1Department for Gastroenterology and Hepatology, University-Hospital Essen, 45133 Essen, Germany; and 2Marion Bessin Liver Research Center and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, 10461
Submitted 3 December 2003 ; accepted in final form 6 February 2004
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ABSTRACT |
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dengue virus; tumor necrosis factor; activator protein-1; casein kinase
Although the overall function of CK2 is not completely understood, it is thought to regulate cell proliferation (32), malignant transformation (12), and apoptosis (31). Several studies have demonstrated that CK2 may protect against cell death. Overexpression of the - but not
-CK2 subunit in the prostate adenocarcinoma cell line PC-3 blocked chemically induced apoptosis (15). A number of solid tumors (12, 43) and lymphoproliferative diseases were demonstrated to express increased levels of the enzyme (26, 42). In addition, in vitro treatment of cancer cells with antisense oligonucleotides that inhibit the expression of the catalytic subunit led to the induction of apoptosis (11, 15). In contrast to the evidence that CK2 functions as a survival factor are recent investigations demonstrating a proapoptotic role for this enzyme. Phosphorylation of p53 at serine392 by CK2 has been suggested to be essential for p53 to induce growth arrest and apoptosis. In HeLa and HCT116 cells, inhibition of CK2
expression compromised the ability of p53 to inhibit cell growth and proliferation and made cells resistant to drug-induced cell death (45).
To further define the function of CK2 in cell death, we examined the role of the newly described CK2" isoform in the regulation of hepatocellular apoptosis. These investigations utilized the CK2
"-deficient membrane trafficking mutant cell line (Trf1), which has been previously isolated by our laboratory from the hepatoma cell line human hepatoma (HuH)-7 (49). The reported consequences of the CK2
" deficiency in Trf1 cells are defects in membrane protein trafficking (48, 50). The effect of CK2
" on liver cell death from dengue virus (DEN), TNF-
, and other apoptotic stimuli was determined. The findings demonstrate resistance of Trf1 cells compared with wild-type HuH-7 (wt-HuH-7) to these varied apoptotic stimuli. Evaluation of different death signaling pathways revealed differential JNK-dependent signaling in Trf1 cells, suggesting a proapoptotic effect of CK2
".
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MATERIALS AND METHODS |
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Wt-HuH-7 and Trf1 cells were cultured in MEM containing 50 µg/ml gentamicin and 10% FBS (Gemini, Woodland, CA). Cell culture reagents were all obtained from Invitrogen/Life Technologies (Carlsbad, CA), plasticware was from Becton Dickinson (Franklin Lakes, NJ), and all other chemicals were from Sigma (St. Louis, MO), unless otherwise indicated. Trf1 cells were transfected with pBK-cytomegalovirus (CMV; Stratagene, La Jolla, CA) as described previously (48) or pcDNA3.1 (Invitrogen, Carlsbad, CA) containing the full-length CK2"-cDNA. Forty-eight hours posttransfection, cells were split into medium containing 600 µg/ml G418 and cultured until G418-resistant colonies became clearly visible. Several cell clones were expanded and tested for CK2
" expression.
Growth and quantification of DEN.
Virus was grown on C6/36 insect cells (American Type Culture Collection), which were cloned from the natural vector Aedes albopticus (21). These cells were cultured in DMEM plus 25 mM HEPES (Sigma), pH 7.55, 1 MEM nonessential amino acids, 10% heat-inactivated FBS, and 50 µg/ml gentamicin in sealed flasks at 28°C. The plaque assay for quantification of infectious virus particles was performed by infection of rhesus monkey kidney (LLCMK2) cells with serial dilutions of the inoculum to be tested. Cells were then overlayed with DMEM containing a mixture of high- and low-viscosity carboxymethylcellulose (Sigma). After 67 days LLCMK2 cells were stained with neutral red (0.35 mg/ml).
Determination and quantification of cell death and apoptosis.
