Correspondence to: Beat W. Schäfer, Division of Clinical Chemistry & Biochemistry, Dept. of Pediatrics, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Tel:41-1-266 75 53 Fax:41-1-266 71 69
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Abstract |
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DRAL is a four and a half LIM domain protein identified because of its differential expression between normal human myoblasts and the malignant counterparts, rhabdomyosarcoma cells. In the current study, we demonstrate that transcription of the DRAL gene can be stimulated by p53, since transient expression of functional p53 in rhabdomyosarcoma cells as well as stimulation of endogenous p53 by ionizing radiation in wild-type cells enhances DRAL mRNA levels. In support of these observations, five potential p53 target sites could be identified in the promoter region of the human DRAL gene. To obtain insight into the possible functions of DRAL, ectopic expression experiments were performed. Interestingly, DRAL expression efficiently triggered apoptosis in three cell lines of different origin to the extent that no cells could be generated that stably overexpressed this protein. However, transient transfection experiments as well as immunofluorescence staining of the endogenous protein allowed for the localization of DRAL in different cellular compartments, namely cytoplasm, nucleus, focal contacts, as well as Z-discs and to a lesser extent the M-bands in cardiac myofibrils. These data suggest that downregulation of DRAL might be involved in tumor development. Furthermore, DRAL expression might be important for heart function.
Key Words: LIM domain protein, transcriptional regulation, p53, apoptosis, subcellular localization
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Introduction |
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Development of cancer results from multiple genetic events occurring in a single cell and culminating in the inactivation and/or malfunction of several key proteins. Among these, the tumor suppressor gene p53 is the most frequently mutated gene in human cancer (
Our previous studies focused on the identification of potential new tumor suppressor genes from a pediatric sarcoma model. For this purpose we performed a subtractive cDNA library screening between normal cultured myoblasts and the human embryonal rhabdomyosarcoma (RMS)1 cell line RD (
The LIM domain is a cysteine-rich domain of 50 amino acids, which folds into two specialized zinc fingers and is involved primarily in proteinprotein interactions (for reviews see
Here, DRAL was identified as a p53-responsive gene. Given the potential unique role of DRAL in tumor biology and to obtain insight into possible functions of this protein, we investigated the effects of ectopic DRAL expression and determined its intracellular localization in a range of cell types.
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Materials and Methods |
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Cell Lines
All cell lines were grown in DME supplemented with 10% FBS (Life Technologies), except for primary myoblasts, which were cultured in F12 medium with 15% FBS; both media contained 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). The nonmuscular cells were maintained in 5% CO2, muscle cells in 10% CO2 at 37°C. The human embryonal RMS cell line RD, NIH 3T3 mouse fibroblasts, and COS-1 African green monkey kidney cells were obtained from American Type Culture Collection. RD-tsp53 (RD cells expressing a temperature-sensitive p53 mutant, amino acid 135 Ala to Val) and RD-Neo cells (vector alone) were generated as described (
Northern Blot Analysis
Total RNA was extracted from different cells by guanidinium-isothiocyanate lysis followed by centrifugation through a 5.7 M caesium chloride cushion. It was then separated on a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred to Nytran nylon membranes (Schleicher & Schuell, Inc.) by capillary transfer. Alternatively, a commercially available human RNA Master BlotTM was used (CLONTECH Laboratories, Inc.). Equal loading of the blots was confirmed by hybridization with ß-actin or ubiquitin, respectively. Probes (inserts of the clones A33-35; nucleotides 64451 of the DRAL cDNA), a 505-bp EcoRIKpnI DRAL fragment for hybridization of the mouse Northern blot, A33-89, A33-124, human EST clone 470149, and mouse EST clone 533961 (p21WAF1) were generated by random priming (Prime-a-gene; Promega) with [32P]dATP (NEN Life Science Products) and used for hybridization at 68°C with QuickHyb Hybridization Solution (Stratagene) according to the manufacturer's instructions. The membranes were exposed to x-ray films (Eastman Kodak Co.) with intensifying screens at -70°C.
