CpG Sites In Promoter Region Research Articles

Increase in Cisplatin Resistance by MeCP2 in Human Gastric Cancer through the Activation of the AKT Pathway by Facilitating PDK-1 Transcription.

Increasing evidence indicates that an imbalance in oncogenes is implicated in cancer chemotherapy resistance. Methyl-CpG binding protein 2 (MeCP2), which acts as a major epigenetic regulator of the expression of various genes, is involved in the carcinogenesis and progression of gastric cancer. However, is it not known whether the role of MeCP2 is vital in acquired cisplatin resistance in gastric cancer. This study aimed to determine whether inhibition of MeCP2 expression could sensitize DDP-resistant GC cells to DDP and elucidate the underlying molecular mechanism. qRT-PCR and western blotting were used to evaluate MeCP2 expression in DDP-resistant GC cells. Subsequently, cell viability, colony formation, cell cycle, apoptosis, and tumorigenicity assays were performed to explore the in vitro and in vivo roles of MeCP2. Chromatin immunoprecipitation- qPCR and luciferase reporter assays were used to identify whether 3-phosphoinositide-dependent protein kinase 1 (PDK-1) was a direct target gene of MeCP2. MeCP2 was upregulated in malignant DDP-resistant cells compared to non-DDP-resistant GC cells or normal gastric epithelial cells. MeCP2 knockdown increased the sensitivity of DDP-resistant GC cells to DDP, resulting in reduced cell growth, G0/G1 phase arrest, and increased apoptosis, whereas MeCP2 overexpression attenuated DDP sensitivity of DDP-resistant GC cells. In addition, MeCP2 knockdown enhanced DDP sensitivity in vivo. MeCP2 elevated PDK-1 expression by binding to CpG sites in promoter regions, and inhibition of PDK-1 reversed the inductive effect of MeCP2 overexpression on DDP resistance in GC cells. These findings indicate that silencing of MeCP2 may potentiate DDP-induced cell death, thereby providing a promising therapeutic strategy for GC.

Elevated methylation of the vault RNA2-1 promoter in maternal blood is associated with preterm birth

BackgroundPreterm birth, defined as parturition before 37 completed weeks of gestation, is associated with an increased risk of neonatal complications and death, as well as poor health and disease later in life. Epigenetics could contribute to the mechanism underlying preterm birth.ResultsGenome-wide DNA methylation analysis of whole blood cells from 10 women (5 term and 5 preterm deliveries) was performed using an Illumina Infinium HumanMethylation450 BeadChips array. We identified 1,581 differentially methylated CpG sites in promoter regions between term and preterm birth. Although the differences were not significant after correcting for multiple tests, seven CpGs on the genomically imprinted vault RNA2-1 (VTRNA2-1; also known as non-coding RNA, nc886 or miR-886) showed the largest differences (range: 26–39 %). Pyrosequencing verification was performed with blood samples from pregnant women recruited additionally (39 term and 43 preterm deliveries). In total, 28 (34.1 %) samples showed hypomethylation of the VTRNA2-1 promoter (< 13 % methylation), while 54 (65.9 %) samples showed elevated methylation levels between 30 and 60 %. Elevated methylation of VTRNA2-1 promoter was associated with an increased risk of preterm birth after adjusting for maternal age, season of delivery, parity and white blood cell count. The mRNA expression of VTRNA2-1 was 0.51-fold lower in women with preterm deliveries (n = 20) compared with women with term deliveries (n = 20).ConclusionsVTRNA2-1 is a noncoding transcript to environmentally responsive epialleles. Our results suggest that elevated methylation of the VTRNA2-1 promoter may result in increased risk of PTB caused by the pro-inflammatory cytokines. Further studies are needed to confirm the association of VTRNA2-1 methylation with preterm birth in a large population, and to elucidate the underlying mechanism.

Whole Genomic DNA Methylation Profiling of CpG Sites in Promoter Regions of Dorsal Root Ganglion in Diabetic Neuropathic Pain Mice.

