RASSF1A, BLU, NORE1A, PTEN and MGMT Expression and Promoter Methylation in Gliomas and Glioma Cell Lines and Evidence of Deregulated Expression of de novo DNMTs

Lorente, Aiala; Mueller, Wolf; Urdangarín, Edurne; Lázcoz, Paula; Laß, Ulrike; Deimling, Andreas von; Castresana, Javier S.

doi:10.1111/j.1750-3639.2008.00185.x

Cited by 46 publications

(36 citation statements)

References 51 publications

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“…In our study, RASSF1A promoter methylation was found in 23.52% of glioblastomas and was not correlated with tumor grade or degree of necrosis, possibly owing to small sample number. However, a recent study investigating RASSF1A methylation in 53 astrocytomas and 10 highgrade glioma cell lines showed 92% of tumor samples being methylated for RASSF1A, suggesting that inactivation of this tumor suppressor gene by promoter methylation is a crucial and frequent event in astrocytoma initiation and progression (11). Furthermore, the fact that RASSF1A promoter methylation occurs rarely in normal tissues, including the brain, renders its analysis a useful diagnostic tool in early tumor detection and disease monitoring.…”

Section: G M T a N D R A R β M E T H Y L A T I O N I N H U M A N A mentioning

confidence: 99%

“…PCR amplification was performed by using 150-200 ng of treated DNA as the template (11). Two sets of primers were used at the same position, one set specific for DNA methylated at CpG sites, and one specific for fully unmethylated DNA.…”

Section: Methylation-specific Pcrmentioning

confidence: 99%

“…RASSF1A binds Ras in a guanosine triphosphate-dependent manner and serves as an effector mediating the apoptotic effects of Ras. Methylation of RASSF1A has been found in a high percentage of various human primary tumors (9,10), including gliomas (11).…”

Section: Introductionmentioning

confidence: 99%

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High Incidence of MGMT and RARβ Promoter Methylation in Primary Glioblastomas: Association with Histopathological Characteristics, Inflammatory Mediators and Clinical Outcome

Piperi

Themistocleous

Papavassiliou³

et al. 2009

Mol Med

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Glioblastomas, the most frequent primary brain tumors in adults, are characterized by a highly aggressive, inflammatory and angiogenic phenotype. Methylation of CpG islands in cancer-related genes may serve as an epigenetic biomarker for glioblastoma diagnosis and prognosis. The aim of this study was to analyze the methylation status of four critical tumor-associated genes (MGMT, RARβ, RASSF1A, CDH13), and investigate possible links with inflammatory (interleukin [IL]-6, IL-8) and angiogenic mediators (vascular endothelial growth factor [VEGF], cyclooxygenase [COX]-2) and clinical outcome in 23 glioma samples (6 grade II astrocytomas, 17 grade IV glioblastomas). RARβ and MGMT genes were more frequently methylated in 70.58% and 58.8% of glioblastomas, respectively. RASSF1A and CDH13 displayed a similar methylation frequency (23.52%) in glioblastomas. No gene methylation was observed in grade II astrocytomas. Tumor grade correlated positively with MGMT and RARβ methylation (P = 0.005 and P = 0.019, respectively) and the extent of necrosis (P = 0.001 and P = 0.003). Interestingly, the marker of chronic inflammation, IL-6, was positively associated with methylation of MGMT (P = 0.004), RARβ (P = 0.002), and RASSF1A (P = 0.0081) as well as the total number of methylated genes (P < 0.0001), indicating the important role of IL-6 in maintaining promoter methylation of these genes. VEGF expression correlated positively with MGMT and RARβ methylation although these relationships were of marginal significance (P = 0.0679 and P = 0.0757). Kaplan-Meier univariate survival analysis indicated an unfavorable survival period in patients with MGMT methylation compared with those without methylation (P = 0.0474). Our study highlights the implication of MGMT and RARβ methylation in the aggressive phenotype of primary glioblastomas. The association of MGMT methylation with clinical outcome indicates its potential prognostic value.

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Section: G M T a N D R A R β M E T H Y L A T I O N I N H U M A N A mentioning

confidence: 99%

Section: Methylation-specific Pcrmentioning

confidence: 99%

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High Incidence of MGMT and RARβ Promoter Methylation in Primary Glioblastomas: Association with Histopathological Characteristics, Inflammatory Mediators and Clinical Outcome

Piperi

Themistocleous

Papavassiliou³

et al. 2009

Mol Med

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“…Bisulfite sequencing (BS) and MSP were done as previously described (13)(14)(15) and used as additional independent techniques for validation and investigation of the DR4 promoter region. MSP primers were designed based on DR4 promoter sequencing data and on previously published data on DR4 promoter methylation (16).…”

Section: Methodsmentioning

confidence: 99%

Epigenetic Silencing of Death Receptor 4 Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

