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Neoplasia. May 2008; 10(5): 471–480.
PMCID: PMC2373908

Decitabine-Induced Demethylation of 5′ CpG Island in GADD45A Leads to Apoptosis in Osteosarcoma Cells1

Abstract

GADD45 genes are epigenetically inactivated in various types of cancer and tumor cell lines. To date, defects of the GADD45 gene family have not been implicated in osteosarcoma (OS) oncogenesis, and the role of this pathway in regulating apoptosis in this tumor is unknown. The therapeutic potential of Gadd45 in OS emerged when our previous studies showed that GADD45A was reexpressed by treatment with the demethylation drug decitabine. In this study, we analyze the OS cell lines MG63 and U2OS and show that on treatment with decitabine, a significant loss of DNA methylation of GADD45A was associated with elevated expression and induction of apoptosis. In vivo affects of decitabine treatment in mice showed that untreated control xenografts exhibited low nuclear staining for Gadd45a protein, whereas the nuclei from xenografts in decitabine-treated mice exhibited increased amounts of protein and elevated apoptosis. To show the specificity of this gene for decitabine-induced apoptosis in OS, GADD45A mRNAs were disrupted using short interference RNA, and the ability of the drug to induce apoptosis was reduced. Understanding the role of demethylation of GADD45A in reexpression of this pathway and restoration of apoptotic control is important for understanding OS oncogenesis and for more targeted therapeutic approaches.

Introduction

Methylation-mediated silencing of genes is one of the most important epigenetic mechanisms implicated in the regulation of normal gene expression. Such changes often affect 5′ regulatory CpG genomic regions and can be associated with aberrant expression of certain genes in cancer (reviewed in the study of Esteller [1]). Epigenetic alterations are considered to contribute in several ways to oncogenesis; for example, by activating oncogenes, by silencing tumor suppressor genes, or by disrupting pathways that contribute to tumorigenesis such as those governing apoptosis [2,3].

There is increasing interest in the use of new epigenetic therapies that might modulate molecular pathways central to tumorigenesis [4,5]. The most frequently used treatment at present is the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (decitabine) [6]. This drug is a cytosine analog that inhibits DNA methylation and can reactivate the expression of transcriptionally silenced genes. Such repression of gene expression in tumors is thought to occur by specific CpG methylation of dinucleotide clusters within CpG islands that are present in the promoters and span exonic and intronic regions at many loci throughout the human genome [7]. Demethylation may remove tumor-specific repression within regulatory regions and permit activation of genes controlling pathways highly pertinent to oncogenesis, such as apoptosis, proliferation, invasion, and so on.

Previously, we showed that decitabine treatment of the osteosarcoma (OS) cell line U2OS led to the upregulation of >50 genes possessing CpG islands at their 5′ region [8]. One of the decitabine-activated genes of importance in OS oncogenesis was GADD45A. This gene belongs to the stress-responsive GADD45 family that was reported to be methylated in multiple tumors [9,10]. GADD45A is relevant to OS tumorigenesis because of its central role in apoptosis and the P53 pathway [11–13]. Moreover, Gadd45a is a central player in the maintenance of genomic stability, and loss of protein function can lead to centrosome amplification, chromosomal instability, and increased aneuploidy [14,15]. Because generalized loss of genome stability is characteristic of OS tumors [16–19], the role of this protein in OS is highly relevant. At the molecular level, the promoter region of GADD45A has a repression-binding site for c-MYC gene [20] that is known to be amplified in OS. Also, GADD45A promoter has a binding locus for P53 in the third intronic region of the gene, and its functionality is linked to the activation of G1/S cell cycle arrest in response to ionizing radiation [21]. Significantly, Gadd45a activation was previously shown to result in the induction of apoptosis in several cancer cell lines including COS, PC-3, DU145, and HeLa cell lines [10,22–24].