Cell death of wt-HuH-7 and Trf1 cells after treatment with different apoptotic stimuli was routinely assessed by 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay as described previously (59). The cell death data derived with MTT were occasionally confirmed by Trypan blue exclusion and Neutral Red assays, measuring different aspects of cellular viability. Trypan blue and Neutral Red assay were performed according to standard protocols (2). For all experiments to examine cell death and apoptosis after DEN infection, wt-HuH-7 and Trf1 cells were infected at a multiplicity of infection of 50 for 1 h in serum-free medium. Regular medium (containing FBS) was then added to the usual culture volume without removing the viral inoculum, and half of the culture medium was changed 24 h postinfection.
Differentiation between necrosis and apoptosis was performed by fluorescent microscopy after costaining of the cells for 2 min with ethidium bromide (1 mg/ml) and acridine orange (2 mg/ml) as described previously (59). Necrosis was indicated by the presence of ethidium bromide staining. Further quantification of apoptotic cells was performed by the assessment of DNA fragmentation via the flow cytometrical measurement of hypoploid cells after staining the cellular DNA with propidium iodide (Sigma). Cells were detached from culture dishes with trypsin/EDTA, centrifuged at 300 g, and fixed for at least 2 h in 70% ethanol at 20°C. Cell membranes were permeabilized with 0.1 M citric acid in 0.2 M sodium phosphate and then incubated with 1 mg/ml propidium iodide and 1 mg/ml RNaseA (Sigma) for 2 h before flow cytometry.
Measurement of virus binding, internalization, degradation.
Assays for binding, internalization, and degradation of DEN were performed with radiolabeled virus as previously described (17). Binding experiments were performed at 4°C, to prevent viral internalization. Viral absorption by wt-HuH-7 or Trf1 cells was initiated by the addition of 1.5 x 105 pfu (2 x 104 counts/min) per 35-mm well in a final volume of 0.5 ml for 45 min with gentle agitation. Cells were washed twice, harvested by scraping in PBS, and lysed in scintillation fluid. Cell-associated radioactivity quantified in a
-scintillation counter (Beckman) was considered to reflect virus specifically bound to cells in addition to a nonspecific component. To quantify internalized virus, cells were shifted after the binding period from 4 to 37°C, thereby allowing internalization (48). At different time points after the temperature shift, noninternalized virus was removed from the cell surface by treatment with 5 mU/ml trypsin for 2 min at room temperature. After trypsin inactivation by washing cells twice with ice-cold binding medium (containing 1% BSA), cells were harvested and cell-associated radioactivity was measured. Kinetics of intracellular degradation of radiolabeled DEN were determined as acid-soluble radioactivity in the culture medium at different time points after initiation of internalization. Radiolabeled virus was bound to cells at 4°C, and unbound virus particles were removed by washing cells twice. Cells were shifted to 37°C, and multiple aliquots of the culture supernatant were precipitated with 20% trichloroacetic acid and 4% phosphotungstic acid (for 20 min at 4°C). After centrifugation, acid-soluble radioactivity was quantified in a
-scintillation counter.
Determination of cytochrome c release.
The determination of cytochrome c release from the mitochondria into the cytosol was done essentially as described previously (19). Briefly, HuH-7 and Trf1 cells were grown in 150-mm dishes and treated with DEN or TNF- for different periods of time. Cells were scraped into the cell culture medium, centrifuged at 300 g for 5 min, and washed once with 30 ml of ice-cold PBS. The cell pellet was resuspended in 1 ml of homogenization buffer (in mM: 50 HEPES, 10 KCl, 100 sucrose, 10 NaCl, 5 MgCl2, 1 PMSF, 1x protease inhibitor cocktail) and homogenized by 20 strokes of a tight-fitting pestle in a Dounce homogenizer. The resulting suspension was centrifuged twice 10 min at 750 g and 4°C to eliminate unbroken cells, large membrane fragments, and nuclei. The supernatant was recentrifuged 20 min at 10,000 g, and the resulting supernatant was collected as the cytoplasmic fraction. The cytoplasmic proteins (30 µg) were resolved on SDS-PAGE and, after transfer to nitrocellulose, were subjected to immunoblot with cytochrome c antibodies, as described below.
Protein isolation and immunoblot analysis.
