CpG Island DNA Research Articles

The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus.

BackgroundHepatitis B virus (HBV) infection correlated with the development of cirrhosis, liver failure and hepatocellular carcinoma (HCC), poses a huge health burden on the global community. However, the pathogenesis of chronic hepatitis B (CHB) remains unclear. Apolipoprotein A1 (ApoA1) mainly secreted by hepatocytes, represents the major protein component of high-density lipoprotein. ApoA1 secretion may be disrupted by HBV infection. In this study, we mainly investigated the molecular mechanism of ApoA1 down regulated by HBV for revealing the pathogenesis of CHB.MethodsApoA1 expression in livers of CHB patients as well as healthy controls were performed by Real-time PCR (RT-PCR) and Western blot. The serum ApoA1 levels were measured by Enzymed-linked immunosorbent assay (ELISA). Expression of ApoA1 mRNA and protein levels were performed by RT-PCR and Western blot in human hepatoma HepG2 cells and subline HepG2.2.15 cells. HBV expression construct, pHBV1.3 were transfected into HepG2, the changes of ApoA1 mRNA and protein expression were detected by RT-PCR and Western blot. To further study the mechanism of ApoA1 down regulation by HBV, 11 CpG islands in ApoA1 promotor were tested for DNA methylation status by MSP. HepG2.2.15 cell lines were treated with DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), then, expression of ApoA1 mRNA and HBV particles in the supernatant, as well as ApoA1 protein levels were detected by RT-PCR and Western blot. Secretion of HBsAg and HBeAg in HepG2 cells cotransfected with pApoA1 and pHBV1.3 constructs was tested by ELISA. Meanwhile, secretion of HBsAg and HBeAg in the supernatant were quantified by ELISA in the HepG2.2.15 cells treated with 5-aza-dC plus ApoA1 siRNA.ResultsExpression of ApoA1 mRNA and protein levels, as well as serum ApoA1 levels in CHB patients were decreased corresponding healthy controls in vivo. In addition, the expression of ApoA1 mRNA and protein levels were down regulated in HepG2.2.15 cells correponding HepG2 cells, 11 CpG islands in ApoA1 promoter were tested for methylation status by MSP in HepG2.2.15 cells compared to HepG2 cells, while two CpG islands were found hypermethylated. Expression of ApoA1 mRNA and protein levels were increased in HepG2.2.15 cells treated with DNA methyltransferase inhibitor 5-aza-dC. Furthermore, overexpression of ApoA1 can enhance HBV expression in HepG2 cells while the inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi.ConclusionsEpigenetic silencing of ApoA1 gene expression by CpG island DNA hypermethylation induced by HBV may contribute to the pathogenesis of CHB.

Impact of Global and Gene-Specific DNA Methylation in de Novo or Relapsed Acute Myeloid Leukemia Patients Treated with Decitabine.

In this investigation, global DNA methylation patterns and the specific methylation status of 5 genes were studied in DNA from peripheral blood (PB) and impact on progression free survival (PFS) and overall-survival (OS) in patients with de novo or relapsed acute myeloid leukemia (AML) treated with decitabine-based regimens waas assessed. DNA was isolated from PB samples at the time of -1, 1, and 7 days of chemotherapy. Global methylation was determined by ELISA, and the CpG island DNA methylation profile of 5 genes using a DNA methylation PCR system. Our data demonstrated that patients with a high level of 5-mC had a poor prognosis after demethylation therapy and those who have low levels of 5-mC in PB achieved higher CR and better SO, but there was no significant correlation found between the 5-mC levels and other clinical features before treatment except the disease status. Higher methylation status of Sox2 and Oct4 genes was associated with differential response to demethylation therapy. A relatively low methylation percentage in one or both of these two genes was also associated with longer OS after decitabine based chemotherapy. We also suggest that global DNA and Oct-4/Sox2 methylation might impact on the pathogenesis of leukemia and play an important role in the initiation and progression. Moreover, dynamic analysis of 5-mC and Oct-4/Sox2 in peripheral blood nucleated cells of leukemia patients may provide clues to important molecular diagnostic and prognostic targets.

MicroRNA Methylation in Colorectal Cancer.

