Methylation-Associated Silencing of the<i>Nuclear Receptor 1I2</i>Gene in Advanced-Type Neuroblastomas, Identified by Bacterial Artificial Chromosome Array-Based Methylated CpG Island Amplification

Misawa, Akiko; Inoue, Jun; Sugino, Yuriko; Hosoi, Hajime; Sugimoto, Tohru; Hosoda, Fumie; Ohki, Misao; Imoto, Issei; Inazawa, Johji

doi:10.1158/0008-5472.can-05-1073

Cited by 64 publications

(74 citation statements)

References 27 publications

(34 reference statements)

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“…To determine whether hypermethylation of CpG sites is associated with transcriptional silencing of the PRTFDC1 gene, we tested the promoter activity of this CpG island in OSCC cells (NA and HSC-2) using a 891-bp fragment (Fragment 1) covering the entire CpG island and 482-bp (Fragment 2) and 409-bp (Fragment 3) fragments covering uncommonly and commonly methylated regions, respectively, in cell lines without expression of PRTFDC1 (Figures 3a and b). We observed a remarkable increase in transcriptional activity of the reporter plasmid containing Fragments 1 and 3 compared with the mock reporter plasmid and reporter plasmid containing Fragment 2 in the plasmidcontaining cell lines (Figure 3b), suggesting that the CpG island around exon 1 of PRTFDC1, especially the region commonly methylated in PRTFDC1-nonexpressing cell lines, contains promoter activity, although few studies, including ours, have shown that promoter activity can be observed in fragments, especially CpG islands, not containing a 5 0 sequence around transcriptional start sites (Kolb 2003;Nakagawachi et al, 2003;Sonoda et al, 2004;Misawa et al, 2005).…”

Section: Identification Of Gene(s) Involved In the Homozygous Deletiomentioning

confidence: 83%

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

et al. 2007

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Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of oral squamouscell carcinoma (OSCC), cell lines for genomic copynumber aberrations by array-CGH using our in-house arrays, we identified a 3-Mb homozygous deletion at 10p12 in 1 of 18 cell lines (5.6%). Among seven genes located within this region, expression of PRTFDC1 mRNA was not detected in 50% (9/18) or decreased in 5.6% (1/18) of OSCC cell lines, but detected in normal oral epithelia and restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2 0 -deoxycytidine. Among 17 cell lines without a homozygous deletion, the hypermethylation of the PRTFDC1 CpG island, which showed promoter activity, was observed in all nine cell lines with no or reduced PRTFDC1 expression (52.9%). Methylation of this CpG island was also observed in primary OSCC tissues (8/47, 17.0%). In addition, restoration of PRTFDC1 in OSCC cells lacking its expression inhibited cell growth in colony-formation assays, whereas knockdown of PRTFDC1 expression in OSCC cells expressing the gene promoted cell growth. These results suggest that epigenetic silencing of PRTFDC1 by hypermethylation of the CpG island leads to a loss of PRTFDC1 function, which might be involved in squamous cell oral carcinogenesis.

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Section: Identification Of Gene(s) Involved In the Homozygous Deletiomentioning

confidence: 83%

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

et al. 2007

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“…BAMCA BAMCA were performed as described elsewhere (Misawa et al, 2005). Test DNA from each of ESCC cell lines was digested first with a methylation-sensitive SmaI and then with methylation-insensitive XmaI.…”

Section: Methodsmentioning

confidence: 99%

“…To identify epigenetic targets in ESCC, we chose an approach using BAMCA for initial screening of ESCC cell lines for aberrantly hypermethylated sequences (Misawa et al, 2005). A summary of our strategy and partial results is provided in Figure 1a and Supplementary Table S1.…”