DNA methylation and demethylation play an important role in neuropathic pain. In general, DNA methylation of CpG sites in the promoter region impedes gene expression, whereas DNA demethylation contributes to gene expression. Here, we evaluated the methylation status of CpG sites in genomic DNA promoter regions in dorsal root ganglions (DRGs) of diabetic neuropathic pain (DNP) mice. In our research, streptozotocin (STZ) was intraperitoneally injected into mice to construct DNP models. The DNP mice showed higher fasting blood glucose (above 11.1mmol/L), lower body weight, and mechanical allodynia than control mice. Whole-genome bisulfite sequencing (WGBS) revealed an altered methylation pattern in CpG sites in the DNA promoter regions in DRGs of DNP mice. The results showed 376 promoter regions with hypermethylated CpG sites and 336 promoter regions with hypomethylated CpG sites. In addition, our data indicated that altered DNA methylation occurs primarily on CpG sites in DNA promoter regions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that differentially methylated CpG sites annotated genes were involved in activities of the nervous and sensory systems. Enrichment analysis indicated that genes in these pathways contributed to diabetes or pain. In conclusion, our study enriched the role of DNA methylation in DNP.

Changes in DNA methylation after 6-week exercise training in colorectal cancer survivors: A preliminary study.

Behavioral interventions such as exercise may induce epigenetic changes. Only few studies investigated the effects of exercise on epigenetic alterations in colorectal cancer survivors. The aim of this study was to explore the changes of genome-wide DNA methylation after 6-week exercise training in colorectal cancer survivors. This preliminary study used a subset of data from a randomized controlled trial in 15 colorectal cancer survivors. Participants were randomized either to the 6-week exercise group or control group. The exercise intervention consisted of a weekly, group-based, supervised resistance exercise program and a home-based same resistance exercise plus walking six times per week. Blood samples were collected at baseline and after the intervention and data from eight subjects were analyzed for genome-wide DNA methylation on 865,918 CpG sites. Compared to the control group, the exercise group shows notable methylation changes in 756 CpG sites (22.7-25.2%). Gene ontology and disease annotation analysis showed that the genes targeting 81 CpG sites in promoter region with significant group-difference were linked in biological process such as immune response and transcription and related to metabolic and immune diseases. Also, hypermethylation on genes related to disease prevention seemed to be inhibited in the exercise group compared to the control group, indicating a likelihood of transcriptional activity of these genes. We found a preliminary evidence of the positive effects of exercise intervention on epigenetic markers in colorectal cancer survivors. Larger scale randomized controlled trials are warranted to further investigate our findings.

The Epigenomics of Pituitary Adenoma.

Background: The vast majority of pituitary tumors are benign and behave accordingly; however, a fraction are invasive and are more aggressive, with a very small fraction being frankly malignant. The cellular pathways that drive transformation in pituitary neoplasms are poorly characterized, and current classification methods are not reliable correlates of clinical behavior. Novel techniques in epigenetics, the study of alterations in gene expression without changes to the genetic code, provide a new dimension to characterize tumors, and may hold implications for prognostication and management.Methods: We conducted a review of primary epigenetic studies of pituitary tumors with a focus on histone modification, DNA methylation, and transcript modification.Results: High levels of methylation have been identified in invasive and large pituitary tumors. DNA methyltransferase overexpression has been detected in pituitary tumors, especially in macroadenomas. Methylation differences at CpG sites in promoter regions may distinguish several types of tumors from normal pituitary tissue. Histone modifications have been linked to increased p53 expression and longer progression-free survival in pituitary tumors; sirtuins are expressed at higher values in GH-expressing compared to nonfunctional adenomas and correlate inversely with size in somatotrophs. Upregulation in citrullinating enzymes may be an early pathogenic marker of prolactinomas. Numerous genes involved with cell growth and signaling show altered methylation status for pituitary tumors, including cell cycle regulators, components of signal transduction pathways, apoptotic regulators, and pituitary developmental signals.Conclusions: The limited clinical predictive capacity of the current pituitary tumor classification system suggests that tumor subclasses likely remain to be discovered. Ongoing epigenetic studies could provide a basis for adding methylation and/or acetylation screening to standard pituitary tumor workups. Identifying robust correlations between tumor epigenetics and corresponding histological, radiographic, and clinical course information could ultimately inform clinical decision-making.