Ionizing Radiation (IR) Treatment
Primary human myoblasts, RD cells, and wild-type and mutant p53 expressing mouse fibroblasts were plated on 10 cm dishes 24 h before treatment, for each time point in duplicate. After exposure to 20 Gy of IR (from a 137Cs source) cells were fed with fresh medium and cultured until harvesting.
Cloning of the Human DRAL Promoter
A human P1 library was screened by PCR (Genome Systems, Inc.) using the DRAL-specific primers DRAL-FOR2 5'-ACCCGCAAGATGGAGTA-3' and DRAL-REV3 5'-GCAGGGCACACAGAAATTCTG-3' under the following cycling conditions: 15 s at 94°C, 30 s at 56°C, and 60 s at 72°C for 30 cycles. Southern blot analysis was carried out either using nucleotide (nt) 64 to nt 150 of the DRAL cDNA or a 2.5-kb PstI subfragment of the P1 clone as probes with QuickHyb solution according to the manufacturer's procedures (Stratagene). Probes were random primed as described above. Subcloning into pUC18 and sequencing was done according to standard procedures. Putative transcription factor binding sites in the promoter region were identified by computer programs (WebSignalScanProgram 4.05 [TFD database] and MatInspector 2.2).
Plasmid Constructions
Two constructs tagging the COOH- and NH2-terminal end of the DRAL coding region with the FLAG epitope DYKDDDDK (DRAL-CF and DRAL-NF, respectively) were constructed by PCR amplification from full-length human cDNA using the ExpandTM High Fidelity PCR System (Roche) and the following primers, which were designed to encode a BamHI (5' end) or Xba I (3' end) restriction site and the FLAG epitope: for DRAL-CF (COOH-terminally FLAG-tagged DRAL): 5'-CGGGATCCGCCACCATGACTGAGCGCTTTGACTGC-3' and 5'-GCTCTAGATCACTTGTCATCGTCGTCCTTGTAGTCGATGTCTTTCC- CACAGTC-3'; and for DRAL-NF (NH2-terminally FLAG-tagged DRAL): 5'-CGGGATCCGCCACCATGGACTACAAGGACGACGATGACAAGACTGAGCGCTTTGACTGC-3' and 5'-GCTCTAGATCAGATGTCTTTCCCACA-3'. Amplified fragments were digested and ligated into pcDNA3 (Invitrogen). All constructs were verified by sequencing before use.
Transfection and Immunofluorescence
RD, NIH 3T3, and COS-1 cells were plated 1 d before transfection on 35-mm dishes (Falcon). For transfections 5 µg of DNA (DRAL, DRALCF, DRALNF, or pFLAGCMV-2BAP as control plasmid; Eastman Kodak Co.) and 5 µl lipofectamine (Life Technologies) for each dish were added to the cells in unsupplemented DME. Medium was changed 68 h after transfection. Cells were fixed with 3.7% formaldehyde, permeabilized with methanol, and then stained with the anti-FLAG primary monoclonal antibody M2 (Sigma-Aldrich; diluted 1:300), followed by a Cy3-conjugated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories; diluted 1:200). DNA was visualized by Hoechst staining. Cells were viewed through a fluorescence microscope (Carl Zeiss, Inc.) and transfected cells were quantified by photographing three representative regions of each plate. Every picture showed 280 to 500 cells, the exact cell number was obtained by counting the Hoechst-stained nuclei. DRAL-positive cells were counted as apoptotic when they were round and displayed condensed nuclei. Three independent experiments were carried out.