Cell monolayers were washed once with ice-cold PBS and scraped into 0.61 ml of lysis buffer (in mM): 10 HEPES, 42 MgCl2, 1 EDTA, 1 DTT, 1 PMSF with 1% Triton, and 1x protein inhibitor cocktail (Sigma). The resulting suspension was incubated for 30 min at 4°C with constant agitation and then centrifuged at 1,500 g for 10 min. The supernatant was harvested, and the protein concentration was determined. Proteins (50 µg) resolved on SDS-PAGE were transferred to nitrocellulose membranes using a semidry blotting chamber (Bio-Rad, Hercules, CA). The membrane was blocked for 90 min at room temperature in TBST (30 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk and then incubated overnight at 4°C with primary antibodies diluted 1:1,000 in TBST, 2% milk against caspase 3, caspase 8, cytochrome c (BD Biosciences, Franklin Lakes, NJ), IB
, phospho-(Ser32)-I
B
(Cell Signaling, Beverly, MA), JNK MAPK (sc-474; Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:6,000 dilution of antibody against protein disulfide isomerase (52). Membranes were washed three times for 10 min and then incubated with horseradish peroxidase-labeled anti-mouse or anti-rabbit antibodies for 60 min at room temperature. Membranes were washed as above and bound antibodies were visualized by chemiluminescence (WestPico kit; Pierce, Rockford, Il), according to the manufacturer's guidelines.
Luciferase reporter gene assay.
For luciferase assays, wt-HuH-7 and Trf1 cells were grown in 35-mm culture dishes. At a density of 6070%, 2436 h after plating, cells were cotransfected with two different reporter plasmids (1 µg), using the lipofectamine plus reagent according to the manufacturer's instructions. The first plasmid contained either NF-B or activator protein-1 (AP-1) promoter sites regulating the transcription of firefly luciferase. The second plasmid contained a Renilla luciferase gene under control of a constitutive CMV promoter. At 2448 h after transfection, cells were infected with DEN or treated with TNF-
for different periods. Firefly and Renilla luciferase were assayed with the dual luciferase kit (Promega, Madison, WI), according to the manufacturer's instructions. The activity of firefly luciferase was normalized to the activity of Renilla luciferase, controlling for different transfection efficiencies.
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RESULTS |
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To initially examine the function of CK2" in hepatocellular apoptosis, cells were infected with DEN. Three cell lines were examined for their sensitivity to DEN-induced apoptosis: wt-HuH-7, the mutant Trf1 line, and Trf1 cells transfected with a CK2
" expression vector (Trf1-
"). It has been previously demonstrated (48) that the mechanism of the Trf1 trafficking-defect phenotype is a deficiency in CK2
" expression that can be reverted to the parental phenotype by transfection of the cells with recombinant CK2
". There was a significant difference in the cell death response after DEN infection among the three cell lines. Death occurred in wt-HuH-7 cells at 3648 h after DEN infection with a majority of cells dying between 48 and 72 h. By 90 h postinfection, 87 ± 6% of the cells had undergone cell death. In contrast, Trf1 cells were markedly resistant to death from DEN infection with only 10 ± 2% of the cells dead by 90 h (Fig. 1A). Consistent with their equivalent quantity of nuclear CK2
" as expressed by wt-HuH-7 cells (Fig. 1B), Trf1-
" cells transfected with recombinant CK2
" were sensitive to DEN-induced cell death (Fig. 1A). Several clones expressing CK2
" were examined with similar findings, indicating that reversion of the Trf1 phenotype was not due to clonal variation (data not shown).