Studying Methylation Status of Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) in Chronic Myeloid Leukemia Patients in the State of Qatar

Background:Chronic myeloid leukemia (CML) is a clonal myeloid stem cell disorder associated with increased myeloid cells spectrum in peripheral blood. The disease is due to the genetic translocation t(9;22)(q34;q11.2) encoding for the BCR-ABL1 fusion oncogene which is the hallmark of CML. The resultant chimeric protein P210 kD of BCR-ABL1 causes a constitutive activation of tyrosine kinase domain that leads to continuous activation of several cell signaling pathways.The CML is currently treated with several generations of tyrosine kinase inhibitors (TKIs). However, treatment failure, resistance and disease progression are still reported and the molecular mechanisms still remain poorly understood.On the other hand, protein tyrosine phosphatase receptor gamma (PTPRG) is the natural regulatory mechanism to tyrosine kinase family, acts as tumor suppressor gene, well documented to be downregulated in CML patients while its re-expression led to reduced tyrosine phosphorylation and induced apoptosis in CML cells.PTPRG is also an important protein in the JAK/STAT pathway, has a vital role in the downstream of BCR-ABL protein kinase. Several studies suggested that the aberrant DNA methylation is involved in TKIs resistance in CML leading to leukemic clone escape and disease propagation.In different types of tumors, several mechanisms have been suggested for the loss of function of PTPRG activities such as mutations, deletions, or loss of heterozygosity LOH and gene silencing by methylation.Aim:In this study, we aimed to evaluate and characterize the methylation status of PTPRG gene function in 15 CML Patients 1- Group A: CML patients at diagnosis and 2- Group B) CML patients failed treatments according to European Leukemia Net (ELN) 2013 criteria and 10 healthy individuals. (Labeled as Normal in the figure 1)Materials and Methods:Thirty subsequent peripheral blood samples have been collected from the 15 CML patients at diagnosis and at time of failure. Genomic DNA was isolated from 400 ml of blood on EDTA tubes using Maxwell DNA Purification Kits, the quality and quantity of DNA were measured on Nanodrop Spectrophotometer and gel electrophoresis. The methylation-specific PCR and bisulfite-sequencing were adapted, EZ DNA Methylation Kit was utilized for bisulfite-conversion. Methylation Primers were designed to target the promotor and intronic CpG islands of the PTPRG gene. Gradient PCR was performed to detect the two bands of 321bp and 218bp for CpG islands of promoter and intron of PTPRG respectively. Bisulfite-specific PCR technique was carried out to amplify a fragment of bisulfite-converted DNA that has been pre-defined by the designed bisulfite-specific primers.The Methylation PCR amplified product was sequenced via Sanger Sequencing (Applied Biosystems 3130 Genetic Analyzer). The methylation analysis was performed to study the methylation status of the CpG islands using the ESME (Epigenetic Sequencing Methylation) Analysis Software.Results:Results of the methylation in 25 CpG sites of Promoter region of PTPRG:There are significant differences in the mean of methylation status amongst CML patients at diagnosis and healthy individuals of CpG promoter of PTPRG (p value is ˂0.04). However, there are no significant differences in the mean of methylation status between patients failed treatments and other groups (Figure1)Results of the methylation in 26 CpG sites of Intronic region of PTPRG:There are also significant differences in the mean of methylation status amongst all groups (p value is ˂0.003). (Figure1)Discussion and Conclusions:This is the first prospective study to evaluate epigenetic changes of PTPRG gene from CML patients in Qatar. We previously documented that the expression level of PTPRG is inversely correlated with the expression of BCR-ABL1 in CML patients.In this study, the mechanism behind the downregulation of PTPRG in our cohorts of CML patients was explored and the aberrant methylation of PTPRG could be a mechanism associated with CML disease progression and resistance to TKIs.However, further studies are still needed including larger patient's cohort and CML patients responding to the TKIs.Finally, the methylation of PTPRG in the CML patients at diagnosis and failed treatment may allow the identification of new therapeutic targets and using methylation inhibitors could be also suggested to prevent or reverse TKIs resistance and restore TKIs sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of ITGB2 (CD18) and SELL (CD62L) genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis.