Neonatal rat cardiomyocytes were transfected as described (-actinin and myomesin were visualized using monoclonal antibodies (clone EA-53 [Sigma-Aldrich] and B4 [
AnnexinV staining was carried out as described previously (
Caspase Activity
Cells were resuspended in lysis buffer (50 mM Pipes, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM PMSF, 1 mM DTT, 1 µg/ml chymostatin, leupeptin, aprotinin, and pepstatin each) and lysed by at least two freezethaw cycles. 150 µg of lysate was incubated with either caspase-3 inhibitor (DEVD-CHO; Bachem) and substrate (DEVD-pNA; Bachem) or substrate alone at 80 µM in 0.1 ml. Substrate cleavage was monitored at 405 nm and background activity subtracted for data presentation.
Confocal Microscopy
Cell lines were cultured in 35-mm plastic dishes (Falcon) or on fibronectin-coated glass coverslips in 24-well plates. Immunofluorescence labeling was done as described above, except that DNA was labeled using Pico Green (Molecular Probes, Inc.; diluted 1:200). Stainings were visualized with a confocal laser scanning microscope consisting of a Leica inverted microscope (DM IRB/E) equipped with an argonkrypton mixed gas laser. Image processing was done on a Silicon Graphics workstation using the software, Imaris (Bitplane AG;
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Results |
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Identification of DRAL as a p53-inducible Gene
Previously, we applied a subtractive cloning procedure to identify molecular changes occurring during progression from normal myoblasts to RMS (
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To confirm and characterize these initial findings in more detail, gene expression was analyzed in a time-dependent manner by extracting RNA at eight different time points after temperature shift (from 2 to 48 h). Northern blot analysis revealed an increase of DRAL mRNA levels specifically in the RD-tsp53 cell line and not in RD-neo cells (Fig 1 B). The increase was highest after 24 h, similar to p21WAF1 used as control. Hence, induction of wild-type p53 in the RD cell line led to the selection of a potential new p53-inducible gene, DRAL, from the initially screened cDNAs.
To determine whether DRAL can also be induced upon activation of endogenous p53, its expression was studied both in primary human myoblasts (wild-type p53) compared with RD cells (mutant p53) and in p53+/+ compared with p53-/- mouse embryo fibroblasts (Fig 1C and Fig D). In untreated samples, the levels of both DRAL and p21 mRNA were strikingly lower in cells containing mutated or no p53 than in wild-type cells. After exposure to 20 Gy of IR increases in p21WAF1 as well as DRAL mRNA levels were observed in wild-type p53 but not in mutant p53 cells. Interestingly, the time course of activation after irradiation is similar for DRAL and p21, although slightly different for the two cell types examined. These data suggest that transcription of endogenous DRAL can be stimulated by irradiation in a p53-dependent manner.
Cloning and Partial Characterization of the DRAL Gene and Its 5' Upstream Region
To gain some insight into the regulation of DRAL expression on the molecular level, the promoter region of the human DRAL gene was cloned. Screening of a human P1 library by PCR using DRAL-specific primers (amplifying nt 472 to nt 597 of the DRAL cDNA) resulted in one positive clone, P1:21280. To ensure that no recombination event occurred in the P1 clone, Southern blot analysis was performed with either human genomic DNA or the P1 clone digested with two different restriction enzymes and using a 2.5-kb PstI fragment from the P1 clone as a probe. Since both DNAs revealed the same hybridization pattern, any gross recombination in the relevant region of the P1 clone can be excluded (Fig 2 B). The 7.8-kb HindIII fragment identified in this Southern blot analysis was then further analyzed in detail and its sequence determined completely (available from Genbank/EMBL/DDBJ under accession number AF211174). The fragment contained 1.5 kb of upstream sequence and exon 1 (63 bp) and exon 2 (51 bp), separated by a 2,276-bp intron of the DRAL gene (Fig 2 A). The sequence of this intron is flanked by consensus splice donor and acceptor sites. In the upstream region, a TATA box is present 72-bp upstream of the start of the cDNA (which was isolated by RACE experiments). Therefore, it is likely that the start of transcription is located further upstream and the cDNA extends another 4050 bp. Additionally, two GC rich sequences (GC-boxes) were identified at positions -132 and -166 (relative to the start of the cDNA). A search for putative sequence elements able to bind upstream regulatory transcription factors within the promoter, identified several sites including one for myocyte enhancer binding factor 2 (MEF-2) at position -890, five E-boxes at positions -480, -508, -708, -878, and -1274 as well as five binding sites for the homeobox protein NKX2.5 at positions -417, -636, -815, -868, and -1426 (only the first four sites for both are indicated in Fig 2 A). The presence of multiple E-boxes might explain expression of DRAL in normal muscle tissue. Additionally, the five NKX2.5 sites are likely to contribute to the strong expression of DRAL in cardiac tissue (see below). Intriguingly, five putative p53-binding sites were identified within the 7.8-kb fragment and designated as p53 I to p53 V (Fig 2 C). The sites p53 I and p53 II are located in the 5' region whereas the sites p53 III, IV, and V are located in intron 2.
