From the Lung Biology Center, San Francisco General Hospital, University of California, San Francisco, California 94143-0854
Received for publication, March 3, 2003 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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Death receptor ligands and DNA damage are considered to activate two separate pathways of apoptosis, each potentially leading to caspase activation and disassembly of the cell (7, 8). Death receptor ligands, such as Fas ligand and TRAIL, trimerize their receptors and thereby recruit and activate an initiator caspase, caspase 8. DNA damaging agents engage apoptosis in a parallel fashion by altering mitochondrial function, thereby releasing pro-apoptotic molecules such as cytochrome c from the intermembranous space, resulting in part in an activation of another initiator caspase, caspase 9. These initiator caspases can then activate downstream caspases such as caspase 3 leading to a cleavage of key cellular components as the terminal and irreversible phase of apoptotic death. Simultaneous activation of the two alternate pathways, also referred to as the extrinsic (death receptor) and intrinsic (damage) pathways, may induce common signaling pathways that mediate cross-talk and amplification of the resultant apoptosis.
One potential apoptotic signaling pathway known to be engaged by both death receptors and by DNA damage is the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway (9). Although the pathway is strongly activated by a variety of apoptotic stimuli, the role of JNK in apoptosis is unclear, because it has been shown to be pro-apoptotic, neutral, or anti-apoptotic in different settings (9, 10). In many circumstances, single agents, such as chemotherapeutics alone or death receptor ligands alone, induce JNK signaling but do not depend on JNK signals for apoptotic responses. For example, a lack of dependence on JNK signals has been shown for apoptosis because of chemotherapeutic agents such as etoposide (11) or doxorubicin (12) or to death receptor activation via TRAIL (13, 14). However, when different stimuli given simultaneously produce amplified responses, the amplification of these two pathways may involve cross-talk utilizing such stress signals. Indeed, in the case of proliferative stimuli, JNK signaling may be involved in synergistic responses (15). In the case of apoptotic stimuli, induction of JNK signaling has been proposed to enhance apoptotic responses to tumor necrosis factor (16) and to Fas (17, 18). JNK signals could thus be important for synergistic apoptotic responses and, if so, could potentially be manipulated to potentiate cancer therapies.
We have previously described synergistic apoptotic responses in
chemoresistant mesothelioma cell lines to the combination of TRAIL and DNA
damaging agents (e.g. chemotherapy or irradiation) that were
not due to up-regulation of TRAIL receptors, DR4 and DR5
(19). In part because
mesothelioma is reported to express wild-type p53
(20), we investigated whether
p53 was involved in either synergy or in a p53-dependent JNK activation. We
found that these cells had nonfunctional p53. Therefore, we asked whether JNK
signals played a p53-independent role in the synergistic apoptotic responses.
We describe a significant role for JNK signals in the synergistic apoptosis
induced by TRAIL and the topoisomerase inhibitor, etoposide.
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EXPERIMENTAL PROCEDURES |
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Plasmids containing dominant-negative or wild-type constructs (empty-pcDNA3, pcDNA3-caspase 9 dominant-negative, pcDNA3-JNK1 wild-type, pcDNA3-JNK1 dominant-negative, pcDNA3-JNK2 wild-type, and pcDNA3-JNK2 dominant-negative) were gifts from Dr. Roger Davis (University of Massachusetts, Boston, MA) (21).
Antibodies against total SAPK/JNK and phosphorylated mitogenactivated protein kinase signaling proteins (phospho-SAPK/JNK (Thr183/Tyr185) and phospho-c-Jun (Ser63)II) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-FLAG antibody (M2) was purchased from Sigma. Antibodies against p53 (DO-1) and p21 (F-5) and secondary antibodies were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA).
Cell Lines and CultureThe human mesothelioma line, M28, was obtained from Dr. Brenda Gerwin (NCI, National Institutes of Health, Bethesda, MD), and the human mesothelioma line REN was obtained from Dr. Steven Albelda (University of Pennsylvania, Philadelphia, PA). Primary human mesothelial cells were obtained from benign pleural effusions as described (22) under approval of the Committee on Human Research and used as controls.
Tumor cell lines and primary cells were cultured in Dulbecco's modified Eagle's medium/RPMI 1640 (1:1), 10% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT), L-glutamine (2 mM; Invitrogen), penicillin (100 units/ml; Invitrogen), and streptomycin (100 µg/ml; Invitrogen).