Systemic sclerosis (SSc), an autoimmune disease of connective tissue, is characterized by inflammation, fibrosis, and vessel endothelial damage. Products of Integrin subunit beta 2 (ITGB2) and selectin L (SELL) genes participate in several functional pathways of immune system. The aim of this investigation was to survey the transcript level of ITGB2 and SELL genes as well as methylation status of CpG sites in promoter region of differently expressed gene in PBMCs of SSc patients. PBMCs were isolated from whole blood of 50 SSc patients and 30 healthy controls. Total RNA and DNA contents of PBMCs were extracted. Gene expression was analyzed by real-time PCR using the SYBR Green PCR Master Mix. To investigate the methylation status of CpG sites, DNA samples were treated by bisulfite, amplified through nested PCR, and sequenced through Sanger difficult sequencing method. ITGB2 gene in PBMCs of SSc patients was overexpressed significantly in comparison to healthy controls. However, no altered SELL expression was observed. Three CpG sites of 12, 13 and 14 were significantly hypomethylated in patients group, despite overall methylation status of ITGB2 gene promoter revealed no significant difference between study groups. There was no statistically significant correlation between methylation status of ITGB2 promoter and the gene expression in patients. Regarding to lack of correlation of increased expression of ITGB2 with its promoter hypomethylation in SSc patients, our study suggests that upregulation of ITGB2 in PBMCs from SSc patients is probably due to another mechanism other than methylation alteration.

Association of methylation status of CpG islands in DAT1 and DRD4 genes with attention deficit hyperactivity disorder

Objective To explore the difference of methylation status of CpG island in promoter region of DAT1 and DRD4 genes between children with attention deficit hyperactivity disorder (ADHD) and normal controls, and further understand the pathogenesis of ADHD from a epigenetics point of view. Methods 111 ADHD patients and 118 normal controls were enrolled in the present study. The demographic data and peripheral venous blood were collected from both groups. Bisulfite genomic sequencing (BGS) was used to confirm the methylation status of every CpG site in promoter region of DAT1 and DRD4 genes. Results No significant differences were found between ADHD patients and normal controls on percentage of methylated CpG sites in total CpG islands for both DAT1 and DRD4 (P>0.05). However, the percentage of methylation in No. 17 CpG site for DAT1 and No. 8 CpG site for DRD4 was higher in ADHD patients (23.42% and 64.86% respectively)compared with that in normal controls(11.86% and 47.46% respectively)(P<0.05). In all samples, the percentage of methylated CpG site in total CpG island for DAT1 was higher in males compared with that in females(P<0.05), whereas that for DRD4 was higher in females compared with that in males (P<0.05); the same gender difference on methylation level for DAT1 was also found in ADHD patients and for DRD4 in normal controls(P<0.05). In all samples and in ADHD patients, percentage of methylated CpG site in total CpG island for DAT1 was higher in individuals over 7 years old compared with that in individuals younger than or equal to 7 years old(P<0.05). Conclusions Methylation status of CpG island in DAT1 and DRD4 genes promoter region might correlate with ADHD susceptibility.Methylation status of CpG island in DAT1 and DRD4 genes show differences in different age span and sex. Key words: Attention deficit hyperactivity disorder; Epigenetics; DNA methylation; Dopamine transporter gene (DAT1/SLC6A3); Dopamine receptor D4 gene (DRD4)

Abstract 2787: Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells

Abstract Expression of Olfactomedin 4 gene (OLFM4) plays a critical tumor suppressor role in human and murine prostate cancer. We have previously reported that loss or reduced OLFM4 expression is associated with the progression of human prostate cancer. We and others have further determined that deletions within the OLFM4 gene are present in about 10 to 25% of human prostate cancer samples. However, we have observed that loss of expression of OLFM4 were found in more than 58% of the samples obtained from advanced prostate cancer patients. Therefore, we sought to study the role of epigenetic factors in the regulation of OLFM4 gene expression in normal and prostate cancer cell lines. Methylation of CpG sites in the promoter region of key genes is an important epigenetic mechanism in cancer development. We examined the methylation status of CpG sites in the OLFM4 promoter region in the benign immortalized prostate epithelial cell line (RWPE1), androgen-responsive cancer cells (LNCaP, VCaP), and androgen-independent cancer cells (PC-3 and DU145). There are eight CpG sites (-681, -666, -562, -486, -91, +4, +34) in the promoter regions of OLFM4 gene. The pattern of methylation status was detected and compared between RWPE1 cell and cancer cell lines LNCaP, VCaP, PC-3 and Du145. To test whether hypermethylation of CpG sites of OLFM4 gene can block the expression of OLFM4 gene, RWPE1 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 μM) from 24h to 96h and PC-3 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 μM) for 3 days and 7 days. The methylation status of CpG sites was compared in the presence or absence of 5-Aza. at different time points. The addition of 5-Aza. consistently reduced the percent methylation of the CpG sites. Correspondingly, the expression of the OLFM4 gene was induced after treatment with 5-Aza. in both RWPE1 and PC-3 cells. These results suggest that expression of OLFM4 is regulated by the methylation status of OLFM4 gene in the RWPE1 and PC-3 prostatic cell lines. Citation Format: Hongzhen Li. Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2787.