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In summary, cloning and characterization of the DRAL promoter region identified five potential p53 target sites, further supporting the observed p53-dependent transcriptional activation of the DRAL gene.
Expression Pattern of DRAL in Normal Tissues
To obtain a broad view of DRAL expression in human tissues, we analyzed its mRNA expression pattern in 50 human adult and fetal tissues. The results confirmed previous observations (
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Ectopic Expression of DRAL Induces Apoptosis
To gain insight into possible functions of DRAL, an expression vector carrying the DRAL cDNA was transfected into human RMS (RD), transformed monkey kidney (COS-1), and normal mouse fibroblast (NIH 3T3) cells. After selection with neomycin only a small number of clones was obtained whereas empty vector readily generated several hundred colonies from a parallel transfection. Surprisingly, none of the clones stably expressed DRAL protein (data not shown). To analyze the possible reason for this phenomenon, transient transfection experiments were performed in the same cells. Since DRAL consists only of LIM domains, the previously raised polyclonal antibody might also interact with other LIM domain proteins. To eliminate this possibility, two plasmids were constructed producing epitope-tagged DRAL protein, namely DRAL-CF (COOH-terminally FLAG-tagged DRAL) and DRALNF (NH2-terminally FLAG-tagged DRAL). As control in these experiments, the pFLAGCMV-2BAP plasmid (FLAG-tagged bacterial alkaline phosphatase) was used. Expression of FLAG-tagged proteins was analyzed by indirect immunofluorescence staining together with Hoechst DNA staining at different time points after transfection. In all cells highest FLAGDRAL expression was detected after 32 h. Interestingly, the number of FLAGDRAL-positive cells rapidly declined afterwards. At the same time labeled remnants were observed in the culture, which were reminiscent of apoptotic bodies (Fig 4). Neither decline in expressing cells nor cell remnants were observed with FLAGBAP, indicating that the effects observed are specific for DRAL. In addition, no difference was observed between DRALCF and DRALNF constructs. To unambigously identify apoptotic cells, caspase-3 activity was measured in transfected cultures as well as simultaneous stainings performed with either pico green labeled DNA (Fig 5) or annexin V. A quantitative summary of these results is shown in Fig 6. A dramatic increase in the number of apoptotic cells (7090% after 72 h depending on cell type) was paralleled by a rapid decline in the number of transfected cells in transfections with both FLAGDRAL constructs. In contrast, expression of pFLAGCMV-2BAP resulted in a constant number of transfected cells over the observation period and no specific increase in apoptotic cells (Fig 6 A). Annexin V stainings on COS-1 cells harvested 42 h after transfection were quantified with the fluorescence-activated cell sorter demonstrating an approximate threefold increase in positively stained cells. This value was obtained over the entire transiently transfected population (transfection efficiency 1520%) (Fig 6 B). To further confirm the induction of apoptosis, caspase-3 activity was measured 28 h after transfection (Fig 6 C). Indeed, expression of DRAL resulted in at least twice as much activity as the controls (vector alone or unrelated protein).