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To determine whether DEN induced mainly apoptotic or necrotic cell death, cells were examined under fluorescent microscopy after being costained with ethidium bromide and acridine orange. Equivalent to the results of MTT assays, at 72 and 90 h postinfection, there were large numbers of apoptotic and necrotic wt-HuH-7 but not Trf1 cells (Fig. 2A). Quantification of the numbers of dying cells at 48 h revealed that of the dead cells in the wt-HuH-7 cell population, twice as many had undergone apoptosis as necrosis (16 ± 3 vs. 8 ± 1%, respectively). Over time, the number of dead cells increased steadily. By 90 h, the percentage of necrotic cells had risen to 56 ± 2%, whereas the number of apoptotic cells reached 43 ± 4% (Fig. 2B). As an additional measure of cell death, cells were examined for the presence of DNA fragmentation. DNA fragmentation was quantified by flow cytometric determination of hypoploid cells after cellular permeabilization and propidium iodide staining. At 72 h postinfection with DEN, 50% of wt-HuH-7 cells were hypoploid, and by 90 h, >70% were hypoploid (Fig. 2, C and D). In contrast, the level of hypoploidy in Trf1 cells was only 6% at 90 h, equivalent to the level in noninfected control cells. Expression of recombinant CK2
" in Trf1 cells converted the degree of hypoploidy to that seen in wt-HuH-7 cells (Fig. 2D).
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Consistent with the previously reported trafficking defects of membrane proteins in the Trf1 mutant, the possibility that resistance of Trf1 cells to DEN-induced cell death may have resulted from an inhibition of viral trafficking was explored. To address this possibility, binding and intracellular trafficking of DEN were examined in the two cell lines. Binding of [35S]DEN was equivalent in wt-HuH-7 and Trf1 cells (Fig. 3A). After binding, the kinetics of viral internalization, as measured by the increase of trypsin-protected virus over time, was also equivalent in the two lines (Fig. 3B). In both cell lines, an almost linear increase in trypsin-protected virus occurred within the first 4 h of infection, indicating a constant rate of internalization. In addition, intracellular viral degradation, representing viral trafficking to lysosomes was compared. Degraded viral proteins measurable 3060 min after the initiation of internalization increased linearly between 60 and 240 min. At 240 min, 60% of internalized virus was degraded in both Trf1 and wt-HuH-7 cells (Fig. 3C).
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Resistance of Trf 1 cells to other death stimuli.
To determine whether Trf1 cell resistance to DEN-induced apoptosis was specific for viral death pathways, the sensitivity of wild-typeand mutant cells to the death stimuli TNF-, hydrogen peroxide (H2O2), menadione (19), okadaic acid (22), acetaminophen (5), and UV irradiation (29) was determined. When cotreated with actinomycin D and TNF-
, 80% of wt-HuH-7 cells underwent cell death within 48 h in contrast to only 20% of the Trf1 cells (Fig. 4A). CK2
"-transfected Trf1 cells were sensitive to TNF-
-induced apoptosis to a similar extent as wild-type cells (Fig. 4A). Similar results were obtained with pcDNA3.1-CK2
" transfected Trf1 cells indicating that reversion of the resistant phenotype was not due to clonal variation. For the other death stimuli, reduction in cell death in Trf1 cells compared with wt-HuH-7 ranged from 43% for acetaminophen to 75% for menadione (Fig. 4B). The increased resistance of Trf1 cells to all five of these death stimuli suggests a common proapoptotic intercept for CK2
" in these pathways.
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Because the caspases are pivotal effector molecules in the signaling cascades leading to death from a variety of stimuli (23), we examined whether a differential activation of these molecules could provide an explanation for the differences in cell death between wt-HuH-7 and Trf1 cells. Both cell lines were treated with DEN or TNF- for different times before immunoblot analysis of procaspase 8 and three levels. TNF-
-treated wt-HuH-7 cells showed decreases in procaspase 8 and 3 after 24 h, whereas, in Trf1 cells, procaspase levels remained unchanged (Fig. 5A). In contrast, DEN-induced cell death occurred in the absence of activation of either procaspase (Fig. 5A), suggesting that this form of cell death was caspase independent. To further examine this possibility, wt-HuH-7 cells were pretreated with either the pancaspase inhibitor Z-VAD-FMK (30 µM) or the caspase 3 inhibitor DEVD-CHO (10 µM) before TNF-
treatment or DEN infection. TNF-
-induced apoptosis after 24 h was effectively inhibited by both Z-VAD-FMK and DEVD-CHO, whereas DEN-related cell death after 72 h was not affected by either compound (Fig. 5B).
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Wt-HuH-7 and Trf1 cells do not differ in their levels of NF-B activation.