Epigenetic alterations such as DNA methylation, histone modifications and non-coding RNA (including microRNA) associated gene silencing have been identified as a major characteristic in human cancers. These alterations may occur more frequently than genetic mutations and play a key role in silencing tumor suppressor genes or activating oncogenes, thereby affecting multiple cellular processes. In recent years, studies have shown that microRNAs, that act as posttranscriptional regulators of gene expression are frequently deregulated in colorectal cancer (CRC), via aberrant DNA methylation. Over the past decade, technological advances have revolutionized the field of epigenetics and have led to the identification of numerous epigenetically dysregulated miRNAs in CRC, which are regulated by CpG island hypermethylation and DNA hypomethylation. In addition, aberrant DNA methylation of miRNA genes holds a great promise in several clinical applications such as biomarkers for early screening, prognosis, and therapeutic applications in CRC.

Promoter Methylation Analysis Reveals That KCNA5 Ion Channel Silencing Supports Ewing Sarcoma Cell Proliferation.

Polycomb proteins are essential regulators of gene expression in stem cells and development. They function to reversibly repress gene transcription via posttranslational modification of histones and chromatin compaction. In many human cancers, genes that are repressed by polycomb in stem cells are subject to more stable silencing via DNA methylation of promoter CpG islands. Ewing sarcoma is an aggressive bone and soft-tissue tumor that is characterized by overexpression of polycomb proteins. This study investigates the DNA methylation status of polycomb target gene promoters in Ewing sarcoma tumors and cell lines and observes that the promoters of differentiation genes are frequent targets of CpG-island DNA methylation. In addition, the promoters of ion channel genes are highly differentially methylated in Ewing sarcoma compared with nonmalignant adult tissues. Ion channels regulate a variety of biologic processes, including proliferation, and dysfunction of these channels contributes to tumor pathogenesis. In particular, reduced expression of the voltage-gated Kv1.5 channel has been implicated in tumor progression. These data show that DNA methylation of the KCNA5 promoter contributes to stable epigenetic silencing of the Kv1.5 channel. This epigenetic repression is reversed by exposure to the DNA methylation inhibitor decitabine, which inhibits Ewing sarcoma cell proliferation through mechanisms that include restoration of the Kv1.5 channel function. This study demonstrates that promoters of ion channels are aberrantly methylated in Ewing sarcoma and that epigenetic silencing of KCNA5 contributes to tumor cell proliferation, thus providing further evidence of the importance of ion channel dysregulation to tumorigenesis.

SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.

Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds. In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations. We found that the SDHBR46Q mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4 ± 63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation.Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylatorphenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines. These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

Prenatal Exposure to Cadmium and Cotinine and CpG island DNA methylation in Mother-Infant Pairs.

A precise biological mechanism by which cadmium acts as a developmental toxicant is unknown but is suggested to include an epigenetic basis. In prior work, we analyzed CpG island methylation levels within gene promoters (n = 16,421) in leukocytes collected from mothers and their infants from a pregnancy cohort in Durham County, North Carolina. The CpG methylation levels were examined in relationship to prenatal exposure to cadmium and/or cotinine to identify genes and pathways influenced by in utero exposure. In the present article, we provide an enhanced description of the data collection and processing to facilitate cross-study comparisons. Data are available within the Gene Expression Omnibus database (GSE67976).

Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy.

Different Mechanisms of Epigenetic Regulation of Gene Expression

In our days it becomes clear that not only the genetic information encoded in the sequence of DNA is responsible for gene expression profile but the epigenetic signature encoded in the pattern of modifications of histones that associate with DNA in chromosomes also plays a very important role in the determination of gene expression. Octomere of histones (H3/H4 and H2A/H2B) and a segment of DNA surrounding histone proteins compose a nucleosome, a basic unit of chromatin that in turn is the first level of chromosome organization. Besides the hypermethylation of CpG islands of DNA that leads to gene repression multiple modifications of histones also influence gene expression either activating or inhibiting it. These histone modifications altogether are called a “histone code” and include such diverse covalent modifications of Lys, Arg, Ser residues of histones as acetylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination that are usually reversible. While acetylation of histones is an activating mark, methylation of histones at particular residues could be either repressive or activating modification. Although changes in histone modification pattern already lead to changes in gene expression, the data were accumulated indicating that complete replacement of whole nucleosome or nucleosome arrays with newly synthesized and modified nucleosomes could induce gene activation or repression. In this review the data on different epigenetic mechanisms of gene expression regulation will be presented. Regulation of interferon-beta gene expression will be described in more details as comprehensively studied example of epigenetic regulation of gene expression.