Rett Syndrome: Reaching for Clinical Trials.

Rett syndrome (RTT) is a syndromic autism spectrum disorder caused by loss-of-function mutations in MECP2. The methyl CpG binding protein 2 binds methylcytosine and 5-hydroxymethycytosine at CpG sites in promoter regions of target genes, controlling their transcription by recruiting co-repressors and co-activators. Several preclinical studies in mouse models have identified rational molecular targets for drug therapies aimed at correcting the underlying neural dysfunction. These targeted therapies are increasingly translating into human clinical trials. In this review, we present an overview of RTT and describe the current state of preclinical studies in methyl CpG binding protein 2-based mouse models, as well as current clinical trials in individuals with RTT.

Quantitative analysis of P16 gene CpG methylation in Uyghur patients with cervical squamous cell carcinoma and its relationship with HPV16 infection.

The methylation of CpG sites in the promoter region of the P16 gene in Uyghur patients with cervical squamous cell carcinoma (CSCC) was quantitatively analyzed and its relationship with human papillomavirus 16 (HPV16) infection was explored. Cervical samples were collected from 20 Uyghur patients with CSCC and 20 Uyghur controls. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was applied to detect methylation of CpG sites in the promoter region of the P16 gene; polymerase chain reaction was performed to assess HPV16 infection in the 2 groups. Among the 16 CpG sites in the P16 gene promoter region, the methylation level of the CpG1-2 and CpG 6 sites, as well as the HPV16 infection rate, was higher in the CSCC group than in the control group (P<0.05). There was no significant correlation between P16 CpG methylation and HPV16 infection in Uyghur patients with CSCC. The P16 gene CpG1-2 and CpG 6 hypermethylation and HPV16 infection, which are independent of each other, play an important role in cervical squamous cell carcinogenesis in Uyghur patients.

High Preservation of CpG Cytosine Methylation Patterns at Imprinted Gene Loci in Liver and Brain of Aged Mice

A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the multiple documented changes with age are likely to involve exceptions to this pattern, possibly associated with specific cellular responses to age-related changes other than genotoxic stress.

A novel DNA-binding feature of MeCP2 contributes to Rett syndrome

GENERAL COMMENTARY article Front. Cell. Neurosci., 09 May 2013Sec. Cellular Neurophysiology Volume 7 - 2013 | https://doi.org/10.3389/fncel.2013.00064

Abstract 4538: Semiquantitative detection of aberrant methylation as a tumor biomarker in the serum and plasma of patients with gastric cancer

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric cancinoma (GC). DNA methylation of multiple CpG sites in promoter regions of several tumor related genes, such as hMLH1, p14, COX2, and APC has been analyzed in GC so for. Circulating tumor DNA in serum/plasma has been demonstrated to reflect the biological characteristics of tumors. Recently, the presence of gene promoter methylation in serum/plasma DNA has also been demonstrated in patients with cancers of lung, head and neck, liver, and breast. So, in the present study, we attempted to determine the feasibility and the clinical correlations of detecting tumor-associated aberrant methylation in the serum/plasma of patients with gastric cancer. Materials and Methods: We examined promoter methylation of 6 genes using methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) in paired serum, plasma and tumor samples of 50 gastric cancer patients. The tumor and the paired serum/plasma were investigated for aberrant methylation in BLU, DAPK1, GADD45G, MGMT, p15 and p16. The sensitivity of the HRM analysis was tested by using dilutions of fully methylated DNA into unmethylated DNA. Results: MSP was used to assess gene methylation in tumor samples. Promoter methylation in BLU, DAPK1, GADD45G, MGMT, p15, and p16 were detected in 47%, 37%, 59%, 32%, 14%, and 43% of tumor tissues by MSP. MS-HRM was used to assess gene methylation in serum/plasma samples concurrently. In the serum/plasma of gastric cancer patients, BLU, DAPK1, GADD45G, MGMT, p15, and p16 were methylated at frequencies of 26%, 21%, 23%, 11%, 9%, and 17%, respectively. Nineteen (38%) gastric tumor samples displayed concurrent methylation in three or more tumor genes. Aberrant methylation in one or more genes was found in 74% (37/50) serum samples and 70% (35/50) plasma samples. However, there was not a significant relationship of gene methylation between gastric tumor tissues and serum/plasma samples.Conclusion: These results suggest that aberrant promoter methylation in serum/plasma can be detected in a substantial proportion of gastric cancer patients. Detection of DNA methylation in serum/plasma may be a biomarker for early detection of gastric cancer. MS-HRM is powerful technique for the analysis of promoter methylation. Application of HRM analysis to large amount of clinical samples proves to be a fast and high-throughput way to investigate the epigenetic status of genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4538. doi:1538-7445.AM2012-4538

