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Carcinogenesis Advance Access originally published online on March 14, 2006
Carcinogenesis 2006 27(9):1758-1767; doi:10.1093/carcin/bgl008
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Plakoglobin is differentially expressed in alveolar and embryonal rhabdomyosarcoma and is regulated by DNA methylation and histone acetylation

Tamara Gastaldi, Paolo Bonvini, Francesca Sartori, Agnese Marrone1, Achille Iolascon1 and Angelo Rosolen*

Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova Via Giustiniani 1, 35128 Padova, Italy
1 CEINGE-Centro di Biotecnologie Avanzate, Università Federico II di Napoli Via Comunale Margherita 482, 80145 Napoli, Italy

*To whom correspondence should be addressed at Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, via Giustiniani 3, 35128 Padova, Italy. Tel: +39 049 8215678; Fax: +39 049 8213510; Email: angelo.rosolen{at}unipd.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plakoglobin ({gamma}-catenin) and ß-catenin are pivotal components of cell–cell adherent junctions that link cadherin receptors to the actin cytoskeleton. Whereas ß-catenin overexpression induces cell proliferation and tumor formation, plakoglobin induces tumor suppressor activity. We investigated the expression of plakoglobin in alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma (RMS) cell lines and tumors, and found that plakoglobin is present both in the cytoplasm and in the nucleus of ERMS cells, whereas it is absent or detectable at extremely low levels in ARMS. As gene silencing can be mediated by methylation and/or deacetylation of promoter regions, we assessed the effects of the DNA demethylating agent 5-Aza-2'-deoxycytidine (5AzadC) and of the histone deacetylase inhibitor Trichostatin A (TSA), and obtained restoration of plakoglobin expression in ARMS cells cultivated in the presence of 5AzadC and TSA. By methylation-specific PCR, ARMS cells were shown to contain methylated CpG dinucleotides in CpG islands located around the transcriptional start site of one or both alleles, whereas ERMS cells did not. Furthermore, we demonstrated that promoter regions (P1–P3) of plakoglobin gene were associated with hypoacetylated H4 histone in ARMS cells RH4, suggesting that aberrant DNA methylation of the 5' CpG island and histone deacetylation play key roles in silencing the plakoglobin gene. These results demonstrate that plakoglobin is differentially expressed in ARMS and ERMS and that its expression depends on the methylation and acetylation status of the gene.

Abbreviations: 5AzadC, 5Aza-2-deoxycytidine; ARMS, alveolar RMS; ChIP, Chromatin Immunoprecipitation; ERMS, embryonal RMS; HDAC, histone deacetylase; MSP, methylation-specific PCR; RMS, rhabdomyosarcoma; TSA, Trichostatin A


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Rhabdomyosarcoma (RMS) includes two major histological subtypes: the more frequent embryonal RMS (ERMS), and the more aggressive alveolar RMS (ARMS) (1,2). The latter harbors a reciprocal chromosomal translocation t(2;13) (q35;q14), or, less frequently, the variant translocation t(1;13)(p36;q14) (3,4) that generates a powerful chimeric transcriptional activator PAX3–FKHR or PAX7–FKHR (5) with oncogenic activity (6). The PAX3–FKHR fusion protein causes aberrant expression of a set of PAX3-target genes normally regulated during development by the wild-type protein, including c-Met (7,8) or the HER/erbB family members (EGFR and erbB2) (9), which have been closely linked with tumor aggressiveness and poor prognosis in many cancer types. These receptor tyrosine kinases, which are expressed in RMS cell lines (7–9), are involved in proliferation, migration and metastasis through the phosphorylation of different proteins contained in cellular junctions with consequent tissue destruction and the increase in invasiveness.

ß-Catenin is a multifunctional protein that plays a pivotal role both in cell–cell interactions and transcriptional activation in Wnt/Wingless signaling during development (10). The regulation of membrane, cytoplasmic and nuclear pools of ß-catenin is crucial for modulating its adhesion and signaling functions (11).

The catenin family member plakoglobin, also known as {gamma}-catenin, is an adhesion molecule with structure, cellular distribution and protein-binding specificity similar to ß-catenin. In addition to its involvement in the desmosome structure, plakoglobin is a component of the adherens junctions and binds E-cadherin and {alpha}-catenin (12).

ß-Catenin expression level is high in cancers of various origin (13), mostly as a result of point mutations in ß-catenin phosphorylation sites necessary for its ubiquitination or in key components of the degradation machinery (14,15). Unlike ß-catenin, plakoglobin mutations are rare. However, the human plakoglobin gene can display the loss of heterozygosity in certain tumors and this has been associated with cancer progression (16). Moreover, plakoglobin overexpression was shown to suppress tumorigenicity in various transformed cell lines (17).

Methylation of cytosine residues at CpG dinucleotides repeats is an important biological phenomenon for gene silencing and regulation of chromatin structure. Cytosine hypermethylation in the promoters of tumor suppressor genes such as RASSF1A and HC1 has been linked to transcriptional repression (18,19). Recent work has demonstrated the importance of post-translational modification of histone proteins as another epigenetic mechanism in the organization of chromosomal domains and gene regulation (20,21). Acetylation of lysine residues on histone H3 and H4 leads to the formation of open chromatin structure (22), whereas deacetylation is associated with repressed chromatin (20,23). There is growing evidence that an interplay exists between cytosine methylation and histone modification. The methyl-CpG binding protein, MeCP2, has been associated with histone deacetylase (HDAC) activity, suggesting that histone modification can be induced by DNA methylation changes (24,25).