The NF-B signaling pathway has been implicated in the regulation of cell death from both TNF-
(34, 40) and DEN (36). CK2 has also been reported to regulate the NF-
B pathway (43, 44). These prior findings suggested that the effects of CK2
" on hepatocellular susceptibility to death stimuli may have been mediated through effects on NF-
B. NF-
B activation was therefore examined in the two cell types after DEN infection and TNF-
treatment by measurements of the phosphorylation of the cytoplasmic NF-
B inhibitor I
B (Fig. 6) (see also Ref. 7). Inactivation of I
B after TNF-
treatment occurred from 5 min to 2 h as demonstrated by the appearance of Ser32-phosphorylated I
B (9) and its subsequent degradation (7). However, there was no significant difference in the timing or extent of phosphorylation between the two cell lines. DEN infection did not lead to any detectable activation of NF-
B in HuH-7 or Trf1 cells up to 48 h (early negative time points are not shown).
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Previous data suggest a significant proapoptotic role for the JNK/AP-1 signaling pathway in several forms of hepatocellular apoptosis (19, 34). These findings prompted an examination of whether differential activation of this pathway in wt-HuH-7 and Trf1 cells correlated with the resistance of Trf1 cells to DEN- and TNF--induced apoptosis. JNK activity as determined by an in vitro kinase assay was increased within 30 min in both cell lines after TNF-
treatment (Fig. 7A). Whereas the absolute level of JNK and c-Jun protein was comparable in the two cell lines as determined by immunoblotting of cell lysates, JNK activity increased to a significantly greater extent in wt-HuH-7 cells than in Trf1 cells. In addition, JNK activation was more prolonged in wt-HuH-7 cells, lasting longer than 6 h, whereas activation in Trf1 cells was limited to 24 h. DEN led to increased JNK activity at 2448 h postinfection in wt-HuH-7 cells, whereas no activation occurred in Trf1 cells.
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These findings suggested that the resistance to cell death in Trf1 might be linked to a failure to activate JNK. As a test of this hypothesis, the effect of the JNK inhibitor SP-600125 (4), on TNF--, DEN-, and UV-light-induced cell death of HuH-7 (Fig. 8A) and Trf1 cells transfected with CK2
" was determined (Fig. 8B). Consistent with a proapoptotic role for JNK (10), pretreatment of cells with SP-600125 reduced cell death by TNF-
DEN or UV-light between 60 and 80%. To test the significance of c-Jun and AP-1 in HuH-7 death signaling, HuH-7 cells were preinfected with a dominant negative c-Jun-expressing adenovirus (Ad5TAM). As we have previously reported for rat hepatocytes (33), Ad5TAM inhibited TNF-
-induced cell death in from 76 ± 6% in control infected wt-HuH-7 cells to 20 ± 4% in Ad5TAM-infected HuH-7 cells. The effect of Ad5TAM on DEN-induced apoptosis could not be determined due to toxicity from long-term adenoviral infection in wt-HuH-7 cells. Consistent with the observations of other investigators (24), a large proportion of adenovirus-infected HuH-7 cells underwent cell death between 36 and 72 h, the same period during which DEN-induced cell death occurred.
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DISCUSSION |
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In the absence of Fas (46) and active p53 (18) expression by HuH-7, the previously proposed relationship of CK2 to these proteins as inducers of apoptosis (14, 44) was not be examined in Trf1. The NF-
B pathway was considered as a possible mechanism for CK2
"-mediated proapoptotic signals in wt-HuH-7 and Trf1 cells in light of various reports suggesting that the CK2 holoenzyme interacts with this pathway. On stimulation, CK2 serves as an upstream kinase of the I
B-kinases
and
(IKK) (25) and cooperates with these kinases during the activation of I
B (20, 27). Other investigators found that CK2 directly phosphorylates the Ser32 and Ser36 residues of I
B independently of the IKKs (51). The p65-subunit of the active transcription factor has been found to be a target for CK2 resulting in an increased transactivation potential of NF-
B (55). In addition to the known interaction of NF-
B and CK2, NF-
B has been demonstrated to be a proapoptotic signal in DEN-induced cell death (36). However, in our study, DEN did not cause NF-
B activation. Whereas TNF-
induced NF-
B activation, the absence of a significant difference in I
B phosphorylation and subsequent degradation (Fig. 6) and NF-
B-dependent reporter activity suggests that the resistance of Trf1 cells to TNF-induced cell death is not related to a NF-
B pathway.