Abstract 2221: DNA methylation of microRNA genes in gastric carcinoma and its clinicopathological association

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric carcinoma (GC). DNA methylation of multiple CpG sites in promoter region of several tumor-related genes, such as hMLH1, p14, p16, and APC has been analyzed in GC so far. MicroRNAs (miRNA) are small, non-coding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies indicate that epigenetic silencing of miRNAs with tumor suppressor activity by CpG island methylation plays an important role in gastric carcinogenesis. To explore the role of epigenetic mechanisms in the down-regulation of miRNA genes (has-miR-9, has-miR-129, has-miR-137), we examined the presence of DNA methylation-associated silencing of miRNAs in GC and observed that aberrant methylation of these miRNAs is associated with expression of target gene products. Materials and Methods: The extent of promoter methylation of 4 miRNA genes (has-miR-9-1, has-miR-9-3, has-miR-129-2, has-miR-137) was assessed using methylation-specific polymerase chain reaction (MSP) in 100 GC tissues (50 early gastric cancers and 50 advanced gastric cancers) and corresponding non-tumor tissues. Twenty patients with benign gastric pathology were also included as a control group. The potential target gene products of miRNAs were studied by immunohistochemistry and the relationship between methylation profile of miRNAs promoter and clinicopathological parameters was analyzed. Results: Methylation of the has-miR-9-3 and has-miR-137 CpG island was frequently observed in tumor tissues (89% and 86%, respectively) and non-tumor tissues in 100 GC patients (70% and 78%), but not in normal gastric tissues (55% and 55%). Methylation of the has-miR-9-1 is slightly higher in tumor tissues (65%) than normal gastric tissue (45%). However, methylation level of the has-miR-129-2 was not shown significant difference in tumor tissues (97%) compared with non-tumor tissues (90%) and normal gastric tissues (90%). Expression of NF-κB and SOX4 protein, which are has-miR-9 and has-miR-129-2 potential target respectively, were inversely correlated with methylation level of miRNAs. The presence of H. pylori infection was significantly associated with aberrant methylation of miRNA genes (p&amp;lt;0.05). But, there was no significant correlation between aberrant methylation of miRNAs and other clinicopathological factors. Conclusion: These results suggest that specific miRNAs methylation in GC could be an important molecular mechanism causing loss of control on its target, and it may be correlated with the high transcriptional activity of target gene. Epigenetic silencing of some miRNAs may involve in the early stage of gastric carcinogenesis and could be used as an efficient diagnostic biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2011-2221

Abstract 157: Quantitative COX2 promoter methylation analysis in gastric carcinoma