Abstract B20: Global hypomethylation of esophageal myofibroblasts is associated with accelerated tumor growth.

Abstract Cancer associated myofibroblasts (CAMs) originating from either tissue-resident fibroblast precursors or from bone marrow derived mesenchymal stromal cells, contribute to the tumor microenvironment by the secretion of growth factors, cytokines, proteases and ECM proteins. Exaggerated CAM reactions have been associated with adverse outcomes in various cancers. The role of altered DNA CpG-island methylation in promoting tumorigenesis has been widely studied in cancer cells; altered DNA methylation in cancer stromal cells including CAMs has also been described but its biological significance remains uncertain. Our present objective was to determine the significance of DNA methylation in influencing the properties of esophageal CAMs. In initial studies we demonstrated different DNA methylation patterns in seven low passage primary myofibroblast lines from esophageal adenocarcinomas (n=3) and squamous cell carcinoma (n=4) patients in comparison to matched myofibroblasts derived from adjacent, macroscopically tumor-free, tissue (ATMs). Further, we found changes in global DNA methylation assessed in LINE1 elements by pyrosequencing analysis between CAMs and matched ATMs. Cluster analysis of the myofibroblast methylome determined by Illumina 450k human DNA methylation array revealed distinct methylation patterns in CAMs and ATMs. In order to generate a model for studying the biological consequences of epigenetic modification of myofibroblasts we treated cultured primary low passage normal esophageal tissue myofibroblasts (NTMs) with the DNA methyl transferase inhibitor, 5'Aza-2'deoxycytidin (DAC). A three-day exposure of NTMs to 0.1μM DAC induced a transient 10% decrease of global DNA methylation assessed by LINE1 element analysis; DAC (0.1 μM) also decreased myofibroblast proliferation by 20 %, and this persisted up to 28 days after treatment, and it stably increased migration in transwell migration assays by 50%. In a nude mouse xenograft model using esophageal squamous carcinoma cells (OE21) inclusion of CAMs stimulated tumor growth more than the inclusion of ATMs; moreover, myofibroblasts pretreated with DAC increased tumor growth compared with xenografts of OE21cells and untreated myofibroblasts. Our data indicate (a) modification of DNA methylation in esophageal cancer myofibroblasts, and (b) that DNA demethylation induced by DAC stimulates migration of CAMs as well as their capacity to promote xenograft growth in vivo. Understanding the mechanisms and consequences of epigenetic changes in esophageal stromal cells may be useful in developing future anticancer strategies. Citation Format: Olivier T. Giger, Benjamin Brown, Triantafillos Liloglou, Puthen V. Jithesh, J Graham Dockray, Andrea Varro. Global hypomethylation of esophageal myofibroblasts is associated with accelerated tumor growth.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B20. doi:10.1158/1538-7445.CHTME14-B20

Analysis of CpG Island DNA Methylation of p15INK4b and RIL in Bone Marrow Samples of Patients with Monoclonal Gammopathies