Prior studies (58) indicated that a balance between the MAP kinases, growth factor-activated ERK and stress-activated JNK, governs whether a cell undergoes apoptosis during neuronal development. The proapoptotic nature of JNK was demonstrated by disruption of the gene encoding the brain-specific jnk3 in mice, thereby preventing stress-induced hippocampal neuron apoptosis (60). Recent data suggest a similar proapoptotic role for JNK in the regulation of hepatocellular cell death (33). The most important target of JNK in this context is the bZIP (basic region leucine zipper)-domain containing protein c-Jun. NH2-terminal phosphorylation of c-Jun by JNK results in dimerization of c-Jun and activation of AP-1. c-Jun/AP-1 promotes pro- or antiapoptotic responses depending on the stimulus and cell type (28). In rat hepatocytes, we have previously demonstrated that c-Jun mediates a proapoptotic function of JNK in TNF--related death signaling (33). Consistent with a proapoptotic role for JNK in TNF-
-induced apoptosis of liver cells (10, 30), inhibition of this kinase by the specific inhibitor SP-600125 prevented TNF-
, DEN infection and UV-induced cell death in wt-HuH-7 and Trf1 cells transfected with CK2
" (Fig. 8), indicating that JNK signaling plays a destructive role in each case. In addition, the extent of c-Jun phosphorylation (9) and AP-1 dependent reporter activity of TNF-
-treated and DEN-infected wt-HuH-7 cells was more than threefold greater than the activity in Trf1 cells. Even with the constraint of a somewhat limited SP-600125 specificity (3), these data suggested a potential involvement of JNK/c-Jun in the resistance of Trf1 cells to death stimuli.
The significance of c-Jun and AP-1 in HuH-7 death signaling was confirmed by preinfection of the cells with a dominant negative c-Jun-expressing adenovirus (Ad5TAM). As we have previously reported for hepatocytes (33, 34), Ad5TAM significantly inhibited TNF--induced apoptosis in wt-HuH-7 cells. Unfortunately, the effect of Ad5TAM on DEN-induced apoptosis could not be determined due to toxicity from long-term adenoviral infection in wt-HuH-7 cells. Whereas both anti- and proapoptotic functions have been suggested for CK2
(31), the data presented provide evidence that regulation of the JNK/c-Jun/AP-1 pathway by CK2
" has a significant function in the execution of TNF-
induced cell death in wt-HuH-7 cells. However, the exact site(s) of interaction of CK2
" within this pathway remains currently unclear.
Recently, a proapoptotic function for CK2 via JNK activation in embryonic hippocampal progenitor cells has been demonstrated (39). In addition, a role for CK2 in regulating the binding of nuclear proteins to the AP-1 site by direct phosphorylation of AP-1 transcription factors has been suggested (44). Other investigators have found an indirect influence of CK2 on AP-1 binding through phosphorylation of the nuclear DNA repair protein APE/REF-1 (13). Similar to many other matrix-bound enzymes (1, 37, 53), association of CK2" with the nuclear matrix (48) and the potential for an interaction with AP-1 indicate a possible connection of CK2
" with transcriptional regulation.
Recent studies (8) suggest the possibility that JNK directly regulates cell death by mechanisms other than through the phosphorylation of c-Jun and activation of AP-1-dependent transcription. Inhibition of cell death by the JNK inhibitor SP-600125 in the absence of significant AP-1-activation in DEN-infected wt-HuH-7 cells is consistent the concept of an alternative JNK-dependent pathway. Whereas there is a substantial cohort of data that supports a role for BH3-only proteins in JNK-dependent apoptosis (56), unlike AP-1 dependent cell death, there has been no proposed connection to CK2 activity. Nevertheless, this study opens the possibility that CK2 might affect intracellular trafficking necessary for BH3-only protein-induced apoptosis.
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GRANTS |
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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