General Experimental ConditionsTRAIL and etoposide were used at concentrations found to induce a synergistic apoptosis (420 ng/ml TRAIL and 315 µg/ml etoposide). For apoptosis studies, the cells were plated at 50,000 cells/well of 12-well plates the day before and exposed to treatments for described times. Ultraviolet irradiation was used as a positive control for JNK signaling; irradiation by UV-C was performed in a Stratalinker UV cross-linker model 1800 (Stratagene, La Jolla, CA) at a dose of 40120 J/m2.
Western Blotting AnalysisProtein analysis was performed on whole cell lysates prepared on ice from 15-cm plates of M28 cells at 70% confluence after the appropriate treatment. The lysis buffer consisted of 20 mM Tris-HCl, pH 8, 137 mM NaCl, 50 mM sodium fluoride, 10% glycerol, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, pH 8, and protease inhibitor mixture (Calbiochem, San Diego, CA). The protein concentrations were determined using the Bio-Rad protein assay.
The samples (40 µg/lane) were boiled for 5 min with sample buffer (0.2 M Tris, pH 6.8, 5% SDS, 3% glycerol, and 0.01% bromphenol blue), separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). The membranes were blocked in 5% nonfat milk (in PBS plus 0.05% Tween 20) for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody in 5% bovine serum albumin (in PBS plus 0.05% Tween 20). The membranes were washed six times for 5 min each in PBS and incubated with the secondary antibody for 1 h at room temperature in 5% nonfat milk (in PBS plus 0.05% Tween 20). After washing, the blots were developed using ECL reagent (Amersham Biosciences) and autoradiography film (Kodak). The molecular weights were determined by comparison with Bio-Rad Kaleidoscope markers.
Annexin V Assay for ApoptosisApoptosis was detected by the binding of fluorescent protein annexin V-Cy3 or annexin V-fluorescein isothiocyanate to the phosphatidylserine residues on the outer leaflet of the apoptotic membrane, as previously described (19). Because fewer than 5% of cells in any condition stained with propidium iodide, necrosis was considered minimal, and all annexin-V positive cells were considered apoptotic.
The cells were trypsinized and stained according to the annexin V-Cy3 or annexin V-fluorescein isothiocyanate kit (Medical & Biological Laboratories, Naka-ku Nagoya, Japan). The cells were analyzed using a FAC-Sort flow cytometer (Becton Dickinson, Franklin Lakes, NJ), with acquisition of a total of 10,000 events/sample to ensure adequate mean data. Data analysis was performed with the CellQuest software, version 3-1 f (Becton Dickinson).
Cell TransfectionDominant-negative caspase 9, JNK1 and JNK2
plasmids, wild-type JNK1 and JNK2 plasmids, and empty pcDNA3 plasmid were
transiently transfected in M28 cells along with a pCMV- galactosidase
vector. The cells were plated in 6-well plates at 0.25 x 106
cells/well. The next day, a total of 1.5 µg of DNA was used at a 5:1 ratio
(pcDNA:pCMV-
-galactosidase). The cells were transfected using
LipofectAMINE and LipofectAMINE PLUS reagents following the manufacturer's
instructions. At 24 h after transfection, the cells were exposed to TRAIL (4
ng/ml) plus etoposide (3 µg/ml) for 6 h. The cells were washed and fixed in
0.05% glutaraldehyde for 1 min. To identify
-galactosidase enzyme
activity, the fixed cells were washed three times with PBS and stained in
buffer containing X-gal (1 mg/ml X-gal in 10 mM
Na3PO4, pH 7, 5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6·3H2O, 2
mM MgCl2, 0.02% Nonidet P-40, and 0.01% SDS) overnight
at 37 °C. Blue cells were counted as a ratio of total cells (blue/total)
as a measure of survival in a blinded fashion and compared among the different
experimental conditions. For each plasmid, the relative survival of
transfected cells was calculated as the survival (blue/total cells) of cells
exposed to TRAIL plus etoposide divided by the survival (blue/total cells) of
cells not exposed.
StatisticsOne-way analysis of variance with Tukey's posthoc test was performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA). Synergy was assessed by isobolographic analysis (23) and by showing that the apoptosis to the combination of agents was significantly greater than additive (e.g. apoptosis to TRAIL plus etoposide was significantly greater than the sum of apoptosis to TRAIL plus apoptosis to etoposide). A p < 0.05 was considered significant.
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RESULTS |
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p53 Is Inactive in M28 and REN Mesothelioma CellsTo determine whether p53 could mediate this synergy, we studied the functional role of p53 in these cells. We exposed M28, REN, and primary mesothelial cells, as a positive control, to a range of UV irradiation shown to induce p53 in many cell lines (24) and harvested the cells 16 h after UV exposure for determining expression of p53. As expected, p53 protein increased in primary human mesothelial cells, but no p53 protein was detected in M28 and REN cells (Fig. 2). As confirmation for nonfunctional p53, we demonstrated p21 up-regulation in response to UV irradiation in primary cells but not in M28 and REN cells (data not shown).