In this study, we report that plakoglobin was downregulated in a subset of RMS and hypothesized that this may be a consequence of hypermethylation of CpG islands and hypoacetylation of histones within plakoglobin promoter region. To test these hypotheses, the expression and the methylation/acetylation status of the plakoglobin promoter were analyzed in RMS cell lines and biopsies.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell cultures and tumor samples
The human RMS cell lines RH30 and RD were purchased from ATCC (Manassas, VA); RH4, RH18 and RH28 were a gift of Dr P.J. Houghton (St Jude Children's Hospital, Memphis, TN); RC2 was provided by Prof. P.L. Lollini (University of Bologna, Bologna, Italy); SMS-CTR, RH36, CCA and the undifferentiated sarcoma cell line A-204 were obtained from Dr M. Tsokos (NCI, Bethesda, MD); the neuroblastoma cell line LAN-5 was a gift of Dr M. Ponzoni (Gaslini Hospital, Genova, Italy). Cells were maintained in RPMI 1640 containing 10% heat-inactivated fetal calf serum (FCS), 2 mmol/l glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin under standard tissue-culture conditions. Specimens were obtained from the Italian Association of Pediatric Hematology and Oncology Soft Tissue Sarcoma Bank at the Department of Pediatrics, University of Padova (Padova, Italy).

Reagents and antibodies
Anti-ß-catenin and anti-plakoglobin ({gamma}-catenin) monoclonal antibodies were purchased from BD Transduction Laboratories (Lexington, KY). Anti-actin IgM monoclonal antibody was obtained from Oncogene Research Products (San Diego, CA). Anti-{gamma}-tubulin was purchased from Sigma-Aldrich (St Louis, MO). Anti-HDCA1 rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit antibodies were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Bicinchoninic acid protein assay reagents and western blot chemiluminescence reagents were obtained from Pierce Chemical (Rockford, IL).

Protein extraction and western blot analysis
Cell lines were treated with either Trichostatin A (TSA) (Sigma-Aldrich) 200 ng/ml for 16 h or 5Aza-2-deoxycytidine (5AzadC) (Sigma-Aldrich) 2 µM for 72 h. Cells were washed twice in ice-cold 1x PBS and incubated on ice for 20 min with lysis buffer [20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 25 mM NaF, 25 mM glycerophosphate, 5 mM sodium orthovanadate, 5 mM EDTA, 5 mM EGTA, 1 mM sodium PPi (pH 7.0), 1 mM DTT, 1.5 mM MgCl2, 10% glycerol, 1 mM phenylmethyl–sulfonyl fluoride (PMSF), 20 µg/ml leupeptin and 20 µg/ml aprotinin] containing 1% Triton X-100. Cell lysates were clarified by centrifugation at 4°C at 14 000 g for 30 min. Protein concentration was determined by the bicinchoninic acid. Aliquots of cell lysates, diluted in 5x reducing Laemmli loading buffer [2% SDS, 100 mM DTT, 60 mM Tris (pH 6.8), 0.01% bromophenyl blue and 10% glycerol], were fractionated by 10% SDS–PAGE, electrotransferred to nitrocellulose membranes and visualized by using specific monoclonal antibodies with the SuperSignal West Pico Chemiluminescent Substrate (Pierce). Membranes were exposed to Hyperfilm (Amersham) and signals were quantified by using the NIH image analysis software.

Semi-quantitative RT–PCR analysis
Total RNA was extracted from cultured cells and tumor specimens using RNAzol-Bee reagent (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. From each sample 1 µg of total RNA was reverse-transcribed using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and random hexamers. The cDNAs were then amplified by PCR using primers specific for plakoglobin (5'-ATGGAGGTGATGAACCTGATGG-3' and 5'-TGGCATAGAACAGGACCGACTC-3') and for ß2-microglobulin, used as an internal control to standardize the amount of DNA in each sample, (5'-GTGGAGCATTCAGACTTGTCTTTCAGCA-3' and 5'-TTCATCCAATCCAAATGCGGCATCTTC-3'). Based on preliminary experiments, we identified the PCR exponential phase and selected 33 cycles for amplification of plakoglobin and 26 cycles for ß2-microglobulin. PCR conditions included initial denaturation at 94°C for 2 min followed by 94°C for 45 s, 61°C for 30 s, 72°C for 45 s, for plakoglobin, and by 94°C for 15 s, 60°C for 15 s, 72°C for 15 s, for ß2-microglobulin, and a final extension step at 72°C for 5 min, on a GeneAmp PCR 9600 System (ABI Applied Biosystems, Foster City, CA). PCR products were electrophoresed through 2% agarose gels and quantified by densitometric analysis of band intensity using Quantity One Quantitation Software (Bio-Rad, Milan, Italy).

Nuclear and cytoplasmic protein fractions
To obtain purified cytoplasmic and nuclear fractions, cells were processed according to the protocol described in ref. 26. Cells grown on Petri dishes were washed three times in ice-cold 1x PBS, frozen on dry ice and scraped into hypotonic lysis buffer (10 mM HEPES, 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 1 mM DTT and a cocktail of protease inhibitors) with 2% NP-40. Cell lysates were incubated on ice for 10 min, passed through a 18 gauge needle and then centrifuged for 2 min at 10 000 g. The supernatant was removed as cytoplasmic fraction and the pellets were resuspended in hypertonic buffer (20mM Hepes, 0.4M NaCl, 1mM EDTA, 1mM DTT and a cocktail of protease inhibitors) and triturated with a pestle. The nuclei were incubated for 30 min on ice and then microcentrifuged at 4°C for 5 min at 10 000 g. The supernatant containing nuclear extract was collected and aliquoted.