Elias

Siegelin

Steinmüller

et al. 2009

Clinical Cancer Research

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Purpose: To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy. Experimental Design: We used methylation-sensitive BeadArray technology to identify novel epigenetically regulated genes associated with apoptosis and with potential therapeutic targets in glioma therapy. To elucidate the functional consequences of promoter methylation in the identified target death receptor 4 (DR4), we investigated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated and anti-DR4-mediated apoptosis in glioma cell lines (U373 and A172) with loss of DR4 and one glioma cell line (LN18) with robust DR4 expression. Results: In human astrocytic tumors, we detected DR4 promoter hypermethylation in 60% (n = 5) of diffuse astrocytomas WHO grade 2, in 75% (n = 8) of anaplastic astrocytomas WHO grade 3, and in 70% of glioblastomas WHO grade 4 (n = 33). DR4 is a cell surface protein restricted to glioma cells and is targeted by TRAIL. Glioma cell lines U373 and A172 harbored heavily methylated DR4 promoters, and 5-aza-2-deoxycytidinemediated demethylation reconstituted DR4 expression in these cell lines. Functional knockdown of DR4 by DR4-specific small interfering RNA in TRAIL-sensitive glioma cell line LN18 significantly mitigated apoptosis induced by an agonistic anti-DR4 antibody. 5-Aza-2-deoxycytidine-mediated demethylation resulted in a functional reconstitution of DR4 on the cell surface of TRAIL-resistant glioma cell line U373 and sensitized U373 to TRAIL-mediated apoptosis. Suppression of DR4 by small interfering RNA in demethylated U373 successfully reestablished the TRAIL-resistant phenotype of U373. Conclusions: DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies. Epigenetic modifications contribute to tumorigenesis. Examples are epigenetically silenced tumor suppressor genes (1). However, the epigenome becomes crucial clinical relevance when epigenetically regulated proteins are identified that themselves constitute therapeutic targets or are responsible for the development of therapy resistance of the tumor. An example for the latter is O 6 -methylguanine DNA methyltransferase (MGMT) in glioma therapy. Epigenetic silencing of MGMT abolishes its capability of chemotherapy resistance and predicts therapeutic response to alkylating drugs in glioma therapy (2-4). Caspase-8 may exemplify a therapeutically targeted epigenetically silenced molecule in gliomas. A downstream target for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, loss of caspase-8 expression, has been associated with epigenetic silencing and TRAIL resistance in these tumors (5). We were interested in identifying additional epigenetically silenced proteins or receptor molecules that are already targeted in glioma therapy. To address this, we implemented single-nucleotide polymorphism-based BeadArra...

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“…Aberrant DNMT expression has been observed in primary tumor specimens (including GBM) relative to normal tissue samples (Ahluwalia et al, 2001;Kanai et al, 2001;Mizuno et al, 2001;Sato et al, 2002;Girault et al, 2003;Fang et al, 2004;Arai et al, 2006;Park et al, 2006;Kim et al, 2006a;Lorente et al, 2008). Genetic approaches that disrupt DNMT activity have been recently employed to unmask epigenetic programs that integrate genesilencing networks in tumor cells (Rhee et al, 2002;Leu et al, 2003;Paz et al, 2003;Robert et al, 2003;Yan et al, 2003;Milutinovic et al, 2004;Suzuki et al, 2004;James et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

DNA methyltransferase-mediated transcriptional silencing in malignant glioma: a combined whole-genome microarray and promoter array analysis

et al. 2009

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Epigenetic inactivation of tumor suppressor genes is a common feature in human cancer. Promoter hypermethylation and histone deacetylation are reversible epigenetic mechanisms associated with transcriptional regulation. DNA methyltransferases (DNMT1 and DNMT3b) regulate and maintain promoter methylation and are overexpressed in human cancer. We performed whole-genome microarray analysis to identify genes with altered expression after RNAi-induced suppression of DNMT in a glioblastoma multiforme (GBM) cell line. We then identified genes with both decreased expression and evidence of promoter CpG island hypermethylation in GBM tissue samples using a combined whole-genome microarray transcriptome analysis in conjunction with a promoter array analysis after DNA immunoprecipitation with anti-5-methylcytidine. DNMT1 and 3b knockdown resulted in the restored expression of 308 genes that also contained promoter region hypermethylation. Of these, 43 were also found to be downregulated in GBM tissue samples. Three downregulated genes with hypermethylated promoters and restored expression in response to acute DNMT suppression were assayed for methylation changes using bisulfite sequence analysis of the promoter region after chronic DNMT suppression. Restoration of gene expression was not associated with changes in promoter region methylation, but rather with changes in histone methylation and chromatin conformation. Two of the identified genes exhibited growth suppressive activity in in vitro assays. Combining targeted genetic manipulations with comprehensive genomic and expression analyses provides a potentially powerful new approach for identifying epigenetically regulated genes in GBM.

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RASSF1A, BLU, NORE1A, PTEN and MGMT Expression and Promoter Methylation in Gliomas and Glioma Cell Lines and Evidence of Deregulated Expression of de novo DNMTs

Cited by 46 publications

References 51 publications

High Incidence of MGMT and RARβ Promoter Methylation in Primary Glioblastomas: Association with Histopathological Characteristics, Inflammatory Mediators and Clinical Outcome

High Incidence of MGMT and RARβ Promoter Methylation in Primary Glioblastomas: Association with Histopathological Characteristics, Inflammatory Mediators and Clinical Outcome

Epigenetic Silencing of Death Receptor 4 Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

DNA methyltransferase-mediated transcriptional silencing in malignant glioma: a combined whole-genome microarray and promoter array analysis

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