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We conclude from these experiments that ectopic expression of DRAL specifically induced apoptosis in all cell types analyzed, including human RMS cells. This notion is consistent with a possible role for DRAL as a potential tumor suppressor molecule.
Subcellular Localization of DRAL
To obtain some insight at potential proteinprotein interactions that might be functionally relevant, the subcellular localization of DRAL was studied after transient transfection of FLAGDRAL into the same cell types used before (RD, COS-1, and NIH 3T3). The previously reported predominant nuclear staining of DRAL (
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Since highest expression of DRAL was found in fetal and adult heart, we also wished to examine the subcellular localization of DRAL in cardiomyocytes. To this end neonatal rat cardiomyocytes were transiently transfected with either FLAGDRAL (Fig 8) or the FLAGBAP plasmid (data not shown). Interestingly, in addition to the already described localization of DRAL in the nucleus and focal contacts, a distinct cross-striated pattern was observed. Double staining of the same cells with the myosin binding protein C and subsequent overlapping of the two staining patterns revealed that DRAL localizes specifically to the Z-discs and, to a lesser extent, to the M-band of myofibrils (Fig 8C and Fig F, small inset). In cardiomyocytes transfected with the FLAGBAP plasmid only completely diffuse labeling could be observed, indicating that the localization is specific for DRAL (data not shown). To confirm these observations, immunofluorescence stainings of the endogenous DRAL in cardiomyocytes were performed as well (Fig 9). Western blot of a cardiomyocyte extract with the anti-DRAL polyclonal antibody shows a major reactive species of about 32 kD, indicating that the antibody reacts specifically (Fig 9 B). Immunofluorescence staining then confirmed localization of endogenous DRAL to the Z-discs and the M-band of cardiac myofibrils (Fig 9 A).
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Hence, DRAL could be unambigously localized in the nucleus, the focal contacts and the Z-discs as well as the M-band of heart myofibrils. Presumably, DRAL will have a different protein interaction partner at each of these locations.
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Discussion |
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This study identifies a member of the FHL subfamily of LIM-only proteins, DRAL, as a novel target gene for the tumor suppressor protein p53. Intriguingly, DRAL protein was capable of efficiently triggering apoptosis in a wide range of cell types upon ectopic expression.
We provide three different experimental criteria suggesting that DRAL might be a direct transcriptional target of p53. First, induction of wild-type p53 in RMS cells through a temperature-sensitive p53 allele specifically increased transcription of endogenous DRAL, in a manner comparable to known p53 target genes like p21WAF1 and thrombospondin-1. Second, exposure of primary myoblasts and fibroblasts to -irradiation induced an increase in DRAL mRNA that paralleled the increase in p21WAF1 mRNA only in cells with wild-type p53. Because of different probes and exposure times, the mRNA levels of p21 and DRAL are not directly comparable. However, cells with mutated or no p53 consistently showed a striking reduction in the expression from both genes whereby basal levels of DRAL were higher in wild-type p53 cells than p21WAF1. This could be a possible explanation for the lower induction of DRAL mRNA after irradiation. Also, the basal expression level of DRAL was higher in fibroblasts than in myoblasts, possibly accounting for the more moderate induction observed in this cell type. Finally, analysis of the genomic structure of DRAL in the promoter region revealed five potential consensus p53-binding sites. Interestingly, the DRAL gene could be identified on recently published draft sequences of the human genome project (GenBank/EMBL/DDBJ accession numbers AC069576 and AC012360) indicating that the entire gene consists of seven exons.