Analysis of plakoglobin gene hypermethylation by MSP and bisulfite DNA sequencing
The plakoglobin gene methylation was studied by methylation-specific PCR (MSP) assay as reported previously (27). Briefly, genomic DNA was isolated using a commercial kit (Qiagen, Hilden, Germany) and bisulfite modification was obtained using CpGenome DNA Modification Kit (Chemicon International, Temecula, CA). The modified DNA was subjected to PCR in separate reactions using the primer pairs specific for the methylated sequence, PG-M (5'-TAGTCGTCGTTCGATCGCGTC-3' and 5'-AACCGAATCGAAATCGAACCG-3'), and for the unmethylated sequence, PG-U (5'-TTGGAGTAGTTGTTGTTTGATTGTGTT-3' and 5'-CAAACCAAATCAAAATCAAACCAA-3') as described previously (28). The PCR mixtures contained 1x PCR buffer, 200 µM dNTPs, 2 mM MgCl2, 10 pmol of each primer, 2.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems) and 200 ng template in a final volume of 50 µl. Amplification was carried out after an initial enzyme activation step (5 min at 95°C) for 40 cycles (30 s at 95°C, 45 s annealing, 30 s at 72°C). Annealing temperatures of 62 and 60°C were used for PG-M and PG-U reactions, respectively. Bisulfite-modified lymphocyte DNA from healthy volunteers served as a positive control for the unmethylated allele. This DNA was methylated in vitro with Sss1 methyltransferase (New England Biolabs, Beverly, MA), subjected to bisulfite modification, and then used as a positive control for amplification of methylated alleles. PCR products were analyzed by gel electrophoresis and ethidium bromide staining on a 3.5% agarose gel. Results were confirmed by repeating the bisulfite reaction and MSP for all samples.

Bisulfite-modified DNA was amplified using Pan-S (5'-GGTTTGGTTGTTAGGTTTTT-3') and Pan-AS (5'-CACTCAATCACCTTAATAAACTACTCC-3') primers. This primer set does not cover any CpG sites within the primer sequences, as reported previously (39). PCR products obtained, were amplified by a second round of nested PCR using primers BD-S (5'-TTGAGTTGTTGTTTTGGTTAGAGTT-3') and BD-AS (5'-TAACTACTAAATATAAAACCTAACC-3') (39). PCR products were then sequenced using the ABI PRISM 377 DNA sequencer (Applied Biosystems) using BD-S and BD-AS primers.

Chromatin immunoprecipitation (ChIP) analysis
Cells grown in 100 mm dishes were treated with or without TSA (200 ng/ml) for 16 h. Before harvesting, the cells were treated with 1% formaldehyde for 10 min at room temperature to crosslink histones to DNA. Crosslinking was quenched by the addition of glycine at a final concentration of 0.125 M and 5 min incubation at room temperature. Cells were scraped with ice-cold PBS containing 0.5 mM PMSF and centrifuged for 10 min at 720 g a 4°C. ChIP assay was performed using ChIP Assay Kit (Active Motif, CA), according to the manufacturer's instructions. Briefly, the chromatin solutions were sonicated to reduce the DNA length to 200–1000 bp, pre-cleared with a salmon sperm DNA/protein A agarose slurry, and then immunocaptured using antiacetyl-H4 antibody (Upstate, Charlottesville, VA) overnight at 4°C with gentle rotation. Aliquots of each chromatin solution were saved for no-antibody controls. Immune complexes were then collected with a salmon sperm DNA/protein A agarose slurry and washed with the kit wash buffer. DNA was eluted twice with elution buffer containing 1% SDS and 0.05 M NaHCO3. Combined eluates were treated with 5 M NaCl to reverse the DNA/histone crosslinks and RNA was removed by RNase treatment overnight at 65°C. Samples were treated with Proteinase K at 42°C for 2 h to digest the proteins. DNA from each eluate was purified through DNA purification mini-columns and collected into microcentrifuge tubes. Specific promoter sequences from no-antibody control and immunoprecipitated samples were detected by PCR analysis using the following primer sets: 5'-AAATGAGAGGAAGGAGGT-3' and 5'-GGTTTGTGAAAGCACTCA-3' for P1, 5'-AAATTTCTAACCTTTAGCAG-3' and 5'-TGATTTTAGGCAAGTTACTT-3' for P2, and 5'-CATCCCTTCCATTGGTTT-3' and 5'-AAGGCAGCAACTCAGTAA-3' for P3.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Plakoglobin protein expression, cellular localization and semi-quantitative analysis of mRNA in RMS cell lines
We studied nine RMS cell lines including three ARMS carrying the translocation t(2;13) (RH4, RH28, RH30); one ARMS with translocation t(1;13) (RC2); one ARMS without translocation (RH18); four ERMS (RD, RH36, SMS-CTR, CCA); an undifferentiated sarcoma cell line (A-204) and a neuroblastoma cell line (LAN-5). Western blot analysis of lysates from log-phase growing cells revealed a consistent and homogeneous ß-catenin expression in all of the cell lines, whereas plakoglobin, detectable in the positive control cell line LAN-5 (29) and in the ERMS cell lines, was absent or barely detectable in ARMS cells (Figure 1A). Unexpectedly, RH18 cells demonstrated a high level of plakoglobin expression similar to ERMS cell lines. The A-204 cells were negative for plakoglobin expression, whereas ß-catenin was almost undetectable.


Figure 1
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  		Fig. 1 Differential expression and cellular localization of catenins in RMS cell lines. (A) Nine RMS cell lines, one ARMS with translocation t(1;13) (RC2), three ARMS with t(2;13) (RH4, RH28, RH30), one ARMS without translocation (RH18) and four ERMS (RD, RH36, SMS-CTR, CCA) were studied for plakoglobin and ß-catenin expression together with the undifferentiated sarcoma cell line A-204 and the neuroblastoma cell line LAN-5. Cells lysates were analyzed by western blot using anti-ß-catenin and anti-plakoglobin ({gamma}-catenin) specific antibodies. (B) Plakoglobin mRNA expression in RMS cell lines was determined by semi-quantitative RT–PCR where ß2-microglobulin transcript was co-amplified. Amplification of plakoglobin and ß2-microglobulin cDNAs originated single bands of 700 and 220 bp, respectively. In the PCR-negative control (H2O) no RNA was added to the reactions, whereas LAN-5 cells represented the positive control. (C) The signal intensity of plakoglobin mRNA of all tumor cell lines, normalized to ß2-microglobulin, was quantified and expressed as arbitrary units as described in Materials and methods. (D) Cytoplasmic (C) and nuclear (N) plakoglobin and ß-catenin were studied by differential cell lysis, followed by immunoblotting of protein lysates. Cell fractions of three ARMS (RH4, RH28, RH30) and three ERMS (RD, RH36, SMS-CTR) cell lines were obtained and 30 µg of protein lysates from each fraction were analyzed. To verify the integrity of the fractions, blots were reprobed for actin and HDAC1 (histone deacetylase), as markers of the cytoplasmic and nuclear compartments, respectively.