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JNK Is Activated in Mesothelioma Cells by TRAIL, Etoposide, and the CombinationTo confirm that JNK can be activated in M28 and REN cell lines, we exposed the cells to UV light (40 J/m2) as a well characterized stimulus for JNK signaling and harvested them 30 min later to measure the phosphorylation of JNK and total JNK expression. We found that JNK was phosphorylated without a change in total JNK protein expression (Fig. 3A).
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We then studied the effect of TRAIL, etoposide, or both. When given alone, TRAIL (20 ng/ml) and etoposide (15 µg/ml) each induced JNK phosphorylation; when given together, these agents induced enhanced JNK phosphorylation (Fig. 3B).
JNK Activation Precedes Apoptosis and Is Not Secondary to Caspase ActivationIn some settings, JNK signaling by TRAIL or by etoposide may be secondary to the apoptotic process itself (11, 13). To address whether activation of JNK by TRAIL plus etoposide in our studies was secondary to apoptosis, we inhibited apoptosis using the pan-caspase inhibitor zVAD-fmk. M28 cells were exposed to the combination of TRAIL and etoposide for different times (216 h) with or without pretreatment with zVAD-fmk (100 µM), which inhibited apoptosis. JNK phosphorylation was not inhibited by zVAD-fmk and, in fact, may have been greater after caspase inhibition, as has been described previously (Fig. 3C) (13). Thus, the JNK signals associated with synergistic apoptosis are proximal to the apoptosis itself.
Inhibition of JNK1, JNK2, and Caspase 9 by Dominant-negative Constructs Increases Survival of M28 Cells after Exposure to TRAIL plus EtoposideTo know whether JNK contributes to the cooperative effect of TRAIL plus chemotherapy in mesothelioma cells, we inhibited JNK by transiently transfected dominant-negative JNK1 and JNK2 plasmids and the combination of the two plasmids. We also transfected a caspase 9 dominant-negative plasmid as a control for blockade of the post-mitochondrial apoptotic pathway. Because of toxicity to REN cells, we were able only to transfect M28 successfully.
First, to confirm successful transfection, we investigated protein expression of FLAG, the peptide tag used for the pcDNA dominant-negative constructs. As a negative control, we used an empty pcDNA plasmid without a FLAG tag. FLAG expression was detected for caspase 9, JNK1, and JNK2 dominant-negative transfected cells at the expected sizes of the tagged construct; no FLAG expression was detected in the cells transfected with the empty plasmid (Fig. 4A).
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Second, to confirm an inhibitory effect of dominant-negative JNK on phosphorylation of its substrate c-Jun, the cells were transiently transfected with several plasmids: pcDNA empty plasmid, dominant-negative caspase 9, JNK1 and JNK2 constructs, and, as controls, JNK1 and JNK2 wild-type constructs and exposed to UV light. At 24 h after transfection, the cells were exposed to UV light (40 J/m2) and harvested 30 min after exposure. A decrease of phospho-c-Jun was shown in the cells transfected with JNK1 and JNK2 dominant-negative constructs compared with those transfected with empty pcDNA, caspase 9 dominant-negative, and JNK1 and JNK2 wild-type constructs (Fig. 4B).
Knowing that the dominant-negative constructs were expressed and inhibited
c-Jun phosphorylation, we investigated the effect of the inhibition of JNK on
the survival of M28 cells exposed to TRAIL plus etoposide. The cells were
co-transfected with various plasmids and pCMV--galactosidase at a 5:1
ratio and, after 24 h, exposed to TRAIL plus etoposide for 6 h, fixed, and
stained for
-galactosidase activity. The blue cells were counted as a
ratio of total cells and compared among the different experimental conditions.
In cells exposed to TRAIL plus etoposide, inhibition of JNK led to a
significant increase of 23-fold in relative survival of the
dominant-negative transfected cells compared with the empty plasmid
transfected cells (Fig.
4C).
We confirmed this increase in survival in further experiments using JNK1 and JNK2 wild-type constructs as additional controls for transfection. Compared with transfection with wild-type plasmids, the dominant-negative plasmids increased survival by at least 2-fold in cells exposed to TRAIL plus etoposide (Fig. 4D).