Introduction: Adding a methyl group on the cytosine residues of CpG dinucleotides (CpGs) is a basic phenomenon in DNA methylation. This modification belongs to a group of epigenetic changes that are thought to play one of the key roles in tumor development and progression. DNA hypermethylation occurs mainly in CpG islands of the promoter region in tumor-supressor (TS) genes, which leads to gene silencing. On the other hand, global DNA hypomethylation leads to a genomic instability. Many specific genes are epigenetically changed in individual malignant diseases and these genes are intensively studied by the scientific community. According to the recent research in plasma cell neoplasms, the global genic hypomethylation is predominant in contrast to hypermethylation events. Epigenetic changes of TS genes may worsen prognosis, drug response and microenvironment interaction in multiple myeloma patients. Our aim was to assess the methylation level of the RIL and p15INK4b gene. RIL protein acts as suppressor of cell proliferation and it sensitizes tumor cells to apoptosis, on the other hand, the p15INK4bplays an important role in inhibiting the cell cycle progression in G1 phase.Methods: Our updated study includes 72 bone marrow aspirate-samples from 68 patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS). 68 samples were acquired at the time of the diagnosis and four patients with MM were assessed also in remission after chemotherapy. Extracted DNA after bisulfite modification was examined by pyrosequencing method (Pyromark Q96, Qiagen, Germany) and the selected gene regions – RIL promotor, p15INK4b promotor and p15INK4b exon were analysed. Nine CpGs in the RIL promoter, thirteen CpGs in the p15INK4b promoter and sixteen CpGs in the p15INK4bexon were studied. Average methylation level (MtL) was generated and samples were sorted according to the mean MtL (%) into four groups for the RIL gene (less than 10% MtL, 11-19% MtL, 21-50% and more than 50%). We used commercially available unmethylated DNA and methylated DNA control. Statistical analysis was performed using Pyromark Software and Wilcoxon rank sum test.Results: Out of the four groups for the methylation level of the RIL gene promoter, the groups with 11-19% MtL and 21-50% MtL, detected significant and highly significant differences respectively at both p-values - p-value ≤0.05 and p-value ≤0.01. There were only 2 samples with MtL level over 50% in patients with active MM, precluding valid statistical analysis. In both regions of the p15INK4b gene, i.e. the promoter and the exon, we did not find any significant differences between the methylation levels - mean MtL ranged from 3% to 6% in p15INK4b promotor and from 3% to 12% in p15INK4b exon. The time-dependend analysis of the DNA methylation level changes in four patients assessed at the time of diagnosis and in remission of the disease after chemotherapy course revealed a significant decrease of CpGs methylation in all MM patients reaching therapeutic response.Conclusions: Updated data on the epigenetic analysis in patients with monoclonal gammopathies confirmed an important role of the RIL gene in the epigenetic network acting in pathogenesis of MM. The presented results suggest possible prognostic value and therapeutic target in patients with MM. In contrast, the p15INK4b gene known as a frequent focus of epigenetic modifications in many tumors, did not show statistically significant differences in our study. Our results indicate that for DNA methylation analyses of MM or MGUS patients, the RIL gene could be more useful marker than the p15INK4b gene. Moreover, the decrease of the methylation level in patients undergoing systemic chemotherapy might be associated with treatment response, suggesting its potential prognostic value.The paper was supported by the grant IGA MZ CR NT14393. DisclosuresNo relevant conflicts of interest to declare.

210 DNA METHYLATION AND HYDROXYMETHYLATION ANALYSIS IN A MODEL OF OOCYTE DIFFERENTIAL DEVELOPMENTAL COMPETENCE IN SHEEP

DNA methylation is an important epigenetic mark that plays a role in gene regulation by the addition of a methyl group to CpG islands in the DNA. Despite being relatively stable in somatic cells, DNA methylation is subject to reprogramming during embryo development and gametogenesis. The aim of this work was to evaluate different aspects of DNA methylation in relation to oocyte quality in the ovine species. A model of differential developmental competence consisting in ovine oocytes and in vitro produced (IVP) blastocysts derived from adult (AD) and prepubertal (PR) donors, was used. The methylation was analysed in terms of: expression of a panel of genes involved in DNA methylation [DNA methyltransferases (DNMTs)] and demethylation [ten-eleven translocation dioxygenases (TET)] in oocytes and blastocysts; global methylation and hydroxymethylation by direct immunofluorescence; locus-specific methylation of 2 imprinted genes by pyrosequencing. Gene relative quantification was performed by RNA reverse transcription followed by real-time PCR. Pools of 10 immature (GV) and in vitro-matured (MII) oocytes and (IVP) blastocysts derived from AD and PR donors (4 replicates per class) were analysed. Lower expression of TET1, TET2, and TET3 was observed in PR GV oocytes (ANOVA; P < 0.05), while no significant differences were found for the enzymes involved in methylation (DNMT1, DNMT3A, DNMT3B; ANOVA; P > 0.05). The levels of all the genes studied showed no significant differences in embryos at blastocyst stage (ANOVA; P > 0.05). Methylation and hydroxymethylation immunostaining were performed in GV and MII oocytes using anti-5-methylcytosine mouse mAb and 5-hydroxymethylcytosine rabbit pAB. High levels of DNA methylation were observed in both AD and PR GV and MII oocytes, while hydroxymethylation immunopositivity was scattered evident throughout the gamete chromatin. Pyrosequencing of bisulfite converted DNA was used to determine the methylation status within differentially methylated regions (DMR) of maternally imprinted H19 (CTCF binding site IV; 11 CpG sites) and paternally imprinted IGF2R (17CpG sites within intron 2). No differences were observed between classes of oocytes for each gene (pools of 40 oocytes per replicate, 3 replicates per class; ANOVA; P > 0.05). Our work shows no differences in the expression of the enzymes involved in methylation, in accordance with the results of global and locus specific methylation analysis. Conversely, we observed lower expression of the TET genes in PR GV oocytes (ANOVA; P > 0.05). TET1, TET2, and TET3, whose expression has never been studied in ovine, generate 5-hydroxymethlcytosine (5hmC) by oxidation of 5-methylcytosine (5mC), and are involved in active DNA demethylation during early embryo development. Our observation of lower expression of the TET genes in lower competence PR GV oocytes suggests that epigenetic mechanisms may affect oocyte quality and paves the way to better understand methylation dynamics during sheep pre-implantation development.