 
A semi-quantitative RT–PCR assay was established to assess the expression of plakoglobin-specific mRNA in the tumor cell lines described above (Figure 1B). Consistent with protein expression shown in Figure 1A, RT–PCR originated a single band of 700 bp that was expressed at lower levels in ARMS compared with ERMS cell lines (Figure 1C). A similar result was observed in the A-204 undifferentiated sarcoma cell line, whereas the plakoglobin mRNA level in RH18 cells was comparable with ERMS, underlining the peculiarity of this ARMS cell line.

ß-Catenin-mediated signaling depends on the balance among its membrane, cytoplasmic and nuclear pools; therefore, we analyzed the subcellular distribution of plakoglobin and ß-catenin in ARMS (RH4, RH28, RH30) and ERMS cell lines (RD, RH36, SMS-CTR). As shown in Figure 1D, ß-catenin was distributed in comparable amounts between the cytoplasm and the nucleus in all of the RMS cell lines tested (with the exception of RH28 cells, where nuclear ß-catenin was significantly lower), whereas in ERMS cells plakoglobin, although detected in both compartments, was mostly localized in the cytoplasmic fraction. As expected, we were unable to detect plakoglobin both in the cytoplasm and in the nucleus of the ARMS cell lines RH4, RH28, RH30 (Figure 1D).

Restoration of plakoglobin expression in ARMS cells treated with the demethylating agent 5AzadC and the HDAC inhibitor TSA
Methylation and acetylation are well-known mechanisms of gene inhibition and activation, respectively (30,31). To assess whether the loss of plakoglobin expression in ARMS was due to methylation, ARMS RH30 cells were treated with different concentrations of the demethylating cytosine analog 5AzadC for 72 h (Figure 2A). Western blot analysis demonstrated partial restoration of plakoglobin expression in 5AzadC-treated RH30 cells in a dose-dependent manner, and this effect appeared specific as the expression of ß-catenin remained unaffected under the same experimental conditions (Figure 2A). In addition, we examined plakoglobin expression in RH30 cells cultured in the presence of the HDAC inhibitor TSA. We observed an upregulation of the protein level that reached the highest level after 16 h. Expression levels decreased when cells were treated for longer time intervals (24 h) possibly because of TSA cytotoxicity (Figure 2B, see ß-catenin steady-state in the lower panel). To confirm these results, the ARMS cell lines RH4, RH30 and RH18, and ERMS RD cells were treated with 5AzadC and TSA alone or in combination, and plakoglobin gene expression was assessed at both mRNA and protein levels. As shown in Figure 2C, combination treatment with 5AzadC and TSA synergistically augmented mRNA and protein expression in the PAX3–FKHR-positive ARMS cells RH4 and RH30, although the effects were quantitatively different in the two cell lines. In contrast, plakoglobin expression was minimally affected in the ERMS RD and in the PAX3–FKHR-negative ARMS RH18 cells, when the two compounds were used alone or in combination. These results support the hypothesis that transcription of plakoglobin gene in RMS cells is regulated by histone acetylation and CpG methylation of its promoter region.


Figure 2
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  		Fig. 2 5AzadC and TSA treatments restore plakoglobin expression in ARMS cells. (A) To determine the role of hypermethylation in plakoglobin expression, RH30 cells were treated with increasing concentrations (0–10 µM) of 5AzadC for 72 h. Total protein extracts containing equal amounts of proteins of treated/untreated cells were analyzed by western blot using antibodies specific for plakoglobin and ß-catenin. RD cell lysate was included as positive control for plakoglobin expression. (B) Time course analysis of plakoglobin protein expression in RH30 cells treated with (+) or without (–) TSA 200 ng/ml. RD cells were used as positive controls. (C) mRNA and protein expression in RMS cell lines. Bar graphs represent plakoglobin expression after treatment with 5AzadC 2 µM for 72 h and with TSA 200 ng/ml for 16 h, alone or in combination, reported as optical density units (mRNA) or as folds of plakoglobin expression levels relative to untreated cells (protein).

 
Analysis of methylation and acetylation status of plakoglobin promoter in RMS cell lines and tumor biopsies
To further investigate plakoglobin expression regulation, genomic DNA of RMS cell lines was extracted, modified by bisulfite treatment and subjected to PCR with primer pairs PG-U and PG-M specific, for unmethylated and methylated promoter-associated CpG islands of plakoglobin gene, respectively. As shown in Figure 3A, RD cells, which endogenously expressed plakoglobin and were 5AzadC unresponsive, contained only unmethylated alleles, whereas plakoglobin-negative and 5AzadC-responsive RH4 and RH30 cells displayed both methylated alleles or coexistence of methylated and unmethylated alleles, respectively. However, the PAX3–FKHR-negative ARMS RH18 cells, which constitutively expressed plakoglobin and were 5AzadC unresponsive, showed a methylated and an unmethylated allele.