In Northern blot experiments, DRAL expression was predominantly found in fetal and adult heart. The five putative NKX2.5-binding sites identified in the promoter region are in concurrence with this expression pattern. In fact the homeobox gene Nkx2.5 represents the earliest known marker of the cardiac lineage in vertebrates and its expression is maintained throughout the developing and adult heart (
Since transcription of DRAL can be induced by wild-type p53, the next question was if DRAL would participate in any of the known functions of p53-like growth arrest or apoptosis. To test this notion, ectopic expression of DRAL was achieved in a range of cell types including human RMS cells. Interestingly, this resulted in efficient induction of apoptosis in all cell types analyzed, which was confirmed on the molecular level by annexin V staining and caspase-3 activity measurements. Indeed, we were unable to generate stable cell lines expressing DRAL. Even RMS cells ectopically expressing Bcl-2 (to protect cells from apoptosis) failed to produce DRAL expressing clones (results not shown). Therefore, DRAL might participate in the apoptotic response following activation of p53. Very recently, another LIM-only protein was described to induce apoptosis in myoblasts upon ectopic expression, namely the paxillin homologue Hic-5 (
To begin to investigate the mechanisms by which ectopic expression of DRAL can induce apoptosis, its intracellular localization was determined. Staining of RD-tsp53 cells with the polyclonal DRAL antibody at the permissive temperature provided the first evidence for localization of DRAL at focal contacts (data not shown). Later, DRAL was found in focal contacts, the cytoplasm, nucleus as well as the Z-discs and the M-band of cardiac myofibrils. These localizations were observed both by staining of the endogenous DRAL protein or staining of an ectopically expressed tagged version of the protein. As a further confirmation, a recently carried out yeast two-hybrid screen using DRAL as a bait identified several transcription factors, myofibrilar proteins and focal contact proteins as potential interaction partners (data not shown). These potential interactions are currently being analyzed in more detail.
Focal contacts are known to establish a transmembrane linkage between the actin cytoskeleton and the extracellular matrix and are implicated in a number of signaling pathways. Some of the proteins at these specialized sites (e.g., focal adhesion kinase, paxillin) serve as scaffolding molecules and can act as signaling centers by providing docking sites for other proteins (
On the other hand, nuclear localization of DRAL might also be responsible or contribute to its pro-apoptotic function. Interestingly, dual localization in focal contacts and the nucleus is known for a number of other protein, e.g., zyxin, Hic-5, and ß-catenin, a multi-functional protein activated by the Wnt signaling pathway (
The Z-disc localization of DRAL is shared with a second LIM domain protein, MLP (
Although the knowledge of the function of FHL proteins is still very limited, they may act either as scaffolding molecules to link several proteins and thus activating distinct signaling pathways or as modulators of transcription by complexing transcription factors (
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Footnotes |
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1 Abbreviations used in this paper: BAP, bacterial alkaline phosphatase; DRAL, down-regulated in rhabdomyosarcoma LIM protein; DRAL-CF, COOH-terminally FLAG-tagged DRAL; DRAL-NF, NH2-terminally FLAG-tagged DRAL; FHL, four and a half LIM domain; IR, ionizing radiation; MEF-2, myocyte enhancer binding factor 2; RMS, rhabdomyosarcoma.
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Acknowledgements |
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We are very grateful to Dr. Sacchi (Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Rome, Italy) for kindly providing RD-tsp53 and RD-Neo cells, and to Dr. M. Pruschy for his help with the -irradiation experiments including the p53+/+, p53-/- mouse fibroblasts. We thank Prof. C. W. Heizmann and Prof. F. E. Würgler for their support, Prof. J.C. Perriard (Institute of Cell Biology, ETH Zürich, Zürich, Switzerland) for making the confocal microscope available for these studies and Dr. A. Lauber-Biason for critical reading of the manuscript.
This work was sponsored by grants from the Krebsforschung Schweiz, the Krebsliga of the Kt. Zug, and the Swiss National Science Foundation (31-46886.96 and 31-56869.99).
Submitted: 19 January 2000
Revised: 14 September 2000
Accepted: 14 September 2000
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References |
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