Section: Methylation Analysis Of Escc Cell Lines By Bamcamentioning

confidence: 99%

Frequent methylation-associated silencing of a candidate tumor-suppressor, CRABP1, in esophageal squamous-cell carcinoma

et al. 2007

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Epigenetic alterations and the resulting inactivation of tumor suppressor genes often contribute to the development of various cancers. To identify novel candidates that may be silenced by aberrant methylation in esophageal squamous-cell carcinoma (ESCC), we analysed ESCC cell lines by a recently developed method known as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA), and selected candidates through BAMCA-assisted strategy. In the course of this program, we identified frequent CpG methylationdependent silencing of the gene encoding cellular retinoic acid binding protein 1 (CRABP1) in our panel of ESCC cell lines. Expression of CRABP1 mRNA was restored in gene-silenced ESCC cells after treatment with 5-aza 2 0 -deoxycytidine. The DNA methylation status of the CRABP1 CpG island with clear promoter activity correlated inversely with expression of this gene. CpG methylation of CRABP1 was frequently observed in primary ESCC tissues as well. Restoration of CRABP1 expression in ESCC cells lacking the protein reduced cell growth by inducing arrest at G 0 -G 1 , whereas knockdown of the gene in cells expressing CRABP1 promoted cell growth. Among 113 primary ESCC tumors, the absence of immunoreactive CRABP1 was significantly associated with de-differentiation of cancer cells and with distant lymph-node metastases in the patients. These results indicate that CRABP1 appears to have a tumorsuppressor function in esophageal epithelium, and its epigenetic silencing may play a pivotal role during esophageal carcinogenesis. Its expression status in biopsies or resected tumors might serve as an index for identifying ESCC patients for whom combined therapeutic modalities would be recommended.

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“…For colony-formation assays, pCMV-Tag3-RGC32, or the empty vector (pCMVTag3-Mock), was transiently transfected into cells as described elsewhere (Imoto et al, 2003;Misawa et al, 2005). Expression of RGC32 protein in transfected cells was confirmed 48 h after transfection by immunoblotting with anti-Myc antibody (Cell Signaling Technology, Beverly, MA, USA) as described below.…”

Section: Chip Assaymentioning

confidence: 99%

RGC32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in G2/M arrest

et al. 2006

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To identify target genes for the hemizygous deletions of chromosome 13 that are recurrently observed in malignant gliomas, we performed genome-wide DNA copy-number analysis using array-based comparative genomic hybridization and gene expression analysis using an oligonucleotide-array. The response gene to complement 32 (RGC32) at 13q14.11 was identified as a deletion target, and its expression was frequently silenced in glioma cell lines compared with normal brain. Levels of RGC32 mRNA tended to decrease toward higher grades of primary astrocytomas, especially in tumors with mutations of p53. Expression of RGC32 mRNA was dramatically increased by exogenous p53 in a p53-mutant glioma cell line, and also by endogenous p53 in response to DNA damage in p53 +/+ colon-cancer cells, but not in isogenic p53 À/À cells. Chromatin immunoprecipitation and reporter assays demonstrated binding of endogenous p53 protein to the promoter region of the RGC32 gene, implying p53-dependent transcriptional activity. Transiently and stably overexpressed RGC32 suppressed the growth of glioma cells, probably owing to induction of G2/M arrest. Immunocytochemical analysis revealed a concentration of RGC32 protein at the centrosome during mitosis. RGC32 formed a protein complex with polo-like kinase 1 and was phosphorylated in vitro. These observations implied a novel mechanism by which p53 might negatively regulate cell-cycle progression by way of this newly identified transcriptional target. Our results provide the first evidence that RGC32 might be a possible tumorsuppressor for glioma, that it is directly induced by p53, and that it mediates the arrest of mitotic progression.

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Methylation-Associated Silencing of theNuclear Receptor 1I2Gene in Advanced-Type Neuroblastomas, Identified by Bacterial Artificial Chromosome Array-Based Methylated CpG Island Amplification

Cited by 64 publications

References 27 publications

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

Frequent methylation-associated silencing of a candidate tumor-suppressor, CRABP1, in esophageal squamous-cell carcinoma

RGC32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in G2/M arrest

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