Figure 3
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  		Fig. 3 Methylation and acetylation status of plakoglobin gene in RMS cell lines. (A) Methylation of plakoglobin gene was characterized in plakoglobin-positive and -negative RMS cell lines using MSP. Genomic DNA of four cell lines (RH4, RH18, RH30, RD) was extracted, modified by bisulfite treatment and subjected to PCR with primer pairs PG-U (UM) and PG-M (M) specific for unmethylated and methylated plakoglobin alleles, respectively. PCR products were analyzed by 3.5% agarose gel electrophoresis. Band size of plakoglobin allele(s) amplicons is indicated. Normal lymphocyte DNA and normal lymphocyte DNA treated with Sss I methyl transferase were used as unmethylated (ctr UM) and methylated gene promoter (ctr M) controls, respectively. (B) Bisulfite mapping of CpG islands in the plakoglobin promoter and gene was carried out on PCR products containing nine CpG sites. All cytosines in the nine CpG islands analyzed are numbered on top of each panel, starting from the transcription start site based on the sequence and GeneBank data (accession no. AF233882). All of the CpG islands in RH4 cells were methylated (the presence of an unmodified ‘C’). In RD cells all CpG islands were non-methylated (‘C’ were converted into ‘T’). RH30 cells showed all CpG islands partially methylated (‘C’ covered with ‘T’). In RH18 all of the CpG islands were unmethylated with the exception of CpG island +54 that was methylated. (C) Acetylation status of histone H4 in plakoglobin promoter in RMS cell lines treated with or without TSA 200 ng/ml for 16 h. Results of a representative ChIP assay carried out at least in triplicate with the antiacetyl-H4 antibody followed by PCR with the indicated primer sets are shown. ProG represents no-antibody control. The amount of DNA used was monitored by a PCR that amplified DNA prepared from the input solution. Ladder 50 (M) was used as DNA size marker. The presence of PCR products demonstrates the association of acetylated H4 histone with a specific promoter region.

 
To assess the methylation status in more detail, a promoter region of plakoglobin gene that allows the study of 9 CpG islands by direct sequencing was amplified by a two-step PCR. As demonstrated by sequence analysis of the amplicons (Figure 3B), the RH4 cell line which showed two methylated plakoglobin alleles, was heavily methylated at all 9 CpG sites tested. In contrast, RD cells that harbor only unmethylated alleles (Figure 3A), completely lacked methylated CpG sites (Figure 3B). RH30 cells showed the presence of both methylated and unmethylated CpG islands. RH18 possessed all of the CpG islands unmethylated except CpG +54 that was methylated. In addition, the sequence analysis of cloned PCR products that allows to study a higher number of CpG islands, showed that CpG island +33, included in the primer sequence used for MSP assay, was methylated in 1/6 RH18 clones (data not shown).

To investigate histone deacetylation as an epigenetic mechanism contributing to gene silencing, we determined the binding of hyperacetylated histones to plakoglobin promoter in TSA-treated RMS cells by ChIP assay. Inhibition of HDAC by TSA causes local hyperacetylation of histones that can be immunoprecipitated; thus, allowing purification of bound DNA sequences. Acetylated histone H4 was immunoadsorbed with a specific monoclonal antibody from TSA-treated and untreated RMS cells, and co-precipitated DNA fragments were assessed by PCR, using three specific primer pairs spanning the P1 (–945–745), P2 (788–646) and P3 (–34+14) 5' flanking promoter regions of the plakoglobin gene (GeneBank accession no. AJ 276892) (32). As shown in Figure 3C, co-precipitation of all three plakoglobin 5' flanking promoter regions (P1–P3) in RH4 cells, which possess both methylated alleles, was obtained only after TSA treatment. When the analysis was extended to RD cells and to the two cell lines displaying the coexistence of methylated and unmethylated alleles (RH30 and RH18), no significant difference was seen between TSA-treated and untreated cells, suggesting that deacetylation of H4 histone is a particularly relevant phenomenon in RH4.

Finally, we studied 11 RMS tumor biopsies: 4 PAX3–FKHR-positive and 2 PAX3–FKHR-negative ARMS and 5 ERMS (Figure 4A). Results in Figure 4A and B confirmed that ß-catenin was expressed at high levels in all specimens, whereas plakoglobin, detected in all of the ERMS biopsies, was barely detectable in the ARMS biopsies. We investigated the methylation status of the plakoglobin gene promoter in 7 of the 11 biopsies by MSP analysis. The hemi-methylated pattern, was predominant irrespective of the histological subtype (Figure 4C) with the exception of sample no. 6 (ARMS) that showed a higher signal intensity for the methylated allele and sample no. 8 (ERMS) where the unmethylated allele was more evident.


Figure 4
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  		Fig. 4 Plakoglobin and ß-catenin expression and plakoglobin promoter status in tumor samples. (A) Plakoglobin and ß-catenin expression were studied in four PAX3–FKHR positive ARMS [ARMS t(2;13)], two PAX3–FKHR negative ARMS (ARMS no. t) and five ERMS (ERMS). Tissue was homogenized in lysis buffer and 30 µg of protein lysates were analyzed by reducing 12% SDS–PAGE. Blots were probed with anti-plakoglobin, anti-ß-catenin and anti-{gamma}-tubulin antibodies. (B) Plakoglobin expression, normalized to {gamma}-tubulin, was quantified using image analysis software (NIH image) and expressed as folds of control (RD). RH30 cell line was used as negative control. (C) MSP of plakoglobin promoter in tumor biopsies. PCR products for the methylated (M) and unmethylated (UM) plakoglobin promoter in seven RMS tumor samples are illustrated. Molecular weight marker (MWM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Plakoglobin overexpression is known to suppress cell proliferation and tumorigenicity, consistent with its ability to act as a tumor suppressor (17,33). Low or absent plakoglobin expression in cancer implies changes in intercellular junctions, and may thus predispose to tumor invasion and metastasis. Indeed, downregulation of plakoglobin is frequently observed in thyroid, lung and bladder cancer (34–36), and the reduced expression of plakoglobin has been correlated with poor outcome in patients with kidney cancer (37) and neuroblastoma (29), as well as with poor tumor differentiation and increased lymph node metastases in primary lung cancer (33,38). However, the mechanisms that contribute to inactivation of plakoglobin expression in malignant cells have not been fully investigated, although CpG island methylation has been proposed as one of the mechanisms contributing to plakoglobin inhibition in cancer cells (39).

Our study was designed to determine the expression and cellular localization of catenins in RMS cells, and to assess whether any relationship exists between the expression of catenins and the RMS histological subtype.