Abstract 18405: Epidrug Treatment Rescues Proliferation and Differentiation in Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients: A Case of Epimetabolic Memory

Introduction: The origin of metabolic/epigenetic memory remains elusive. It, however, may be at the basis of chronic diseases and functional alteration associated to ageing and cardiovascular disease. HYPOTHESIS: Diabetes is known to alter cellular metabolism causing defects in tissue/organ regeneration. In this study we asked whether human cardiac stromal cells from diabetic and normoglycaemic voluntary donors revealed differences associated to their original metabolism and whether the alterations could be rescued by epigenetically active drugs. METHODS and RESULTS: D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of histone H3 lysine 9 and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5- related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-a general control of amino acid synthesis (GCN5a), determining a relative decreasein total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. CONCLUSIONS: This study investigated the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Abstract 1373: Comprehensive analysis of HBV DNA methylation in liver tissues of hepatitis B, cirrhosis and hepatocellular carcinoma patients

Abstract Hepatitis B virus (HBV) is a hepatotropic virus causing hepatitis, cirrhosis and hepatocellular carcinoma. In patients, HBV DNA circulates in blood as virion DNA and exists in hepatocytes both as nuclear form (episomal cccDNA and integrated DNA) and as cytoplasmic core DNA form. It has been reported that HBV infection up-regulates DNMTs and thus induces epigenetic changes in the host cells. Understanding the methylation status of the HBV DNA in its different forms can potentially provide insight into the pathogenesis of HBV-related liver diseases including hepatocarcinogenesis. Previous studies conducted in patient serum and in cell cultures have demonstrated that the CpG islands of HBV virion DNA are in the unmethylated state, and cccDNA methylation has been extensively studied for its role in the regulation of transcription, but very few studies have investigated the methylation status of integrated HBV DNA in human liver tissues. To better understand the methylation status of HBV DNA in hepatitis B, cirrhosis and HCC, a comprehensive methylation profile of three CpG islands in the HBV genome was assessed by BS-sequencing, and methylation of each of the 3 CpG islands was compared by using bisulfite specific PCR and methylation specific PCR assays in a larger sample size. We observed that the methylation of CpG island 1 and 3 of HBV DNA from liver tissues was significantly higher in HCC as compared to hepatitis and cirrhosis. We also obtained, for the first time, the evidence of rare non-CpG methylation in the CpG island 1 and 2 of HBV genome in liver tissues. The overall co-relation of HBV genome methylation and disease progression was also investigated. In addition, no co-relation between the methylation levels of the HBV genome and host genome was found, suggesting independent mechanisms regulate the methylation of host and viral genomes. The mechanisms of HBV genome methylation in liver disease and its regulation are discussed. Citation Format: Surbhi Jain, Sitong Chen, Batbold Boldbaatar, Selena Y. Lin, Ran Yan, Chi-Tan Hu, Haitao Guo, Timothy M. Block, Wei song, Ying-Hsiu Su. Comprehensive analysis of HBV DNA methylation in liver tissues of hepatitis B, cirrhosis and hepatocellular carcinoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1373. doi:10.1158/1538-7445.AM2014-1373