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric cancinoma. DNA methylation of multiple CpG sites in promoter regions of several tumor related genes, such as hMLH1, p14, p16, and APC has reported in gastric carcinoma. Recent studies indicate that aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in a subset of gastric carcinomas (GC). To determine the occurrence of CpG island hypermethylation in GC in relation to H. pylori infection, the status and level of CpG methylation in promoter region of COX-2 was analyzed in early and advanced gastric carcinomas as opposed to normal gastric tissues. Materials and Methods: The extent of promoter methylation of COX-2 gene was assessed quantitatively using Pyrosequencing (PS) in 60 early gastric cancers (EGC) and 60 advanced gastric cancers (AGC) samples harvested upon gastrectomy, and 40 normal gastric mucosa samples from patients with benign gastric pathology. Giemsa stain was performed to detect H. pylori in neoplastic and non-neoplastic gastric mucosa. Gene product was studied by immunohistochemistry and the relationship between methylation profile of gene promoter and clinicopathological parameters was analyzed. Results: The PS analysis of GCs revealed a high frequency of COX-2 methylation in 30.0% (36/120). The methylation frequency for COX-2 gene by PS techniques was significantly higher in EGCs than in AGCs (40.0% and 20.0%, respectively, p&amp;lt;0.05) There was a significant difference of COX-2 methylation in between GCs and normal gastric tissues (30.0% vs. 10.0%, respectively by PS, p&amp;lt;0.05). The mean methylation level in 8 CpG sites of COX-2 promoter by PS was 19.9%, 33.5%, and 18.5% in normal gastric tissue, EGCs, and AGCs respectively. COX-2 gene methylation was significantly associated with H. pylori infection (p&amp;lt;0.001). However, the methylation of COX-2 promoter by PS was not correlated with reduced or absent expression of COX-2 protein (p&amp;gt;0.05). Conclusion: we found that COX-2 promoter methylation was significantly higher in the tumor tissues and was early event for gastric carcinoma development, but not in progression of GC. These results suggest that COX-2 gene methylation may be important in the initial development of gastric carcinogenesis and the H. pylori infection is associated with promoter methylation of COX-2 gene. Furthermore, standard PCR followed by PS could be a more specific and quantitative method providing a more comprehensive picture of the distribution of DNA methylation throughout the promoter regions of specific genes, which will be of benefit in oncology research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 157.

Inhibition of DNMT1 with 5‐aza‐2′‐deoxycytidine induces expression of tumor antigens (GAGE, MAGE, PAGE, and CT45) in MCF7 cells

Cancer cells often express tumor antigens that are recognized by host cytolytic T lymphocytes. Such antigens include GAGE (G antigen), MAGE (melanoma specific antigen), PAGE (prostate specific antigen), and CT45 (cancer/testis antigen). Repression of tumor antigens is a mechanism by which cancer cells may evade recognition by the immune system. Methylation of CpG sites in promoter regions of specific genes leads to recruitment of methylated DNA binding proteins and co‐repressors that cause chromatin compaction and gene repression. This is a key mechanism contributing to abnormal gene expression patterns observed in cancer cells. We are investigating the effect on gene expression of 5‐aza‐2′‐deoxycytidine (ADC), an inhibitor of DNA methyltransferase 1 (DNMT1), in the human breast cancer cell line, MCF7. Cells were treated in vitro for 72 hours with 1 µM ADC in DMSO. RNA was then isolated for microarray analysis (Affymetrix). Compared with cells treated with DMSO alone, ADC‐treated cells exhibited increased expression (≥2‐fold) of 38 genes. Ten of these genes were members of the GAGE, MAGE, PAGE, or CT45 families, with all 10 exhibiting &gt;5‐fold increased expression. These results suggest that tumor antigens are repressed epigenetically in MCF7 cells. De‐repression of these antigens using drugs that target epigenetic factors may provide an effective strategy for cancer treatment. American Cancer Society

Pyrosequencing® assays to study promoter CpG site methylation of the O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A, and APC1A genes

DNA methylation of CpG sites in promoter regions of several cancer related genes, such as O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A and APC1A, has been actively explored recently. Much of the data has been obtained using a variation of an allele-specific PCR assay known as methylation specific PCR. This technique is objectionable for a number of methodological limitations and drawbacks. We wanted to study the promoter regions of the above mentioned genes using bisulfite-treated genomic DNA amplified by PCR, using primers designed to bind only to CpG-free sequences. The methylated fraction (%) of each CpG site was measured by Pyrosequencing technology. For three of the genes, O6-MGMT, hMLH1 and p14ARF, two amplicons were designed to cover all relevant CpG sites. Several of the amplicons were analyzed by two different Pyrosequencing assays. In all, we designed nine optimized PCR protocols and 13 Pyrosequencing assays covering the promoters of all six studied genes. In all cases, standard PCR generated sufficient quantities of pure amplicons to be further analyzed by Pyrosequencing technology. Thus, a total of 119 CpG sites in six genes could be quantified. We conclude that standard PCR followed by Pyrosequencing is a workable, more specific and quantitative alternative to 'methylation specific' PCR. This approach provides a more comprehensive picture of the distribution of DNA methylation throughout the promoter regions of the studied set of six genes, which will be of benefit in oncological research.

