MeCP2/H3meK9 are involved in IL-6 gene silencing in pancreatic adenocarcinoma cell lines

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-α, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2′-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions –666 to –426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2

Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting

Prognostic Value of Three Different Methods of MGMT Promoter Methylation Analysis in a Prospective Trial on Newly Diagnosed Glioblastoma

Hypermethylation in the promoter region of the MGMT gene encoding the DNA repair protein O6-methylguanine-DNA methyltransferase is among the most important prognostic factors for patients with glioblastoma and predicts response to treatment with alkylating agents like temozolomide. Hence, the MGMT status is widely determined in most clinical trials and frequently requested in routine diagnostics of glioblastoma. Since various different techniques are available for MGMT promoter methylation analysis, a generally accepted consensus as to the most suitable diagnostic method remains an unmet need. Here, we assessed methylation-specific polymerase chain reaction (MSP) as a qualitative and semi-quantitative method, pyrosequencing (PSQ) as a quantitative method, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a semi-quantitative method in a series of 35 formalin-fixed, paraffin-embedded glioblastoma tissues derived from patients treated in a prospective clinical phase II trial that tested up-front chemoradiotherapy with dose-intensified temozolomide (UKT-05). Our goal was to determine which of these three diagnostic methods provides the most accurate prediction of progression-free survival (PFS). The MGMT promoter methylation status was assessable by each method in almost all cases (n = 33/35 for MSP; n = 35/35 for PSQ; n = 34/35 for MS-MLPA). We were able to calculate significant cut-points for the continuous methylation signals at each CpG site analysed by PSQ (range, 11.5 to 44.9%) and at one CpG site assessed by MS-MLPA (3.6%) indicating that a dichotomisation of continuous methylation data as a prerequisite for comparative survival analyses is feasible. Our results show that, unlike MS-MLPA, MSP and PSQ provide a significant improvement of predicting PFS compared with established clinical prognostic factors alone (likelihood ratio tests: p<0.001). Conclusively, taking into consideration prognostic value, cost effectiveness and ease of use, we recommend pyrosequencing for analyses of MGMT promoter methylation in high-throughput settings and MSP for clinical routine diagnostics with low sample numbers

Genetic and epigenetic alterations on the short arm of chromosome 11 are involved in a majority of sporadic Wilms' tumours

Wilms' tumour is one of the most common solid tumours of childhood. 11p13 (WT1 locus) and 11p15.5 (WT2 locus) are known to have genetic or epigenetic aberrations in these tumours. In Wilms' tumours, mutation of the Wilms tumour 1 (WT1) gene at the WT1 locus has been reported, and the WT2 locus, comprising the two independent imprinted domains IGF2/H19 and KIP2/LIT1, can undergo maternal deletion or alterations associated with imprinting. Although these alterations have been identified in many studies, it is still not clear how frequently combined genetic and epigenetic alterations of these loci are involved in Wilms' tumours or how these alterations occur. To answer both questions, we performed genetic and epigenetic analyses of these loci, together with an additional gene, CTNNB1, in 35 sporadic Wilms' tumours. Loss of heterozygosity of 11p15.5 and loss of imprinting of IGF2 were the most frequent genetic (29%) and epigenetic (40%) alterations in Wilms' tumours, respectively. In total, 83% of the tumours had at least one alteration at 11p15.5 and/or 11p13. One-third of the tumours had alterations at multiple loci. Our results suggest that chromosome 11p is not only genetically but also epigenetically critical for the majority of Wilms' tumours

Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast

Most long non-coding RNAs (lncRNAs) encoded by eukaryotic genomes remain uncharacterized. Here we focus on a set of intergenic lncRNAs in fission yeast. Deleting one of these lncRNAs exhibited a clear phenotype: drug sensitivity. Detailed analyses of the affected locus revealed that transcription of the nc-tgp1 lncRNA regulates drug tolerance by repressing the adjacent phosphate-responsive permease gene transporter for glycerophosphodiester 1 (tgp1(+)). We demonstrate that the act of transcribing nc-tgp1 over the tgp1(+) promoter increases nucleosome density, prevents transcription factor access and thus represses tgp1(+) without the need for RNA interference or heterochromatin components. We therefore conclude that tgp1(+) is regulated by transcriptional interference. Accordingly, decreased nc-tgp1 transcription permits tgp1(+) expression upon phosphate starvation. Furthermore, nc-tgp1 loss induces tgp1(+) even in repressive conditions. Notably, drug sensitivity results directly from tgp1(+) expression in the absence of the nc-tgp1 RNA. Thus, transcription of an lncRNA governs drug tolerance in fission yeast