Methylation within the 5′ region of GADD45A is likely to be a major mechanism of repression and inactivation of the protein's apoptotic function. There is a region with dense repetitive CG sequence (CpG island) near the transcription start site (TSS) of GADD45A that spans 1357 bp and covers the first three exons of the gene [8,25]. Methylation of this CpG island was reported in breast cancer tissues [25]. A cluster of eight CpG dinucleotides within the first intron of GADD45A was found to be methylated in the OS cell line U2OS in vitro and in xenografts [8]. Our previous study demonstrated that induction of apoptosis in U2OS followed decitabine treatment, but the precise role of GADD45A CpG island demethylation and the specificity of Gadd45a expression in the induction of apoptosis were not defined. The present study was designed to examine the role of decitabine-dependent GADD45A CpG island demethylation on the expression of the gene and on subsequent induction of apoptosis in OS cell lines. These analyses will help determine whether GADD45A is a potential therapeutic effector and useful biomarker of apoptosis for future clinical trials in OS that involve genome-wide demethylation.

Materials and Methods

Cell Cultures and Treatment

The human OS cell lines U2OS (ATCC # HTB-96) and MG63 (ATCC # CRL-1427) and cervical adenocarcinoma (HeLa) cell lines (ATCC # CCL-2) were purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in ATCC recommended medium. Normal osteoblasts are a primary osteoblasts from the hipbone of normal male donor that were purchased from PromoCell, Heidelberg, Germany (catalogue # C-12760) and maintained in medium provided by the manufacturer and used at culture passage 3. Treatment with decitabine was performed as described by Liang et al. [26]. Briefly, 5 x 105 cells were plated in 56-cm2 culture plates with 10 ml of growth medium. At 12 hours after plating, they were treated with freshly prepared decitabine (Sigma Chemical Co., St Louis, MO) to a final concentration of 1 µM without changing the medium. Decitabine was added to the medium only once to minimize the drug toxicity that is not resulting from drug effects on DNA methylation [26]. Three days after initiating the treatment, cells were harvested for RNA and DNA extraction or Hoechst 33342 staining. The use of a single-dose decitabine to significantly reactivate methylation-silenced genes was reported in bladder cancer cells [26]. Other treatment schedules analyzing demethylation in lung, head, and neck tumors have involved fresh decitabine when culture media is replaced [27].

U2OS Xenografts and Treatment

Decitabine treatment of U2OS xenografts was previously described [8]. Immune-deficient mice were bred and maintained, and xenograft experiments were performed by the Animal Resource Centre at the British Colombia Cancer agency, Vancouver, Canada. Xenografts were established under the renal capsule, and host mice were treated intraperitoneally with three doses (2-day intervals) of decitabine (2.5 mg/kg body weight) dissolved in saline (0.9% w/v NaCl) or saline alone (control). Mice were sacrificed 5 days after the last treatment, and the xenograft tissues were snap-frozen or were prepared in paraffin and sectioned using standard procedures [28].

Knockdown By Gadd45a-siRNA and Transient Transfection of GADD45A

Silencer predesigned short interference RNA (siRNA) for human GADD45A (siRNA ID: 146174) and control nontargeting RNA (ctRNA) were obtained from Ambion, Foster City, CA. Transfection of OS cells with siRNA was performed using siPORT Amine transfection agent (Ambion) in six-well plates according to manufacturer's protocol at the concentration of 60 nM. Decitabine (1 µM) treatments were done 12 hours after plating. RNA and protein was extracted at specified time points. For the transient transfection experiments, 2 µg of pCMV(GADD45A) or the empty pCMV vectors (TrueClone; OriGene, Rockville, MD) with 10 µl of Lipofectamine 2000 (Invitrogen, Burlington, Ontario, Canada) were used in the transfection experiments in six-well plates according to the manufacturer's protocol. Three days later, protein was extracted for Western blot analysis.

Quantitative Bisulfite Pyrosequencing (Pyro-Q-CpG)

Pyro-Q-CpG primers and protocol were previously described [8]. Genomic DNA from the control and decitabine treatment was bisulfite-treated using the Zymo DNA Methylation Kit (Zymo Research, Orange, CA). Bisulfite-treated DNA was PCR-amplified and sequenced according to standard protocol (Biotage, Kungsgatan, Sweden). The criteria for a CpG island was based on those outlined by Takai and Jones [29], also see [8], where the GC ≥ 55%, Obs/Exp ≥ 0.65, and length >300 bp which was reported to exclude most Alu-repetitive elements. We identified the genes that harbored CpG island within a 2000-bp window upstream or downstream from the TSS-based Human Genome Browser database (http://genome.ucsc. edu/). To be certain that there were no CpG island closer to the TSS and gene promoter regions, we submitted the sequences of interest (including a 2000-bp window upstream and downstream from TSS) to the CpG search engine available in Reference [29].