Inhibition of the JNK Pathway by Chemical Inhibitor SP600125 Decreases Apoptosis of M28 and REN Cells after Exposure to TRAIL plus EtoposideTo confirm our results using transient transfection and to extend the study to REN cells, we used the JNK inhibitor, SP600125, to investigate its effect on the apoptotic synergistic response of TRAIL plus etoposide in M28 and REN cells (25, 26). First, to confirm the effect of the inhibitor, we treated M28 cells with or without SP600125 (20 µM) for 1 h before exposing the cells to UV (40 J/m2) and 30 min later harvested the cells for detection of phospho-c-Jun. We found that UV induction of phospho-c-Jun was inhibited by SP600125. There was no effect on total JNK protein expression (Fig. 5A).
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Then M28 and REN cells were pretreated with SP600125 (10, 20, or 50 µM) or Me2SO for 1 h and exposed to TRAIL or TRAIL plus etoposide for 16 h. We observed a significant decrease in the apoptosis induced by TRAIL plus etoposide in cells treated with SP600125 (Fig. 5, B for M28 and C for REN cells). These results confirmed a role of JNK signaling in the synergistic response of TRAIL plus etoposide. There was no effect of JNK inhibition on the response to TRAIL alone.
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DISCUSSION |
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Our study is unusual in that it addresses the role of JNK specifically in the setting of synergistic apoptosis. In most studies, the activation of JNK and its role in the apoptotic response of the cell has been determined in response to single stimuli. It is known, for example, that the JNK pathway is activated by TRAIL and other death ligands as well as by etoposide and other chemotherapeutic agents; in most cases, JNK signals in response to the individual stimuli are independent of the resultant apoptosis (11, 13, 14). In this study, we have confirmed that TRAIL alone or etoposide alone induces JNK phosphorylation; however, it is the combination of stimuli that induces apoptosis that can be inhibited by specific inhibition of JNK. In effect, one apoptotic stimulus appears to prime the cell to respond to the other stimulus. As such, it raises the possibility that activating JNK signals could be a means of sensitizing cells to apoptosis without the need for more toxic treatments, such as those that cause DNA damage.
The reported role of JNK in cell death continues to be complex and, at times, contradictory. The JNK pathway has been shown both to mediate survival or death in response to stress, perhaps depending on the particular cell type or on the presence of other mitogenic or environmental signals (9). Particularly in transformed or tumor cell lines, JNK signaling may function as a pro-survival pathway (2731). In other cases, JNK signaling has been unrelated to cell fate (11, 13), possibly because JNK signals were induced by the apoptotic process itself. Finally, there is strong evidence that JNK can function as a pro-apoptotic signal (3236). In these studies, blockade of JNK signals by antisense, dominant-negative constructs or genetic deletion has shown a role for JNK in apoptosis, whether in response to ultraviolet radiation, anticarcinogenic isothiocyanates, oxidative stress, or ceramide.
Because mesothelioma is a tumor highly resistant to therapy, strategies
that would enhance its response to chemotherapy could provide a useful
therapeutic adjunct (37).
Mesothelioma most likely has abnormal function of the p53 pathway,
e.g. because of mutation or loss of p14ARF
(38) or to interaction with
simian virus 40 large T antigen
(39), even though the tumor
often expresses wild-type p53
(20). Thus, as for most
tumors, therapeutic approaches successful in p53 inactive cells would be
desirable. In our p53 inactive mesothelial cells, which consistently fail to
respond to single agents, the use of combination therapy with TRAIL and with
DNA damaging agents (chemotherapy, irradiation, or ultraviolet light)
has shown promise (19). To
investigate a possible role for JNK in the synergy, we confirmed that JNK was
phosphorylated by TRAIL and by etoposide separately, as described by others,
but also showed that JNK phosphorylation was enhanced by the combination.
Inhibition of caspase activity had no effect on JNK activation, demonstrating
that JNK was activated proximal to apoptosis and was not caspase-dependent.
Blocking the JNK pathway was then essential for determining the contribution,
if any, of JNK signaling to the apoptosis.