Results demonstrated that plakoglobin is abundantly expressed in ERMS cell lines and tumor samples, but is absent or expressed at very low levels in the alveolar subtype (ARMS) that is more aggressive and has a higher tendency to metastasize. In contrast, we found ß-catenin consistently expressed at high levels in all RMS cell lines and tumor samples.

Owing to the fact that the cytoplasmic accumulation of ß-catenin in tumor cells increases its nuclear localization and the transcriptional activity of the Lef-1/Tcf transcription factor family members, causing the upregulation of genes involved in tumor progression and metastasis (40,41), we determined the subcellular distribution of catenins. Plakoglobin and ß-catenin localized both in the cytoplasmic and nuclear fractions. This is partly in contrast with a recent paper reporting that catenin expression was restricted to cell membrane and cytoplasm in RMS cells (42), but may be explained by the different experimental approach used.

Semi-quantitative RT–PCR analysis demonstrated that plakoglobin mRNA was expressed at lower levels in ARMS compared with ERMS cell lines (Figure 1B and C), with the exception of RH18 that, originally described as an alveolar RMS cell line lacking both PAX3–FKHR and MyoD1 expression, may probably represent an ERMS cell line (43) or, alternatively, an ‘atypical’ ARMS cell line.

DNA promoter methylation has gained considerable importance as an epigenetic mechanism for gene silencing that contributes to tumor initiation and progression when tumor suppressor genes are involved. Although little is known about gene methylation in childhood cancer, methylation and silencing of HIC1 and RASSF1A tumor suppressor genes, or increased expression of DNA methyltransferase have been reported in pediatric tumors including RMS (19,18). Histone methylation and deacetylation are also associated with transcriptionally inactive chromatin. Acetylation of selected aminoacids is required for chromatin relaxation and gene expression; whereas histone methylation correlates with DNA promoter methylation and gene silencing. However, dense CpG island methylation of flanking promoter regions is dominant for the stable maintenance of a silent status (44), as treatment with the DNA methyltransferase inhibitor 5AzadC restores gene expression; whereas the HDAC inhibitor TSA has only very limited effect unless used in combination with 5AzadC (45,46). Furthermore, in the absence of dense CpG island methylation, histone deacetylase expression can be sufficient to inhibit gene expression, as recently shown for plakoglobin in fibrosarcoma HT1080 cells (32), and HDAC inhibitors exert antitumor activity in human RMS in vitro and in vivo (47,48).

In accordance with this scenario, we observed restoration of plakoglobin expression in PAX3–FKHR-expressing RH30 cells following treatment with either the demethylating agent 5AzadC or the deacetylase inhibitor TSA (Figure 2C). Owing to the fact that TSA treatment increased plakoglobin expression, although ChIP assay did not show any difference between TSA-treated and untreated cells, we cannot exclude that other histones (i.e. H3) or a partial acetylation of histone H4 may be involved.

Interestingly, the two single agents 5AzadC and TSA had a less pronounced effect on plakoglobin protein expression in RH4 cells that possess both hypermethylated alleles, but were highly effective when used in combination, suggesting a marked increase of the acetylation/methylation ratio achieved synergistically by the two agents in a mechanism involving both histone methylation/acetylation status and methylated plakoglobin promoter regions, as previously reported (44). In contrast, treatment with 5AzadC and/or TSA had only a limited effect on RMS cells that constitutively expressed plakoglobin, such as RD and RH18 cells, independent of the histological subtype.

From further studies based on MSP assay and sequencing, lack of unmethylated allele as in RH4 cells would suggest that gene inactivation might be the consequence of the methylation of both alleles, possibly associated with a deacetylation of histone H4 that may increase plakoglobin gene silencing. However, we cannot exclude that the presence of one methylated allele, and loss of the other may also occur.

The PAX3–FKHR positive RH30 cell line showed a loss of plakoglobin expression despite the presence of methylated and unmethylated alleles. DNA sequencing of a promoter region that enabled us to study 9 CpG islands confirmed these findings. In this case, we may hypothesize that inactivation of the unmethylated allele might be achieved through different mechanisms such as nonsense or frame-shift mutations, or deletion. In addition we cannot rule out uniparental isodisomy. This is also suggested by the observation that the PAX3–FKHR negative RH18 cell line harboring both unmethylated and methylated alleles does express plakoglobin, although the methylation status of plakoglobin gene promoter region analyzed by sequencing differs from RH30 cells.

The RD cell line did not show either methylation or acetylation of the plakoglobin promoter in agreement with the expression profile.

Finally, in the tumor samples we found both unmethylated and methylated alleles as in some RMS cell lines (Figures 4C and 3A). Interestingly, cases no. 6 (ARMS) and no. 8 (ERMS) showed a higher intensity of the methylated and unmethylated alleles, respectively, in agreement with the plakoglobin protein expression. In other tumors though, plakoglobin expression was very low despite the presence of both alleles similar to the case of RH30 cell line. In this regard, we need to consider that mechanisms other than selected promoter methylation may influence plakoglobin expression in vivo. In addition, the heterogeneity of tumor biopsies, due to the presence of some normal cells intermixed with tumor cells, may also influence the results of the MSP assay making it more difficult to fully appreciate the methylation status in vivo.

Given the relationships and interactions between catenins and cadherins, their role in proliferation and transformation, and the recent findings suggesting that impairment of cadherin-mediated adhesion might contribute to the rapid growth and metastatic properties of RMS (42), our results highlight a further differential feature that may contribute to the more aggressive behavior of ARMS compared with ERMS. The availability of drugs that can modify the methylation and acetylation status of the cell should encourage their preclinical evaluation as novel therapeutic agents in RMS.