Reverse Transcription and Quantitative Real-Time PCR

Total RNA from U2OS, MG63, and NHOst cells was extracted using TRIzol method (Invitrogen, Osaka, Japan). Total RNA was reverse-transcribed with the GeneAmp kit (Applied Biosystems; ABI, Foster City, CA). About 20 ng of the resulting cDNA was used for TaqMan real-time PCR GADD45A expression assay (Hs00169255_m1; ABI). All reactions were done in triplicate in a 384-well plate using the 7900 Sequence Detector System (ABI). Data analysis was performed by applying the ΔΔCt method relative to nontreated osteoblast with β-actin as reference gene.

Immunohistochemistry and Image Analysis

Xenograft tissue and pelleted paraffin-embedded formalin-fixed HeLa cell sections were deparaffinized and rehydrated before incubation at room temperature in 3% H2O2 in PBS for 10 minutes. Slides were washed in PBS three times for 3 minutes each. Antigen retrieval was performed at 95°C for 30 minutes in 10-mM sodium citrate (pH 6.0). After cooling in a running water bath for 5 minutes and PBS wash, the slides were blocked in 3% skim milk for 30 minutes. Blocking solution was rinsed briefly, and slides were incubated at 4°C overnight with 500x dilution of primary anti-human Gadd45a-Ab (Abnova, Taipei City, Taiwan). After the PBS wash, subsequent steps were performed using DakoCytomation Kit (Dako, Glostrup, Denmark) as per manufacturer's protocol. Hematoxylin was used for counterstaining, and slides were mounted and scanned by ScanScope CS (Aperio Technologies, Vista, CA) as previously described [8]. Positive controls were generated by exposing HeLa cells to ultraviolet (UV) radiation at 10 µJ/cm2 [24]. These cells were then mounted on slides and processed identically to the experimental xenografts. The extent of positivity was determined by comparing the xenograft tissue sections to the control slides derived from HeLa cells. To confirm that each section retained immunoreactivity, a positive finding for the Ki67 antibody stain indicated that adjacent xenograft sections were positive and informative.

Western Blot Analysis

Cells were lysed in culture plates on ice using radioimmunoprecipitation buffer. Lysates were sonicated briefly on ice and centrifuged at high speed for 15 minutes at 4°C. Total protein was quantitated using Bradford reagent (Bio-Rad, Hercules, CA). About 50 µg of total protein/well was separated on acrylamide gel and was transferred to polyvinyl derivative membranes at 25 V at 4°C overnight. Membranes were blocked with 5% BSA for 8 hours at 4°C, followed by a 16-hour primary antibody incubation [1:12,000 mouse monoclonal anti-human Gadd45a-Ab (Abnova) or 1:24,000 rabbit polyclonal anti-human β-actin-Ab (Abcam, Cambridge, MA)]. The blots were washed with TBS-Tween 20 five times for 10 minutes each and a final wash with TBS. The blots were incubated with perioxidase-labeled secondary antibody (ECL-Plus kit; Amersham, Buckinghamshire, UK) as per manufacturer's instructions at a dilution of 1:20,000 and were scanned on the Typhoon 9410 scanner (Amersham) at normal sensitivity, 600 laser, and 200-µm resolution. Quantitation was performed using ImageJ software on blots from three replicates, and significance was estimated using t test at P < .05 and β-actin levels served as a control.

Hoechst 33342 Staining for Apoptotic Nuclei

The cells were trypsinized and resuspended in 1 ml of PBS. Staining was performed in 5 ml of 4% formalin solution with Hoechst 33342 dye (0.01-µg/µl final concentration). Aliquots of the samples were bar-coded before spreading the cells on glass slides. Hoechststained cells were blindly analyzed using a fluorescence microscope. One hundred cells were scored per slide, and the percentage of stained (apoptotic nuclei) out of total was recorded. Triplicate experiments were analyzed using Student's t test at P < .05.

Statistical Test

Student's t test was applied to analyze the differences between treatment groups.