Abstract 2299: An epigenetic study of DNA methylation and histone modification of brain cancer Glioblastoma multiforme

Abstract Epigenetic modifications to the genome affect gene expression, causing increased risk for cancers and other diseases. DNA methylation and modification of histone proteins are ways in which this occurs. The specific aim of this study was to identify DNA methylation percentages in Glioblastoma multiforme (GBM) samples compared against normal astrocyte genomic DNA(gDNA). Within the context of DNA methylation, a global genome methylation analysis was performed to determine gDNA methylation differences between GBM and a normal astrocyte samples using an Imprint® Methylated DNA Quantification Kit (Catalog # MDQ1, Sigma®, Saint Louis USA). . Methyl-sensitive cut counting analysis (MSCC) technique was used further estimate and compare methylation of cancer and normal gDNA. DNA samples were digested using the restriction endonuclease HpaII, which is methyl-sensitive and will cut DNA at unmethylated CCGG sites. It is estimated that generally 90% of CpG islands in genomic DNA have at least one HpaII site . HpaII sites must be greater than 40 bases from another HpaII site to be designated an individual HpaII site. In this case, the identified gene sequence is considered to be a single site. DNA (2 μg) was digested using 20 units of HpaII.An adaptor with the recognition site for the MmeI restriction enzyme was added using T4 DNA ligase. The DNA was then ethanol precipitated and nicks were repaired using an 8 unit Bst DNA polymerase. MmeI (2 units) was added to the DNA, which served to cleave at 18 bases adjacent to the HpaII sites. These segments were then ligated to another adapter for PCR amplification and sequencing. Tag sizes were purified using a 10% PAGE gel and amplified with quantitative PCR and Bio-Rad iProof high-fidelity polymerase. An Illumina HiSeq 2000 Genome Analyzer at the University of Nebraska Medical Center Sequencing Core Facility was used for tag sequencing. Tag sequences were paired against the human genome (h19) library using Bowtie, a short sequence aligner . Sequence tags, representing an unmethylated HpaII site, were counted in GBM and normal astrocyte gDNA. The change in tag counts between the two samples represented a change in the methylation status of the HpaII site. The results showed GBM genomic DNA(gDNA) possessed lower gDNA methylation percentages compared to normal samples. Methyl-sensitive cut counting analysis (MSCC) showed fold decreases in GBM gDNA methylation sites for genes PBK, KIF23, COL6A3 and LOX.Results could be used as prognostic indicators for glioblastoma therapeutic strategy. ACKNOWLEDGEMENT:Supported by a NIH-RISE GRANT to ELIZABETH CITY STATE UNIVERSITY and a TMCF-DOE award to Dr.Hirendra Banerjee. Citation Format: Hirendra N. Banerjee, GWYN HYMAN, Jeffrey Rousch, VINOD MANGALIK, Deidre Vann, Christopher Krauss, sabrina sharpe, DAVID KLINKEBIEL, SANTOSH MANDAL, MUKESH VERMA. An epigenetic study of DNA methylation and histone modification of brain cancer Glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2014-2299

Abstract 2298: Methylated gene markers discovered in nasopharyngeal carcinoma by differential methylation hybridization (DMH) in high density CpG island DNA chips

