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Bisulfite Sequencing (bisulfite + sequencing)
Selected AbstractsBone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer developmentGENES, CHROMOSOMES AND CANCER, Issue 6 2008Kim Loh Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well-described functions in bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down-regulated as assessed by real-time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hypermethylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aberrant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01), the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001). Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re-introduction of BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tumor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early and frequent event in colorectal tumors progressing via the serrated and traditional pathways. © 2008 Wiley-Liss, Inc. [source] Widespread disruption of genomic imprinting in adult interspecies mouse (Mus) hybridsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 3 2005Wei Shi Abstract Mammalian interspecies hybrids exhibit parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit opposite phenotypes, especially in growth. This was also observed in hybridization with the genus Mus. These parent-of-origin effects suggested that imbalance in the expression of imprinted genes, which are expressed differentially, depending on their transmission through the maternal or paternal germline, and/or differential loss-of-imprinting (LOI) could underlie these opposite growth phenotypes in reciprocal mammalian hybrids. Here we report that tissue-specific LOI occurs in adult Mus hybrids. Contrary to expectations, LOI patterns were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Bisulfite sequencing revealed that reactivation of maternal alleles of Peg3 and Snrpn in specific tissues was accompanied by partial demethylation at their potential imprinting control regions. We propose that abnormal reprogramming after fertilization and during preimplantation development is in part responsible for hybrid dysgenesis, for which a strong epigenetic basis has been demonstrated. genesis 43:100,108, 2005. © 2005 Wiley-Liss, Inc. [source] Identification of differentially methylated CpG islands in prostate cancerINTERNATIONAL JOURNAL OF CANCER, Issue 5 2004Yasushi Yamada Abstract Epigenetic change such as DNA methylation is one important mechanism for regulating gene expression as genetic change, such as mutation or loss of heterozygosity. Methylation of cancer-related genes has been shown to play an important role in carcinogenesis and tumor progression. Using methylated CpG island amplification (MCA)/representational difference analysis (RDA), we identified four CpG islands in neurotrophin tyrosine kinase receptor type 2 (NTRK2), Protocadherine Flamingo1 and MFPC (Methylated Fragments in Prostate Cancer) 7 and 8. Bisulfite sequencing revealed that 2 regions of NTRK2 as well as MFPC7 and MFPC8 were aberrantly methylated in prostate cancer cell lines, and COBRA showed that 48 (76.24%), 37 (58.7%) and 14 (22.2%) of 63 prostate cancer tissues were methylated, respectively, for these sites. On the other hand, none of 13 benign prostate samples were methylated, except for 1 (7.7%) with NTRK2. For NTRK2, mRNA expression was negative in prostate cancer cell lines (LNCaP and DU145) but was recovered on a methyltransferase inhibitor (5-Aza-CdR) treatment. The role of NTRK2 within NTRK remains unclear. Our results suggest that these 3 hypermethylated DNA fragments also may be markers of prostate cancer detection. © 2004 Wiley-Liss, Inc. [source] RASSF1A, BLU, NORE1A, PTEN and MGMT Expression and Promoter Methylation in Gliomas and Glioma Cell Lines and Evidence of Deregulated Expression of de novo DNMTsBRAIN PATHOLOGY, Issue 2 2009Aiala Lorente Abstract Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5,-aza-2,-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A, 30%,57% for BLU and 47% for MGMT, suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A. No association between methylation and loss of expression could be established for PTEN. We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas. [source] Aberrant methylation of the vascular endothelial growth factor receptor-1 gene in prostate cancerCANCER SCIENCE, Issue 6 2003Yasushi Yamada Transcriptional silencing of cancer-related genes by DNA methylation is observed in various cancers. To identify genes controlled by methylation in prostate cancer, we used cDNA microarray analysis to investigate gene expression in prostate cancer cell lines LNCaP and DU145 treated with a methyltransferase inhibitor alone or together with a histone deacetylase inhibitor. We detected significant changes (3.4,5.7%) in gene expression in prostate cancer cell lines with the drug treatments. Among the affected genes, that for the vascular endothelial growth factor receptor 1 (VEGFR-1) was re-expressed in LNCaP and DU145 after the drug treatments. Bisulfite sequencing revealed the promoter and exon 1 of the VEGFR-1 to be hypermethylated in the cell lines. These results support the idea that methylation is associated with loss of VEGFR-1 mRNA expression in prostate cancer cell lines. Combined bisulfite restriction analysis (COBRA) showed the gene to be methylated in 24 (38.1%) of 63 primary local prostate cancer samples, while in all 13 benign prostate samples it was not. These findings indicate that methylation of VEGFR-1 is related with prostatic carcinogenesis. [source] Aberrant promoter methylation of the TPEF gene in esophageal squamous cell carcinomaDISEASES OF THE ESOPHAGUS, Issue 7 2008B.