Results

CpG Methylation of GADD45A in OS Cells

GADD45A repression of expression in U2OS in vitro and in xenografts was shown previously by our group to be associated with 5′ CpG island hypermethylation of a cluster of eight CpG dinucleotides, within the first intronic region of the gene 620 bp downstream of the TSS (Figure 1A) [8]. In this study, quantitative bisulfite pyrosequencing (Pyro-Q-CpG) showed that ~80% of the sequenced alleles from the eight CpG dinucleotides in GADD45A of MG63 were methylated (Figure 1B). Exposure of MG63 to 1-µM decitabine significantly decreased methylation (P < .001) to ~55% of sequenced alleles at the eight CpG dinucleotides (Figure 1B). Moreover, methylation repression of GADD45A in MG63 was implicated because mRNA expression was found to be reduced at levels slightly lower than those of normal low-passage osteoblasts (Figure 2A). Importantly, loss of DNA methylation was associated with a 3.5-fold increase in GADD45A mRNA expression relative to normal osteoblasts and a six-fold increase relative to untreated MG63 cells as detected by quantitative real-time PCR (Figure 2A). Similar observations were found in U2OS cells, which confirmed the previous results published by our group [8]. Interestingly, very low methylation (range, 0.1–2.9% methylation of sequenced alleles) was observed at all eight CpG dinucleotides in both treated and untreated osteoblasts (Figure 1B). Moreover, GADD45A mRNA expression in osteoblasts was not induced after decitabine treatment (Figure 2A).

Figure 1
GADD45A CpG methylation in osteosarcoma cells. (A) Schematic of GADD45A genomic locus. 5′ CpG island spanning the first three exonic regions of GADD45A is shown. An amplicon of ~250 bp was amplified, and a cluster of eight CG dinucleotides ...
Figure 2
Repression of GADD45A expression. (A) Induction of GADD45A mRNA expression in U2OS and MG63 after decitabine treatment. Total RNA was extracted on day 3 after decitabine treatment was initiated. cDNA was then made as detailed in the Materials and Methods ...

To confirm that induction of mRNA expression led to a concomitant increase in Gadd45a protein levels, Western blot analyses were performed on cell extracts from both decitabine-treated and untreated U2OS and MG63 cells (Figure 2, B and C). In both cell lines, the levels of Gadd45a protein induction after decitabine treatment were similar to that observed in the mRNA levels.

Decitabine Induces Significant Levels of Gadd45a Protein in OS Cells

To investigate the level of Gadd45a protein induction in U2OS xenografts, six xenografts (three from no treatment and three from decitabine-treated) were analyzed by immunohistochemistry using a human Gadd45a antibody. Sections from cell pellets prepared from the Gadd45a-activated UV-treated HeLa cells were used as positive controls. The staining of xenograft nuclei was quantitated using the intensity of staining and the proportion of positive tumor nuclei. As shown in Figure 3, A and B, xenograft sections from the untreated control mice exhibited low nuclear staining for Gadd45a, whereas the staining was stronger and more frequent in nuclei from xenografts in decitabine-treated mice (P < .05). Induction of Gadd45a was also found to be associated with increased apoptosis as shown in a previous analysis on the same U2OS xenografts [8]. In addition, caspase-9 activation was assessed by immunohistochemistry in U2OS xenografts using an antibody specific for cleaved (activated) caspase-9. In this analysis, there was no significant induction of cleaved caspase-9 (P > .05) as a result of decitabine treatment when compared to no-treatment xenografts.

Figure 3
Induction of Gadd45a protein in OS xenografts by decitabine treatment. (A) Induction of Gadd45a protein levels in U2OS xenografts. Analysis of the relative levels of nuclear Gadd45a protein within xenografts derived from representative control untreated ...