Abstract Nasopharyngeal carcinoma (NPC) is a prevalent cancer in southern China and south east Asian countries. A variety of tumor suppressor (TS) genes including p16, RASSF1A, EDNRB, DAP-Kinase, RIZ1, E-Cadherin were found to be hypermethylated in NPC. This, however, could only be the tip of an iceberg. We made use of the high throughput differential methylation hybridization (DMH) technique in screening for methylated DNA markers in 244K CpG island DNA chip in three NPC xenografts (C15, C17, 99-186), a NPC tumor cell line (C666) versus an immortalized NP non-cancer cell line (NP69). This DMH screening discovered 100, 213, 372 and 977 genes respectively in 99-186, C17, C15 and C666 with ≥ 5 CpG sites in their gene promotors being methylated. Among these genes, 28 of them were found to have DNA methylation in all the 4 NPC xenografts/ tumor cell line. Bisulphite sequencing was performed in 17 pairs of tumor and normal biopsy samples from 17 NPC patients. This confirmed tumor specific methylation in the promotor regions of 8 gene markers, namely, NRP3, PCDH10, PENK, ADHFE1, IRX4, GSC, PFN2 & GFI1 with positive methylation ratios (+ve MR) in tumor biopsies of 86.7%, 85.7%, 80%, 72.7%, 54.5%, 50%, 41.7% &23.1% respectively and +ve MR of 18.2%, 0%, 0%, 0%, 0%, 37.5%, 12.5% & 0% respectively in normal biopsies. Methylation of CpG sites appears to occur either all or none with 15 to 45 consecutive CpG sites in these genes either all methylated or non-methylated in tumor biopsies probably indicating the need of methylation of a full cluster of CpG sites in order to affect the gene transcription of the discovered genes. Functions of the DMH identified genes includes cell adhesion (PCDH10), signal transduction (PENK), chloride ion binding (NPR3), regulation of DNA dependent transcription (IRX4) and xenobiotic metabolism (ADHFE1). In fact, PCDH10, PENK and GFI1 genes have been reported to be closely associated with NPC/esophageal carcinoma, pancreatic adenocarinoma and T-cell lymphoma respectively. Further analyses in these methylated markers could be useful in the diagnosis and study of etiology of NPC. Citation Format: Wai Wai Cheng, Roger K.C. Ngan, Dora L.W. Kwong, Tim H.M. Huang, Victor W.S. Ma, Stephen C.K. Law, Loretta Tse, Pierre Busson, George S.W. Tsao, Maria Li Lung, Timothy T.C. Yip. Methylated gene markers discovered in nasopharyngeal carcinoma by differential methylation hybridization (DMH) in high density CpG island DNA chips. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2298. doi:10.1158/1538-7445.AM2014-2298

Abstract LB-127: Maternal folate gene polymorphisms define distinct DNA methylation clusters in children with acute lymphoblastic leukemia

Abstract BACKGROUND. Acute lymphoblastic leukemia (ALL) is the most common form of pediatric cancer and occurs as early as infancy, supporting a role of intrauterine exposures. Maternal polymorphisms in genes regulating folate metabolic pathway have recently been reported to modify ALL risk in the child; however, the mechanistic basis underlying this association remains unknown. Folate metabolic pathway is indispensible for DNA methylation and synthesis during embryogenesis and early development. Gene polymorphisms affecting the mother's folate metabolism capacity may exert influence on the offspring's DNA methylome and predispose the child in utero with an increased risk of ALL. Promotor CpG island DNA methylation is a well-recognized phenomenon for silencing tumor-suppressor genes in cancers. Recent studies have discovered subsets of cancer with high degrees of methylation. This phenomenon, termed CpG methylator phenotype (or CIMP), has been validated in different adult cancers. However, the origin of CIMP remains unidentified. Additionally, very few studies have examined the DNA methylome of pediatric ALL and the discovery of CIMP was mainly based on candidate-gene approach. Therefore, in the current study we examined genome-wide DNA methylation in pediatric ALL and determined whether maternal folate gene polymorphisms were associated with distinct clusters of DNA methylation in their offspring. METHODS. We collected bone marrow (BM) specimens from 53 cases whose mothers were genotyped previously on 67 folate SNPs and seven BM specimens from healthy donors. We classified ALL patients as high-risk or low-risk based their mother's folate genotype. Methylated CpG amplification coupled with Hi-Sequencing was performed to determine genome DNA methylation profiles. One-way ANOVA was applied to assess DNA methylation differences among the high-risk, low-risk, and normal groups. Samples were clustered hierarchically using Euclidean distance and complete linkage, and analyzed using Principal Component Analysis (PCA). RESULTS. We have sequenced and analyzed 21 samples (15 cases and 6 normal controls). A mean of 1,401,690 unique reads per sample were matched to human genome hg19. A total of 62,425 sites were identified in these samples. Of these, we identified 320 unique sites with significant differences among the three groups (FDR<0.05). Genes hyper-methylated in normal tissues were generally hypo-methylated in ALL patients, regardless of their risk groups. However, genes normally hypo-methylation showed various degrees of methylation among cancer samples. Especially, high-risk patients displayed intense hyper-methylation across all genes, when compared to the low-risk group. CONCLUSIONS. This is the first study showing that variation in the mother's folate metabolism capacity might explain the CpG methylator phenotypes in pediatric ALL and has an important public health impact in cancer prevention. Citation Format: Teresa Tingting Yiu, Spiridon Tsavachidis, Philip J. Lupo, Rabin Karen, Melissa L. Bondy, Michael E. Scheurer, Lanlan Shen. Maternal folate gene polymorphisms define distinct DNA methylation clusters in children with acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-127. doi:10.1158/1538-7445.AM2014-LB-127