   Acknowledgments
 
This research was supported by Fondazione Cittá della Speranza; by AIRC (Associazione Italiana per la Ricerca sul Cancro) and by MIUR (Ministero Istruzione Universitá e Ricerca). Paolo Bonvini is supported by a grant from Istituto Oncologico Veneto (IOV). We are grateful to E. Tosato for technical assistance and to Dr Martina Boscaro (Education and Communication Office— IOV) for help in editing the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wexler L.H. and Helman L.J. (1997) Rhabdomyosarcoma and the undifferentiated sarcoma. In Pizzo P.A. and Poplack P.G. (Eds.). Principles and Practice of Pediatric Oncology (Raven-Lippincott, Philadelphia) pp. 799–829.
  2. Newton W.A.J., Gehan E.A., Webber B.L., et al. (1995) Classification of rhabdomyosarcomas and related sarcomas. Pathologic aspects and proposal for a new classification—an Intergroup Rhabdomyosarcoma Study. Cancer 76:1073–1085.[CrossRef][ISI][Medline]
  3. Barr F.G. (1997) Molecular genetics and pathogenesis of rhabdomyosarcoma. J. Pediatr. Hematol. Oncol. 19:483–491.[CrossRef][ISI][Medline]
  4. Davis R.J., D'Cruz C.M., Lovell M.A., Biegel J.A., Barr F.G. (1994) Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res. 54:2869–2872.[Abstract/Free Full Text]
  5. Fredericks W.J., Galili N., Mukhopadhyay S., Rovera G., Bennicelli J., Barr F.G., Rauscher F.J. III. (1995) The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol. Cell. Biol. 15:1522–1535.[Abstract]
  6. Bennicelli J.L., Edwards R.H., Barr F.G. (1996) Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma. Proc. Natl Acad. Sci. USA 93:5455–5459.[Abstract/Free Full Text]
  7. Ginsberg J.P., Davis R.J., Bennicelli J.L., Nauta L.E., Barr F.G. (1998) Up-regulation of MET but not neural cell adhesion molecule expression by the PAX3–FKHR fusion protein in alveolar rhabdomyosarcoma. Cancer Res. 58:3542–3546.[Abstract/Free Full Text]
  8. Ferracini R., Olivero M., Di Renzo M.F., Martano M., De Giovanni C., Nanni P., Basso G., Scotlandi K., Lollini P.L., Comoglio P.M. (1996) Retrogenic expression of the MET proto-oncogene correlates with the invasive phenotype of human rhabdomyosarcomas. Oncogene 12:1697–1705.[ISI][Medline]
  9. Ricci C., Landuzzi L., Rossi L., et al. (2000) Expression of HER/erbB family of receptor tyrosine kinases and induction of differentiation by glial growth factor 2 in human rhabdomyosarcoma cells. Int. J. Cancer 87:29–36.[CrossRef][ISI][Medline]
  10. Morin P.J. (1999) Beta-catenin signaling and cancer. Bioessays 21:1021–1030.[CrossRef][ISI][Medline]
  11. Papkoff J. (1997) Regulation of complexed and free catenin pools by distinct mechanisms—Differential effects of Wnt-1 and v-Src. J. Biol. Chem. 272:4536–4543.[Abstract/Free Full Text]
  12. BenZeev A. and Geiger B. (1998) Differential molecular interactions of beta-catenin and plakoglobin in adhesion, signaling and cancer. Curr. Opin. Cell Biol. 10:629–639.[Medline]
  13. Polakis P. (1999) The oncogenic activation of beta-catenin. Curr. Opin. Genet. Dev. 9:15–21.[CrossRef][ISI][Medline]
  14. Polakis P. (2000) Wnt signaling and cancer. Genes Dev. 14:1837–1851.[Free Full Text]
  15. Sadot E., Simcha I., Iwai K., Ciechanover A., Geiger B., BenZe'ev A. (2000) Differential interaction of plakoglobin and beta-catenin with the ubiquitin-proteasome system. Oncogene 19:1992–2001.[CrossRef][ISI][Medline]
  16. Aberle H., Bierkamp C., Torchard D., et al. (1995) The human plakoglobin gene localizes on chromosome 17q21 and is subjected to loss of heterozygosity in breast and ovarian cancers. Proc. Natl Acad. Sci. USA 92:6384–6388.[Abstract/Free Full Text]
  17. Simcha I., Geiger B., YehudaLevenberg S., Salomon D., BenZeev A. (1996) Suppression of tumorigenicity by plakoglobin: an augmenting effect of N-cadherin. J. Cell Biol. 133:199–209.[Abstract/Free Full Text]
  18. Harada K., Toyooka S., Maitra A., et al. (2002) Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines. Oncogene 21:4345–4349.[CrossRef][ISI][Medline]
  19. Rathi A., Virmani A.K., Harada K., Timmons C.F., Miyajima K., Hay R.J., Mastrangelo D., Maitra A., Tomlinson G.E., Gazdar A.F. (2003) Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms. Cli. Cancer Res. 9:3674–3678.
  20. Litt M.D., Simpson M., Gaszner M., Allis C.D., Felsenfeld G. (2001) Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 293:2453–2455.[Abstract/Free Full Text]
  21. Nakayama J., Rice J.C., Strahl B.D., Allis C.D., Grewal S.I. (2001) Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292:110–113.[Abstract/Free Full Text]
  22. Tazi J. and Bird A. (1990) Alternative chromatin structure at CpG islands. Cell 60:909–920.[CrossRef][ISI][Medline]
  23. Strahl B.D., Ohba R., Cook R.G., Allis C.D. (1999) Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc. Natl Acad. Sci. USA 96:14967–14972.[Abstract/Free Full Text]
  24. Nan X., Ng H.H., Johnson C.A., Laherty C.D., Turner B.M., Eisenman R.N., Bird A. (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393:386–389.[CrossRef][Medline]
  25. Jones P.L., Veenstra G.J., Wade P.A., Vermaak D., Kass S.U., Landsberger N., Strouboulis J., Wolffe A.P. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19:187–191.[CrossRef][ISI][Medline]