We inhibited the JNK pathway in two main ways, by dominant-negative constructs and by a specific JNK inhibitor, SP600125 (26). In the first approach, the dominant-negative blockade of JNK had limitations: the act of transfection induced some sensitization to the cells leading to an increased apoptotic response to TRAIL alone and was not possible in the REN cells because of toxicity. Nonetheless, the dominant-negative approach showed that the inhibition of JNK1 and JNK2 significantly increased survival of transfected cells exposed to TRAIL plus etoposide. Inhibition of both JNK1 and JNK2 had no more effect than of each alone, suggesting that a maximal effect had been achieved. In addition, a maximal effect was also suggested by the similar effect with dominant-negative JNK as with a dominant-negative construct to caspase 9, the initiator caspase activated by the mitochondrial apoptotic pathway. In our second approach, the use of SP600125 allowed us to avoid the confounding effect of toxicity so that the synergy of TRAIL plus etoposide could be studied in both cell lines. The contributory role of JNK in the response to TRAIL plus etoposide, but not to TRAIL alone, was confirmed using SP600125. Interestingly, the effect of JNK inhibition was greater for M28 cells than for REN cells, perhaps because M28 cells showed a greater degree of synergy than did REN cells. At the highest dose of SP600125 (50 µM), the apoptosis induced by TRAIL increased, suggesting some toxicity; nonetheless, with JNK inhibition, the degree of apoptosis caused by TRAIL plus etoposide was reduced to the level induced by TRAIL alone. We conclude that JNK mediates the stress signals that enable a synergistic apoptotic response.
JNK is thought to mediate apoptosis via either transcriptional or nontranscriptional means. By phosphorylation of its target c-Jun, JNK may initiate transcription of apoptotic genes, a function shown to be important in certain stress- or ceramideinduced apoptosis (40, 41) and that may include expression of a pro-apoptotic Bcl-2 homology molecule, Bim(EL) (42). JNK may also function in nontranscriptional fashion by directly phosphorylating and possibly inhibiting anti-apoptotic proteins, such as Bcl-2 (43) or Bcl-xL (44). In addition, JNK may interact with p53 pathways by phosphorylating p53 and enhancing its stability and function in response to stress (45); nonetheless, JNK has been shown to induce apoptosis in cells without p53 and thus can serve to mediate apoptotic signals independent of the p53 pathway (46, 47).
The mechanism by which JNK signals mediate apoptotic synergy is not yet
clear. Such synergy could possibly result from enhanced JNK signaling, because
of either an increased activity or a prolonged duration of activity, which by
itself was sufficient to induce apoptosis. For example, enhanced activity of
JNK may result by combining signals that separately activate the JNK kinase
MKK7, such as tumor necrosis factor, and MKK4, such as stress or DNA damage
(48,
49). MKK7 and MKK4
phosphorylate JNK preferentially on threonine and tyrosine, respectively, and
both are required for optimal JNK activity
(50). Although Fas ligand has
been shown to activate MKK7
(51), the ability of TRAIL to
activate MKK7 has not been reported
(14). If indeed, TRAIL
activated MKK7 and etoposide activated MKK4, the combination would activate
JNK better than each alone and perhaps lead to apoptotic synergy. In addition,
an increased duration of JNK signaling may be important for JNK-induced
apoptosis (32); such an
increased duration can result if one stimulus blocks an inhibition, such as by
NF-B, to the alternate stimulus
(52). Apoptotic synergy could
further be explained by actions distal to JNK as, for example, if JNK signals
sensitized the mitochondria to respond to TRAIL-induced signals via caspase
8-mediated cleavage of the Bcl-2 homology molecule, BID. In primary
fibroblasts with targeted gene disruption of JNK1 and JNK2, JNK-induced
apoptosis has been shown to depend on its actions at the mitochondria
(36) and to require the
presence of the pro-apoptotic members of the Bcl-2 family, Bax and Bak
(53). We have previously shown
that mesothelial cell lines, including M28 and REN, highly express Bax and
postulated that this would allow the cells to respond to appropriate apoptotic
signals directed to the mitochondria
(37). In that case, perhaps,
enhanced JNK signals caused by TRAIL plus etoposide would be sufficient to
sensitize the mitochondria to respond to TRAIL-induced BID signals, mediating
synergy. Understanding the role of JNK in apoptotic synergy will be useful in
understanding how JNK functions in apoptosis generally and how its function
could be manipulated to amplify apoptosis of tumor cells.
Activation of two distinct apoptotic pathways can overcome resistance to each separately in a way that involves signaling via the stress-activated pathway, JNK/SAPK. Understanding the role of JNK signals may provide insight into the cellular response to therapy, especially in a system without p53. Such understanding may lead to ways to manipulate signals to enhance responses to chemotherapy or even to replace chemotherapy with equally effective and less toxic downstream signals.
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FOOTNOTES |
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To whom correspondence should be addressed: Lung Biology Center, Box 0854,
University of California San Francisco, San Francisco, CA 94143-0854. E-mail:
sfcourt{at}itsa.ucsf.edu.
1 The abbreviations used are: TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand; JNK, c-Jun N-terminal kinase; SAPK,
stress-activated protein kinase; MKK, mitogen-activated protein kinase kinase;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; PBS,
phosphate-buffered saline.
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ACKNOWLEDGMENTS |
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REFERENCES |
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