Gadd45a-Specific Induction of Apoptosis

Decitabine treatment at 1 µM significantly induced apoptosis in vitro (P < .05) in both cell lines in comparison to no-treatment cells (Figure 4A). Similarly, transient transfection of Gadd45a overexpression vector significantly induced apoptosis (P < .05) in untreated U2OS and MG63 in comparison to transfection with an empty vector (Figure 4A). In both experiments, Western blot analysis demonstrated activation of the Gadd45a protein (Figure 4, B and C). To investigate the specificity of Gadd45a induction on decitabine-induced apoptosis, GADD45A mRNAs were disrupted using specific siRNA in U2OS and MG63 cells. The effect of 1-µM decitabine treatment on GADD45A mRNA and protein levels 3 days after treatment was analyzed alone or in combination with 60 nM Gadd45a-siRNAs. Transfection of Gadd45a-siRNA against a decitabine-treated background reduced GADD45A mRNA levels in both cell lines by >70% knockdown efficiency (Figure 5A). This knockdown efficiency was consistently observed in the protein level in U2OS and MG63 cells as analyzed by Western blot analysis (Figure 5, B and C). Decitabine treatment resulted in a five-fold induction of GADD45A mRNA in U2OS, and six-fold in MG63 cells. Similar levels of induction were seen in the presence of 60 nM of scrambled ctRNA in decitabine-treated cells.

Figure 4
Apoptosis induction by pCMV-GADD45A in U2OS and MG63. (A) Percentage of apoptotic nuclei as detected by Hoechst stain in U2OS and MG63. Columns are mean of three replicas, and error bars are standard deviation from the mean. (B) Induction of Gadd45a protein ...
Figure 5
Reduction of GADD45A expression by GADD45A-siRNAs in decitabine-treated U2OS and MG63 cells. (A) Effects of GADD45A-siRNA on mRNA levels in U2OS and MG63. Total RNA was extracted on day 3 after the decitabine treatment was initiated. Preparation of cDNA ...

To investigate the effect of knocking down Gadd45a protein on apoptosis levels, nuclei from U2OS and MG63 were stained with Hoechst 33342 dye at day 3 after treatment in the decitabine-alone and combination experiments (Figure 6A). Fluorescent microscopy demonstrated that the fractions of fragmented nuclei caused by 1-µM decitabine treatment were 14% in U2OS and 15% in MG63, whereas the no-treatment cells or the ctRNA (60 nM) cells (in the presence of a transfection agent) had a fragmented nuclei (apoptotic nuclei) percentage of less than 4% in both cell lines (Figure 6B). Decitabine also induced apoptosis to similar levels in U2OS and MG63 when used in combination with same amounts of ctRNA and transfection agent. Interestingly, when Gadd45a-siRNA (60 nM) was used in combination with decitabine, apoptotic nuclei were reduced from 14% to 6% (P < .05) in U2OS and from 15% to 8% in MG63 (P < .05) (Figure 6, A and B), indicating that decitabine's ability to induce apoptosis was reduced specifically by Gadd45a-siRNA.

Figure 6
Gadd45a-siRNA treatment abolishes decitabine-induced apoptosis. (A) A representative image of apoptotic nuclei (arrows) from decitabine-treated MG63 stained with Hoechst 33342 dye. (B) One hundred nuclei were counted per slide, and three slides were prepared ...

Discussion

Osteosarcoma is a particularly aggressive cancer in which current treatment modalities result in a 5-year event-free survival in 60% to 70% of patients [30,31]. The chemotherapeutic drugs most commonly used in the treatment of OS include doxorubicin, high-dose thotrexate, cis-platinum, and ifosfamide either alone or with etoposide (reviewed in the study of Uchida et al. [32]). Unfortunately, there is a poor response to chemotherapy in a significant subgroup of OS patients, and some patients have a high drug toxicity profiles [33]. Attempts at changing chemotherapy regimens for poor responders have generally failed to improve outcome [34].

In recent years, novel therapeutic approaches involving genomewide epigenetic modification have been introduced (reviewed in the study of Issa [6]). Decitabine is one of the most popular in this class of new drugs, and it has been approved by the US Food and Drug Administration [35]. The results of early clinical trials using this drug are promising, but there is little detail available concerning the molecular pathways leading to tumor response (reviewed in the study of Kihslinger and Godley [36]). The mechanisms of response to decitabine may vary in different patients, but they are thought to include induction of senescence, differentiation, and apoptosis (reviewed in the study of Issa [6]).