  26. Rosette C. and Karin M. (1995) Cytoskeletal control of gene expression: depolymerization of microtubules activates NF{kappa}B. J. Cell. Biol. 128:1111–1119.[Abstract/Free Full Text]
  27. Herman J.G., Graff J.R., Myohanen S., Nelkin B.D., Baylin S.B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA. 93:9821–9826.[Abstract/Free Full Text]
  28. Potter E., Braun S., Lehmann U., Brabant G. (2001) Molecular cloning of a functional promoter of the human plakoglobin gene. Eur. J. Endocrinol. 145:625–633.[Abstract]
  29. Amitay R., Nass D., Meitar D., Goldberg I., Davidson B., Trakhtenbrot L., Brok Simoni F., Ben Ze'ev A., Rechavi G., Kaufmann Y. (2001) Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma. Am. J. Path. 159:43–49.[Abstract/Free Full Text]
  30. Santi D.V., Norment A., Garrett C.E. (1984) Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc. Natl Acad. Sci. USA. 81:6993–6997.[Abstract/Free Full Text]
  31. Walia H., Chen H.Y., Sun J.M., Holth L.T., Davie J.R. (1998) Histone acetylation is required to maintain the unfolded nucleosome structure associated with transcribing DNA. J. Biol. Chem. 273:14516–14522.[Abstract/Free Full Text]
  32. Shim J.S., Kim D.H., Kwon H.J. (2004) Plakoglobin is a new target gene of histone deacetylase in human fibrosarcoma HT1080 cells. Oncogene 23:1704–11.[CrossRef][ISI][Medline]
  33. Winn R.A., Bremnes R.M., Bemis L., Franklin W.A., Miller Y.E., Cool C., Heasley L.E. (2002) Gamma-catenin expression is reduced or absent in a subset of human lung cancers and re-expression inhibits transformed cell growth. Oncogene 21:7497–7506.[CrossRef][ISI][Medline]
  34. Cerrato A., Fulciniti F., Avallone A., Benincasa G., Palombini L., Grieco M. (1998) Beta- and gamma-catenin expression in thyroid carcinomas. J. Pathol. 185:267–272.[CrossRef][ISI][Medline]
  35. Pirinen R.T., Hirvikoski P., Johansson R.T., Hollmen S., Kosma V.M. (2001) Reduced expression of alpha-catenin, beta-catenin, and gamma-catenin is associated with high cell proliferative activity and poor differentiation in non-small cell lung cancer. J. Clin. Pathol. 54:391–395.[Abstract/Free Full Text]
  36. Syrigos K.N., Harrington K., Waxman J., Krausz T., Pignatelli M. (1998) Altered gamma-catenin expression correlates with poor survival in patients with bladder cancer. J. Urol. 160:1889–1893.[CrossRef][ISI][Medline]
  37. Buchner A., Oberneder R., Riesenberg R., Keiditsch E., Hofstetter A. (1998) Expression of plakoglobin in renal cell carcinoma. Anticancer Res. 18:4231–4235.[ISI][Medline]
  38. Pantel K., Passlick B., Vogt J., Stosiek P., Angstwurm M., SeenHibler R., Haussinger K., Thetter O., Izbicki J.R., Riethmuller G. (1998) Reduced expression of plakoglobin indicates an unfavorable prognosis in subsets of patients with non-small-cell lung cancer. J. Clin. Oncol. 16:1407–1413.[Abstract/Free Full Text]
  39. Breault J.E., Shiina H., Igawa M., et al. (2005) Methylation of the gamma-catenin gene is associated with poor prognosis of renal cell carcinoma. Clin. Cancer Res. 11:557–564.[Abstract/Free Full Text]
  40. He T.C., Sparks A.B., Rago C., Hermeking H., Zawel L., DaCosta L.T., Morin P.J., Vogelstein B., Kinzler K.W. (1998) Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512.[Abstract/Free Full Text]
  41. Shtutman M., Zhurinsky J., Simcha I., Albanese C., D'Amico M., Pestell R., Ben Ze'ev A. (1999) The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl Acad. Sci. USA 96:5522–5527.[Abstract/Free Full Text]
  42. Charrasse S., Comunale F., Gilbert E., Delattre O., Gauthier-Rouviere C. (2004) Variation in cadherins and catenins expression is linked to both proliferation and transformation of rhabdomyosarcoma. Oncogene 23:2420–30.[CrossRef][ISI][Medline]
  43. Jankowski K., Kucia M., Wysoczynski M., et al. (2003) Both hepatocyte growth factor (HGF) and stromal-derived factor-1 regulate the metastatic behavior of human rhabdomyosarcoma cells, but only HGF enhances their resistance to radiochemotherapy. Cancer Res. 63:7926–7935.[Abstract/Free Full Text]
  44. Kondo Y., Shen L., Issa J.P. (2003) Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer. Mol. Cell. Biol. 23:206–215.[Abstract/Free Full Text]
  45. Cameron E.E., Bachman K.E., Myohanen S., Herman J.G., Baylin S.B. (1999) Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21:103–107.[CrossRef][ISI][Medline]
  46. Eden S., Hashimshony T., Keshet I., Cedar H., Thorne A.W. (1998) DNA methylation models histone acetylation. Nature 394:842.[CrossRef][Medline]
  47. Kutko M.C., Glick R.D., Butler L.M., Coffey D.C., Rifkind R.A., Marks P.A., Richon V.M., LaQuaglia M.P. (2003) Histone deacetylase inhibitors induce growth suppression and cell death in human rhabdomyosarcoma in vitro. Clin. Cancer Res. 9:5749–5755.[Abstract/Free Full Text]
  48. Jaboin J., Wild J., Hamidi H., Khanna C., Kim C.J., Robey R., Bates S.E., Thiele C.J. (2002) MS-27–275, an inhibitor of histone deacetylase, has marked in vitro and in vivo antitumor activity against pediatric solid tumors. Cancer Res. 62:6108–6115.[Abstract/Free Full Text]
Received September 27, 2005; revised November 12, 2005; accepted March 3, 2006.


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