The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Serrated polyps and colorectal cancer risk

SUMMARY Colorectal cancer is among the three most common cancers in the World with a global incidence exceeding 1.2 million new cases and 600,000 deaths per year; yet most cases could be prevented by endoscopic removal of precursor lesions. Efficient screening and follow-up are the best options to reduce the cancer burden caused by colorectal cancer (CRC). Therefore, it is essential to know which colorectal polyps have the potential to progress into cancer. During the last decade, serrated polyps have emerged as the second most important group of CRC precursor lesions, in addition to conventional adenomatous polyps. It has been estimated that up to 30–35% of CRCs have their origin in serrated polyps. Carcinogenesis via serrated polyps, known as the serrated pathway, requires activation of the MAPK pathway by mutations of BRAF and KRAS, and low- or high-level methylation of CpG islands of DNA (CpG island methylator phenotype), and subsequent high-level DNA microsatellite instability in a third of cases. The mechanisms involved in carcinogenesis via the serrated pathway profoundly differ from those of conventional adenoma–carcinoma pathway, and the biology and behavior of serrated polyps also differ from that of adenomatous polyps, which are characterized by chromosomal instability and loss of tumor suppressor genes.

Methylation by DNMT1 is more Efficient in Chronic Lymphocytic Lymphoma Cells than in Normal Cells

Binding of activators and repressors plays a significant role in transcriptional regulation of gene expression. The systems biology approach has been applied to predict and determine the efficiency of this process; however, most of the work is done in prokaryotic systems. Epigenetic regulation provides another dimension of transcriptional regulation, which does not involve changes in the coding region of genes. Methylation of specific CpG residues in the CpG islands of DNA by the enzyme DNA methyl transferase 1 (DNMT1) causes gene silencing. In cancer cells, the enzyme DNMT1 is highly expressed, and the methylation level at specific sites of genes, including tumor suppressor genes, is elevated. This results in the silencing of tumor suppressor genes for carcinogenesis. However, it is not known whether the increase in DNMT1 expression is proportional to the increase in the level of methylation. This paper developed a systems biology approach to determine whether DNMT1 acts more efficiently in cancer cells than in normal cells. The cooperativity of DNMT1 binding to eight selected genes was determined in chronic lymphocytic leukemia (CLL) cells using a modified Hill equation and compared these values with those obtained from normal lymphocytes and granulocytes. The cooperativity of DNMT1 was found to be positive in four cancer-associated genes, whereas their values were negative in both normal lymphocytes and granulocytes. These results show for the first time how to apply systems biology in the analysis of transcriptional gene regulation. This approach will be beneficial in determining the efficiency of genome-wide methylation in development and in diverse types of diseases.

Gene markers in brain tumors: what the epileptologist should know.

Gene markers or biomarkers can be used for diagnostic or prognostic purposes for all different types of complex disease, including brain tumors. Prognostic markers can be useful to explain differences not only in overall survival but also in response to treatment and for development of targeted therapies. Multiple genes with specific types of alterations have now been identified that are associated with improved response to chemotherapy and radiotherapy, such as O(6)-methylguanine methyltranferase (MGMT) or loss of chromosomes 1p and/or 19q. Other alterations have been identified that are associated with improved overall survival, such as mutations in isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2) or having the glioma CpG island DNA methylator phenotype (G-CIMP). There are many biomarkers that may have relevance in brain tumor-associated epilepsy that do not respond to treatment. Given the rapidly changing landscape of high throughput "omics" technologies, there is significant potential for gaining further knowledge via integration of multiple different types of high genome-wide data. This knowledge can be translated into improved therapies and clinical outcomes for patients with brain tumors.