects that may contribute to its toxicity: activation of caspase-3 by
a unique mechanism independent of caspase-8, -9, and -12 and cytochrome
c release from mitochondria and inhibition of
NF-B-mediated survival gene expression. The latter leads to sensitization to TNF-
-induced apoptosis.
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ACKNOWLEDGEMENTS |
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We thank the Cell Culture Subcore of the University of Southern
California Research Center for Liver Diseases (P01 DK-48522) for
isolation of mouse hepatocytes. We also thank Dr. E. Zandi for
providing GST-IB-
fusion protein and helpful advice.
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FOOTNOTES |
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This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA-09526, project I of the Alcohol Center, and American Liver Foundation postdoctoral fellowship (to G. Feng).
Address for reprint requests and other correspondence: N. Kaplowitz, Keck School of Medicine, 2011 Zonal Ave., HMR 101, Los Angeles, CA 90033 (E-mail: kaplowit{at}hsc.usc.edu).
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. Section 1734 solely to indicate this fact.
First published January 16, 2002;10.1152/ajpgi.00467.2001
Received 1 November 2001; accepted in final form 9 January 2002.
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