Comprehensive Promoter Methylation Analysis of Genes on 5q31.1 in MDS and AML.

Interstitial deletion or loss of chromosome 5 is a frequent chromosomal abnormality in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Previous studies uncovered a 1.5–2Mb region on 5q31.1 to be a common deleted region (CDR) in those diseases. It is believed that there exists one or more of critical tumor suppressor gene(s) in this CDR. Mutation analysis, however, failed to identify target gene(s). With a hypothesis that epigenetic silencing may play a significant role as a “second hit” in MDS/AML, we performed a comprehensive methylation analysis of genes in this CDR on 5q31.1. Among 25 genes that are located in this region (SPOCK-AF116688, covering CDR determined by Horrigan et al. Blood 2000 and Fang et al. Genomics 2000), 14 genes had promoter regions in CpG islands that were therefore susceptible to methylation dependent silencing. Methylation status of multiple CpG sites in promoter regions of these those 14 genes (SPOCK, HNRPA1, PKD2L2, C5orf5, KIF20A, GFRA3, CDC25C, C5orf6, JMJD1B, C5orf19, EGR1, ETF1, HSPA9B, CTNNA1, SIL1) was analyzed using bisulfite-PCR followed by pyrosequencing. Analyzed samples were 13 leukemic cell lines as well as peripheral blood mononuclear cells from 5 patients with AML with 5q deletion (5qAML), 9 AML without 5q deletion (non-5qAML), 17 MDS with 5q deletion (5qMDS), 9 MDS without 5q deletion (non-5qMDS) and 10 individuals without leukemia. Significant hypermethylation (>15% by pyrosequencing) was observed in 5 genes in patient samples (SPOCK, PKD2L2, GFRA3, HSPA9B, and ETF1, see Table 1) and 3 other genes in cell lines only (CDC25C, C5orf19, CTNNA1). In 5qMDS, the most frequently methylated genes were ETF1 (5/17), GFRA3 (4/17), and HSPA9B (11/17), but HSPA9B also showed methylation in normal blood. We then evaluated the expression levels of 5 genes that showed significant methylation in patients' samples using quantitative RT-PCR. SPOCK, PKD2L2, GFRA3 were not expressed in normal PBMC. HSPA9B expression levels did not correlate with the degree of methylation. On the other hand, ETF1 expression was inversely correlated with the degree of methylation. ETF1 is a eukaryotic translation termination factor, which plays a role in recognizing stop codon in mRNA and terminates translation. Based on specific hypermethylation and silencing in 5qMDS, we propose ETF1 as a candidate tumor suppressor gene in the 5q31.1 region. Functional analysis of ETF1 in leukemic cells is underway.Frequency of promoter CpG sites methylationSamplesNSPOCKPKD2L2GFRA3HSPA9BETF1Cell lines1362%46%77%38%0%5qAML50%0%0%20%0%Non-5qAML933%0%0%0%0%5qMDS176%6%24%65%29%Non-5qMDS911%11%33%33%11%Normal100%0%0%90%0%

Analysis of CpG methylation in the killifish CYP1A promoter

Fundulus heteroclitus (Atlantic killifish or mummichog) inhabiting a creosote-contaminated Superfund site on the Elizabeth River (VA, USA), exhibit a lack of induction of cytochrome P4501A (CYP1A) mRNA, immunodetectable protein, and catalytic activity after exposure to typical inducers. This “refractory CYP1A phenotype” is not explained by alterations in mRNA expression of known CYP1A transcription factors. Furthermore, the refractory phenotype is lost progressively during development in laboratory-reared F 1 generation fish. Thus, while heritable, the refractory CYP1A phenotype does not appear to be genetically based. To test the hypothesis that cytosine methylation at CpG sites in the promoter region of CYP1A underlies the refractory CYP1A phenotype, we employed bisulfite sequencing to compare the methylation status of CpG sites in the CYP1A promoter region of DNA from killifish from the Elizabeth River and a reference site. We examined genomic DNA both from livers of wild-caught adult killifish and from pools of F 1 generation embryos raised in the laboratory. In fish from both the contaminated and the reference site, cytosine methylation was not detectable at any of the 34 CpG sites examined, including 3 that are part of putative xenobiotic response elements.