-J. Zhao SUMMARY., Aberrant methylation of tumor suppressor genes plays an important role in the development of esophageal squamous cell carcinoma (ESCC). The purpose of the present study was to identify the epigenetic changes in ESCC. Methylation-sensitive arbitrarily primed polymerase chain reaction (MS AP-PCR) analysis was used on 22 matched ESCC tumors and adjacent normal tissues. Through this screen we identified a frequently methylated fragment that showed a high homology to the 5,-CpG island of the gene encoding a transmembrane protein containing epidermal growth factor and follistatin domains (TPEF). The methylation status of the TPEF gene was then detected by bisulfite sequencing and the levels of TPEF mRNA were detected by RT-PCR. In addition, the effects of a methylation inhibitor 5-aza-2,-deoxycytidine on TPEF mRNA expression was determined in cells of ESCC cell lines. Hypermethylation of the 5,-CpG island of TPEF was found in 12 of 22 (54.5%) primary tumors. Reverse transcription PCR analysis demonstrated that TPEF mRNA expression was significantly lower in tumors than in adjacent normal tissues, which is associated with promoter hypermethylation. In addition, treatment of ESCC cell lines with 5-aza-2,-deoxycytidine led to re-expression of the TPEF transcript. In conclusion, we observed promoter of TPEF gene is frequently hpermethylated, and is associated with the loss of TPEF mRNA expression in ESCC samples. Promoter hypermethylation of TPEF gene may play a role in the development of ESCC. [source] Identification of novel DNA methylation markers in cervical cancer,INTERNATIONAL JOURNAL OF CANCER, Issue 1 2008Hung-Cheng Lai Abstract Testing for DNA methylation has potential in cancer screening. Most previous studies of DNA methylation in cervical cancer used a candidate gene approach. The aim our study was to identify novel genes that are methylated in cervical cancers and to test their potential in clinical applications. We did a differential methylation hybridization using a CpG island (CGI) microarray containing 8640 CGI tags to uncover methylated genes in squamous cell carcinomas (SCC) of the uterine cervix. Pooled DNA from cancer tissues and normal cervical swabs were used for comparison. Methylation-specific polymerase chain reaction, bisulfite sequencing and reverse transcription polymerase chain reaction were used to confirm the methylation status in cell lines, normal cervices (n = 45), low-grade lesions (n = 45), high-grade lesions (HSIL; n = 58) and invasive squamous cell carcinomas (SCC; n = 22 from swabs and n = 109 from tissues). Human papillomavirus (HPV) was detected using reverse line blots. We reported 6 genes (SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1) more frequently methylated in SCC tissues (81.5, 94.4, 89.9, 80.4, 77.8 and 20.4%, respectively) than in their normal controls (2.2, 0, 6.7, 11.9, 11.1 and 0%, respectively; p < 0.0001). Parallel testing of HPV and PAX1 methylation in cervical swabs confers an improved sensitivity than HPV testing alone (80% vs. 66%) without compromising specificity (63% vs. 64%) for HSIL/SCC. Testing PAX1 methylation marker alone, the specificity for HSIL/SCC is 99%. The analysis of these novel DNA methylations may be a promising approach for the screening of cervical cancers. © 2008 Wiley-Liss, Inc. [source] Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF-dependent transcription in medulloblastomasINTERNATIONAL JOURNAL OF CANCER, Issue 2 2007Arend Koch Abstract Medulloblastomas (MBs) represent the most common malignant brain tumors in children. Most MBs develop sporadically in the cerebellum, but their incidence is highly elevated in patients with familial adenomatous polyposis coli. These patients carry germline mutations in the APC tumor suppressor gene. APC is part of a multiprotein complex involved in the Wnt signaling pathway that controls the stability of ,-catenin, the central effector in this cascade. Previous genetic studies in MBs have identified mutations in genes coding for ,-catenin and its partners, APC and AXIN1, which cause activation of Wnt signaling. The pathway is negatively controlled by the tumor suppressor AXIN2 (Conductin), a scaffold protein of this signaling complex. To investigate whether alterations in AXIN2 may also be involved in the pathogenesis of sporadic MBs, we performed a mutational screening of the AXIN2 gene in 116 MB biopsy samples and 11 MB cell lines using single-strand conformation polymorphism and sequencing analysis. One MB displayed a somatic, tumor-specific 2 bp insertion in exon 5, leading to carboxy-terminal truncation of the AXIN2 protein. This tumor biopsy showed nuclear accumulation of ,-catenin protein, indicating an activation of Wnt signaling. In 2 further MB biopsies, mutations were identified in exon 5 (Glu408Lys) and exon 8 (Ser738Phe) of the AXIN2 gene, which are due to predicted germline mutations and rare polymorphisms. mRNA expression analysis in 22 MBs revealed reduced expression of AXIN2 mRNA compared to 8 fetal cerebellar tissues. Promoter hypermethylation could be ruled out as a major cause for transcriptional silencing by bisulfite sequencing. To study the