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Bisulfite Modification (bisulfite + modification)
Selected AbstractsDNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 11 2009Ko Hashimoto Objective To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1, (IL-1,) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. Methods IL-1,, tumor necrosis factor , (TNF,) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4,5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green,based quantitative reverse transcription,polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B -expressing and IL1B -nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (,299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1, into the culture media was assessed by enzyme-linked immunosorbent assay. Results Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100,1,000-fold by treatment with TNF,/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1, and was almost abolished by TNF,/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. Conclusion These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes. [source] Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancersGENES, CHROMOSOMES AND CANCER, Issue 1 2002Taiji Furukawa It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high-frequency MSI (MSI-H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI-H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI-H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI-H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation. © 2002 Wiley-Liss, Inc. [source] Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissuesINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Eri Arai Abstract To evaluate the significance of altered DNA methylation during renal tumorigenesis, tumorous tissues (T) and corresponding nontumorous renal tissues (N) from 94 patients with renal tumors, and normal renal tissues (C) from 16 patients without renal tumors were investigated. DNA methylation status on CpG islands of the p16, human MutL homologue 1 (hMLH1), von-Hippel Lindau (VHL) and thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT) -1, -2, -12, -25 and -31 clones and DNA methyltransferase (DNMT) 1 expression were examined by bisulfite modification and immunohistochemistry, respectively. The average number of methylated CpG islands was significantly higher in N than in C, and was even higher in T. The average number of methylated CpG islands in N was significantly correlated with a higher histological grade of corresponding conventional renal cell carcinomas (RCCs). The average number of methylated CpG islands in RCCs was significantly correlated with macroscopic configuration with extranodular or multinodular growth, higher histological grade, infiltrating growth pattern and vascular involvement. The recurrence-free survival rate of patients with RCCs showing accumulation of DNA methylation was significantly lower than that of patients not showing this feature. The incidence of nuclear immunoreactivity for DNMT1 tended to be higher in proximal tubules from N than in those from C, and was significantly higher in RCCs. From the viewpoint of altered DNA methylation, N is at the precancerous stage, and N showing accumulation of DNA methylation may generate more malignant RCCs. Regional DNA hypermethylation may be associated with renal tumorigenesis from a precancerous condition to malignant progression and become a predictor of patient prognosis. © 2006 Wiley-Liss, Inc. [source] Influence of methylated p15 INK4b and p16 INK4a genes on clinicopathological features in colorectal cancerJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 8 2006Atsushi Ishiguro Abstract Background and Aim:, Genetic silencing by promoter methylation has attracted attention in the carcinogenesis of colorectal cancer. Methylation of the p16INK4a gene has been found in primary colorectal cancer. Although the p15INK4b gene displays high homology to the p16INK4a gene in the amino acid sequence, methylation of p15INK4b has not been fully studied. We investigated p15INK4b methylation status in patients with colorectal cancer to verify the association between the methylation of p15INK4b and clinicopathological features compared with p16INK4a. Methods:, DNA samples from the tissues of primary colorectal cancer and corresponding adjacent normal colon mucosa were obtained from surgical resections of 88 patients (47 males and 41 females, aged 29,83 years). Methylation-specific polymerase chain reaction was used to analyze p15INK4b and p16INK4a methylation status after bisulfite modification. Cumulative survival rates (mean follow-up period: 53.2 months) were calculated by the Kaplan-Meier analysis. Results:, Methylations of p15INK4b and p16INK4a genes were detected in 23 (26.1%) and 20 (22.7%) colorectal cancers, respectively. Methylation of p15INK4b was not associated with any clinicopathological features. Compared with normal mucosa, the methylation of p15INK4b was more prominent in tumor tissue (P < 0.001). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that p15INK4b methylaton decreased mRNA expression. Kaplan-Meier analysis showed that patients with stage I-II had a significant difference in survival rate between those with and without methylated p15INK4b (P = 0.018). Conclusions:, Our results suggest that methylation of the p15INK4b gene contributes to the process of carcinogenesis in colorectal cancer as well as p16INK4a and is useful as a prognostic factor in the early stage. [source] Quantitative assessment of RUNX3 methylation in neoplastic and non-neoplastic gastric epithelia using a DNA microarrayPATHOLOGY INTERNATIONAL, Issue 10 2006Kanji So Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber-type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end-labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non-neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non-neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non-neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation-positive by conventional methylation-specific polymerase chain reaction (MSP). This microarray-based methylation assay is a promising method for the quantitative assessment of gene methylation. [source] | ||||||||||