Aberrant Hypermethylation of p14ARF and O6‐methylguanine‐DNA Methyltransferase Genes in Astrocytoma Progression

Watanabe, Takao; Katayama, Yoichi; Yoshino, Atsuo; Yachi, Kazunari; Ohta, Takashi; Ogino, Akiyoshi; Komine, Chiaki; Fukushima, Takao

doi:10.1111/j.1750-3639.2006.00030.x

Cited by 54 publications

(38 citation statements)

References 24 publications

(52 reference statements)

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“…A methylation of DNA regulatory elements -mainly promoters, is one of the mechanisms of tumour suppressor genes silencing (Watanabe et al, 2007). It was shown that a similar proportion of gliomas with and without P53 mutation present P53 promoter methylation (Amatya et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…Exons 5 -8 of the P53 gene were amplified by PCR as described before and sequenced using the dideoxy termination method and SequiTherm Excel DNA Sequencing Kit (Epicentre Technologies) (Zakrzewska et al, 2005).…”

Section: P53 Dna and Cdna Sequencingmentioning

confidence: 99%

“…In this context, the occurrence of heterozygous mutations of P53 remains enigmatic, leading to a question of whether mechanisms other than P53 mutations or deletions are involved in the elimination of the wild-type P53 protein. Several nongenomic mechanisms of protein elimination or aberration have been described, including processes operating at the level of transcription (e.g., methylation) or translation (e.g., miRNA) (Voorhoeve et al, 2006;Watanabe et al, 2007). We examined whether glioblastoma cells with heterozygous mutations of P53 contained a mixture of wild-type and mutated P53 mRNA, or predominantly the mutated P53 mRNA.…”

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Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

et al. 2008

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We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene. (Nigro et al, 1989;Dittmer et al, 1993). However, the effects of at least some heterozygous mutations cannot be explained only by the gain of P53 function (Park et al, 1994). In case of P53 mutations such as R249S or R273H, at least three mutated monomers per tetramer appeared to be required to inactivate the transactivation of MDM2 and p21 CIP1/ WAF1 promoters (Chan et al, 2004). In case of R280T mutations, heterotetramers consisting even of three mutated and one wild-type P53 monomer showed partially but not completely abolished activity compared to P53 homotetramers consisting of wild-type monomers only (Sun et al, 1993). In this context, the occurrence of heterozygous mutations of P53 remains enigmatic, leading to a question of whether mechanisms other than P53 mutations or deletions are involved in the elimination of the wild-type P53 protein. Several nongenomic mechanisms of protein elimination or aberration have been described, including processes operating at the level of transcription (e.g., methylation) or translation (e.g., miRNA) (Voorhoeve et al, 2006;Watanabe et al, 2007). We examined whether glioblastoma cells with heterozygous mutations of P53 contained a mixture of wild-type and mutated P53 mRNA, or predominantly the mutated P53 mRNA. Additionally, we also checked the methylation status of the P53 promoter. MATERIALS AND METHODS Tumour samplesThe study included 50 cases of glioblastoma, diagnosed at Department of Pathology, Medical University of Lodz, according to the World Health Organization criteria for classification of brain tumours (Louis et al, 2007). The group consisted of 25 females and 25 males, aged from 15 to 76 years (median 59.5). DNA and RNA isolationThe investigations were performed using snap-frozen tissues stored at À801. DNA was isolated from tumour tissues and blood samples from each patient. DNA and RNA were coextracted by means of Macherey-Nagel DNA/RNA purification kit. RNA samples were treated with DNAase. RNA and DNA concentrations were measured spectrophotometrically. In all 100 ng of total RNA was reverse-transcribed into single-stranded cDNA in a final volume of 40 ml containing 50 mM DTT, 1.5 mg oligo(dT), 0.5 mM dNTP, 40 units RNase inhibitor and 200 units M-MLV reverse transcriptase (Promega). Loss of heterozygosity and microsatellite instability analysesLoss of heterozygosity (LOH) and microsatellite instability (MSI) analyses were performed using paired tumour specimens and corresponding peripheral blood samples, to recognise tumour samples with minimal contamination by normal cells. The following LOH and MSI markers were used:

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Section: Discussionmentioning

confidence: 99%

Section: P53 Dna and Cdna Sequencingmentioning

confidence: 99%

mentioning

confidence: 99%

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Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

et al. 2008

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“…Interestingly, all secondary GBM (4/4) showed a methylated MGMT promoter and an unmethylated p15 promoter which underlines that distinct molecular pathways constitute the primary and the secondary glioblastomas and are responsible for their different biological behavior and in clinical outcome. This corresponds to the finding that methylation of MGMT promoter is mutually exclusive of p14 ARF methylation and the latter is associated with a shorter patient survival, except in one case of low grade astrocytoma who underwent progression or recurrence (38).…”

Section: Discussionmentioning

confidence: 99%

p15 promoter methylation - A novel prognostic marker in glioblastoma patients

et al. 2009

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Abstract. Glioblastomas are the most frequent and malignant brain tumors in adults. Surgical cure is virtually impossible and despite radiation and chemotherapy the clinical course is very poor. Epigenetic silencing of MGMT has been associated with a better response to temozolomide-chemotherapy. We previously showed that temozolomide increases the median survival time of patients with tumors harbouring deletions on 9p within the region for p15 (INK4b), p16(INK4a), and 10q (MGMT). The aim of this study was to investigate the methylation status of p15, p16, p14 ARF and MGMT in glioblastomas (n=27) and to correlate the results with the clinical data. Only patients with KPS >70, radical tumor resection, radiation and temozolomide-chemotherapy after recurrence were included. We observed promoter methylation of MGMT in 56% and of p15 in 37% of the tumors, whereas methylation of p16 and p14 ARF were rare. Interestingly, methylation of p15 emerged as a significant predictor of shorter overall survival (16.9 vs. 23.8 months, p=0.025), whereas MGMT promoter methylation had no significant effect on median overall survival under this treatment regimen (22.5 vs. 22.1 months, p=0.49). In the presence of other clinically relevant factors, p15 methylation remains the only significant predictor (p=0.021). Although these results need to be confirmed in larger series as well as under different treatment conditions, our retrospective study shows clear evidence that p15 methylation is an important prognostic factor for survival and underlines that this tumor suppressor, involved in cell cycle control, is an attractive candidate for therapeutic approaches in glioblastomas.

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“…Hypermethylation of p14 ARF has been associated with progression of astrocytomas (Watanabe et al, 2007) and has been described as a factor of poor prognosis of grade II diffuse gliomas (Watanabe et al, 2003), although its prognostic value in oligodendroglioma is more controversial (Korshunov and Golanov, 2001;Kamiya and Nakazato, 2002). p16 INK4A deletion is a factor of poor prognosis of these tumours (Jeon et al, 2007).…”

Section: Expressionsmentioning

confidence: 99%

Prognostic molecular markers with no impact on decision-making: the paradox of gliomas based on a prospective study

et al. 2008

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This study assessed the prognostic value of several markers involved in gliomagenesis, and compared it with that of other clinical and imaging markers already used. Four-hundred and sixteen adult patients with newly diagnosed glioma were included over a 3-year period and tumour suppressor genes, oncogenes, MGMT and hTERT expressions, losses of heterozygosity, as well as relevant clinical and imaging information were recorded. This prospective study was based on all adult gliomas. Analyses were performed on patient groups selected according to World Health Organization histoprognostic criteria and on the entire cohort. The endpoint was overall survival, estimated by the Kaplan -Meier method. Univariate analysis was followed by multivariate analysis according to a Cox model. p14 ARF , p16 INK4A and PTEN expressions, and 10p 10q23, 10q26 and 13q LOH for the entire cohort, hTERT expression for high-grade tumours, EGFR for glioblastomas, 10q26 LOH for grade III tumours and anaplastic oligodendrogliomas were found to be correlated with overall survival on univariate analysis and age and grade on multivariate analysis only. This study confirms the prognostic value of several markers. However, the scattering of the values explained by tumour heterogeneity prevents their use in individual decision-making.

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Aberrant Hypermethylation of p14^ARF and O⁶‐methylguanine‐DNA Methyltransferase Genes in Astrocytoma Progression

Cited by 54 publications

References 24 publications

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

p15 promoter methylation - A novel prognostic marker in glioblastoma patients

Prognostic molecular markers with no impact on decision-making: the paradox of gliomas based on a prospective study

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