Previously, we showed that decitabine treatment of the OS cell line U2OS led to the upregulation of >50 genes possessing CpG islands at their 5′ region [8]. GADD45A was one of the decitabine-activated genes that were known to be involved in apoptosis, and importantly, the function of the protein is relevant to OS oncogenesis. Interestingly, the GADD45 stress-response family has been shown to be inactivated by methylation in several types of tumors [9] (reviewed in the study of Zerbini and Libermann [10]). The protein has been implicated in regulating genome stability (reviewed in the study of Hollander and Fornace [15]), DNA damage response [11], DNA repair [37,38], apoptosis [24], and most recently, when overexpressed, DNA demethylation [39]. Despite the impact of GADD45A gene on all these processes, and its frequent methylation in some tumors, there is limited information regarding the role of GADD45A in the response to epigenetic modifications, in general, and to demethylation treatment, in particular.

In this study, we found that extensive methylation of the 5′ CpG region of GADD45A was present in the MG63 and U2OS cell lines, and this epigenetic change was associated with the reduced expression of GADD45A in OS. Furthermore, it was found that exposure of both OS cell lines to decitabine significantly decreased the methylated alleles to ~55% in this region of the gene. The relationship between loss of DNA methylation in this region, and elevated gene expression was demonstrated by a six-fold increase in both GADD45A mRNA and protein levels. The role of Gadd45a on cell cycle arrest is well established. The protein has been shown to play a role in G2-M checkpoint in response to DNA damage. Gadd45a activates p53-dependent G2-M arrest, providing a link between p53-dependent cell cycle checkpoint and DNA repair. In this regard, it is noteworthy that p53 inactivation is one of the most common aberrations observed in human OS (reviewed in Kansara and Thomas [40]).

The role of Gadd45a in apoptosis is not as well understood as is its role in cell cycle arrest. GADD45A overexpression in normal human fibroblasts and human cancer cells causes G2-M arrest [25,41,42]. Gadd45a-dependent induction of apoptosis has been observed frequently in cancer cell lines [10,24], but its role in apoptosis induction in normal cells has been controversial. Gadd45a has been shown to induce cell cycle arrest in normal fibroblasts but fail to induce apoptosis [41]. Others, however, have shown Gadd45a-dependent induction of apoptosis in normal epithelial cells [43]. In the present study, we show that Gadd45a reexpression correlates with a significant increase in apoptosis in OS cells. In vivo effects of decitabine treatment were studied by establishing xenografts and showing that tumor sections of xenografts from untreated control mice exhibited low nuclear staining for Gadd45a protein, whereas the nuclei from xenografts in decitabine-treated mice exhibited much higher levels of Gadd45a protein. As with the in vitro studies, an increase in Gadd45a protein levels was associated with a significant increase in apoptosis.

To show the specificity for Gadd45a in decitabine-induced apoptosis, GADD45A mRNAs were disrupted using siRNA. This approach has been used previously in other cell types, and inhibition of GADD45A led to disruption of functions including its ability to induce apoptosis [10,44,45]. GADD45A activation in OS was found to be central to the decitabine-induced apoptosis, and knockdown by siRNA demonstrated the specificity of this effect. This observation, however, does not eliminate the possibility of the involvement of the other apoptotic factors and pathways in the decitabine-induced apoptosis in OS cells [8].

Osteosarcoma is known for having high levels of genomic instability with multiple chromosomal breakpoints and increased incidence in genomic aberrations [17–19,46]. Inhibition of the DNA damage response gene such as GADD45A by DNA hypermethylation could be one explanation on how OS cells escape apoptosis and undergo survival, despite the numerous DNA breakpoints required to generate the complex karyotypes that characterize the OS genome. Interestingly, Gadd45a has recently been shown to actively demethylate downstream target genes by promoting DNA repair [39] thus linking both processes. Finally, the findings of a potential role for GADD45 repression by methylation in OS oncogenesis may encourage the development of novel therapeutic strategies that take advantage of improved understanding of the genomics and epigenomics of this tumor.

Acknowledgments

We thank Annie Huang and Rod Bremner for their thoughtful discussion and helpful suggestions. We thank Meihua Li and Rajesh Gubta for their technical assistance.

Abbreviations

ctRNA
control nontargeting RNA
Decitabine
5-aza-2-deoxycytidine
OS
osteosarcoma
Pyro-Q-CpG
quantitative methylation pyrosequencing
siRNA
short interference RNA

Footnotes

1This work has been supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and Terry Fox Foundation. K. A.-R. was supported by a scholarship from King Faisal Specialist Hospital and Research Centre.

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