functional role of AXIN2 in MBs, wild-type AXIN2 was overexpressed in MB cell lines in which the Wnt signaling pathway was activated by Wnt-3a. In this assay, AXIN2 inhibited Wnt signaling demonstrated in luciferase reporter assays. In contrast, overexpression of mutated AXIN2 with a deleted C-terminal DIX-domain resulted in an activation of the Wnt signaling pathway. These findings indicate that mutations of AXIN2 can lead to an oncogenic activation of the Wnt pathway in MBs. © 2007 Wiley-Liss, Inc. [source] Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma,INTERNATIONAL JOURNAL OF CANCER, Issue 3 2005Joseph Kwong Abstract Tazarotene-induced gene 1 (TIG1) and Tazarotene-induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation-specific PCR (MSP). We have detected dense methylation of TIG1 5,CpG island in the 5 TIG1 -negative NPC cell lines and xenograft (C666-1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1 -expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5-aza-2,-deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis. [source] Reduced expression of the Rassf1a gene and its aberrant DNA methylation in pancreatic duct adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamstersMOLECULAR CARCINOGENESIS, Issue 2 2008Kyoko Shimizu Abstract Alterations of the Rassf1a gene were investigated in pancreatic duct adenocarcinomas (PDAs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters. Female Syrian golden hamsters received 70 mg/kg BOP, followed by repeated exposures to an augmentation pressure regimen consisting of a choline-deficient diet combined with a sequential course of DL -ethionine, L -methionine, and 20 mg/kg BOP. A total of 15 PDAs were obtained, and total RNAs were assessed by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Expression of the Rassf1a was significantly reduced in PDAs (P,<,0.005) compared with normal pancreatic tissues. For analysis of methylation status, bisulfite sequencing was performed. Normal tissues were all unmethylated in the 5, upstream region of Rassf1a. In contrast, four PDAs were highly methylated, correlating with reduced expression of the Rassf1a gene. Using reverse transcription (RT)-polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, mutations were detected in 3 out of 15 PDAs (20%). These results suggested that alterations of the Rassf1a gene may be involved in development of PDAs induced by BOP in hamsters. © 2007 Wiley-Liss, Inc. [source] Overexpression of the growth arrest and DNA damage,induced 45, gene contributes to autoimmunity by promoting DNA demethylation in lupus T cellsARTHRITIS & RHEUMATISM, Issue 5 2010Yaping Li Objective Demethylation of CD11a and CD70 regulatory regions in CD4+ T cells contributes to the development of autoreactivity and overstimulation of autoantibodies. Because growth arrest and DNA damage,induced 45, (GADD45,) reduces epigenetic silencing of genes by removing methylation marks, this study examined whether the gadd45A gene could contribute to autoimmunity by promoting DNA demethylation in T cells from patients with systemic lupus erythematosus (SLE). Methods Levels of GADD45,, CD11a, and CD70 messenger RNA (mRNA) and protein were detected by real-time reverse transcription,polymerase chain reaction and Western blotting or flow cytometry. Global DNA methylation was evaluated using Methylamp global DNA methylation quantification kits. Detection of CD4+ T cell proliferation and autologous B cell IgG antibodies was performed using commercially available kits. CD11a and CD70 promoter methylation was determined with bisulfite sequencing. Results Elevated gadd45A mRNA expression and global DNA hypomethylation were observed in CD4+ T cells from SLE patients. The levels of gadd45A mRNA were inversely proportional to the levels of DNA methylation. Positive correlations were found between gadd45A and CD11a/CD70 mRNA levels. Expression of gadd45A mRNA was increased in CD4+ T cells following ultraviolet B irradiation, and this was accompanied by increased levels of CD11a and CD70 mRNA. Moreover, increased expression of gadd45A, CD11a, and CD70 mRNA was accompanied by increased autoreactivity and excessive B cell stimulation in gadd45A -transfected CD4+ T cells. CD11a promoter methylation was also significantly reduced in transfected cells. Transfection of gadd45A small interfering RNA inhibited the autoreactivity of SLE CD4+ T cells and led to significant increases in the methylation levels of the CD11a and CD70 promoter regions. Conclusion These findings indicate that gadd45A may contribute to lupus-like autoimmunity by promoting DNA demethylation in SLE CD4+ T cells. [source] DNA hypomethylation in rheumatoid arthritis synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 12 2009Emmanuel Karouzakis Objective Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification. Methods Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5-azacytidine (5-azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry. Results In situ and in vitro, RASF DNA had fewer 5-methylcytosine and methylated CG sites upstream of an L1 open-reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5-azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty-six genes were up-regulated >2-fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix-degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs. Conclusion DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies. [source] | ||||||||||