Partial Methylation Research Articles

DrosophilaSet1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.

Studies on separation, purification and structure characteristics of a polysaccharide LTC-Ⅱfrom Pyrola corbieri

To characterize the structure of polysaccharide LTC-II obtained from Pyrola corbieri. The polysaccharide was extracted from P. corbieri by hot water and ethanol precipitation. Crude polysaccharide was purified by DEAE-Cellulose chromatography and Sephacryl S-300 HR column chromatography. The purity and molecular weight of polysaccharide was determined by gel permeation chromatography. UV, IR, optical rotation, complete acid hydrolysis, periodate oxydation, Smith degradation, partial acid hydrolysis and methylation analysis were applied to determine the structural features. A homogeneous fraction LTC-II was obtained and its relative molecular mass was 22 000 Da. It consisted of arabinose, mannose, glucose, galactose in the molar ratio of 35. 2: 1.0: 13. 4: 4. 2. LTC-II had a backbone consisting glucose, mannose, galactose and mainly contained (1 --> 6)-linkaged glucose. The side chain possessed arabinose, glucose, galactose and mainly contained (1 --> 5)-linkaged arabinose. The terminal sugar were mainly glucose and galactose. Studies on the preliminary characterization of polysaccharide LTC-II from P. corbieri for the first time.

Structural characterization and in vitro inhibitory activities in P-selectin-mediated leukocyte adhesion of polysaccharide fractions isolated from the roots of Physalis alkekengi

Selectin-mediated leukocyte initial attachment and rolling over vessel endothelial surface are crucial steps for inflammatory responses. As P-selectin is a promising target for anti-inflammation therapeutic strategy, recent works have focused on searching for more potent and non-toxic P-selectin antagonists among various natural carbohydrate products. Here, we isolated three water-soluble polysaccharide fractions (PPS-1, PPS-2 and PPS-3) from the roots of Physalis alkekengi by DEAE-cellulose and Sephacryl S-200 chromatography. Their physicochemical and structural characterizations were determined by chemical methods, GC (gas chromatography), HPLC (high performance liquid chromatography), FT-IR (Fourier transform infrared spectrometry), partial acid hydrolysis, methylation and GC–MS (gas chromatography–mass spectrometry) analyses. The inhibitory capacity of the polysaccharide fractions in P-selectin-mediated leukocyte adhesion was evaluated by flow cytometric, static adhesion and laminar flow assays. Results showed that different polysaccharide fractions possess distinct physicochemical and structural properties, including carbohydrate, protein and uronic acid contents, molecular weight, monosaccharide composition and glycosidic linkage type. Among the polysaccharide fractions, PPS-2 could effectively block the interaction between P-selectin and its native ligand.

Unfavorable clinical implications for hypermethylation of RUNX3 in patients with salivary gland adenoid cystic carcinoma

To elucidate the potential etiological role of RUNX3 in the development of salivary gland adenoid cystic carcinoma (ACC), we analyzed the methylation status of RUNX3 in a series of 114 ACC tissues and 3 ACC cell lines. Results showed that the methylated rate of RUNX3 was 50.9 and 3.5% in the 114 ACC samples and the corresponding normal salivary glands, respectively, achieving a significant difference (P<0.001). There was a significant correlation between RUNX3 methylation and various clinicopathological parameters of ACCs, such as perineural invasion, lymph node involvement, T-stage and distant metastasis (P<0.001). RUNX3 methylation was a significant predictor of the 5-year disease-free survival in ACC patients after surgery. Partial methylation was found in all 3 ACC cell lines, and the reactivation and more potent expression of RUNX3 was induced by 5-triazole-2-deoxycytidine. Our findings indicate that RUNX3 methylation may occur as a common event in the development of ACC and that methylation may be a major mechanism for inactivation of RUNX3 in ACC.

Abstract 4813: Aberrant promoter methylation of TMS1 gene in patients with colorectal carcinoma in Saudi Arabia

Abstract Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration. It serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Several genes have been shown to be epigenetically inactivated in a wide range of tumors. TMS1 is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to be hyper-methylated in various cancers. Therefore, we studied the methylation status of TMS1 using a panel of colorectal cell lines and 15 CRC cases. Methylation status of the promoter region of TMS1 gene was determined using methylation specific PCR in colorectal cancer cell lines. Seven out of 13 colorectal cell lines showed to be methylated for TMS1 gene (54%). Initial analyses with 15 clinical colorectal cases demonstrated 53% methylation. Real time PCR and western bolt analysis was employed to assess the expression levels. 5-AZA-2′-deoxycytidine was used to demethylate colorectal cancer cell lines. Four out of 7 methylated colorectal cell lines were completely methylated and 3 out of 7 showed partial methylation for TMS1 gene resulting in loss of TMS1 expression. Treating methylated colorectal cell lines with the methyltransferase inhibitor, 5-aza-2’ deoxycytidine alone resulted in partial demethylation and re-expression of TMS1 gene, suggesting the role of methylation in silencing the gene. TMS1 gene has shown tumor suppressive effects after demethylation in thyroid cancer cell. Additionally the oncogenic role of TMS1 has been demonstrated in different cancers. We will further evaluate the role of TMS1 gene after demethylation for apoptosis. After demethylation of TMS1 gene with 5-aza-2’ deoxycytidine we will treat our colorectal cancer cell lines with increasing doses of TRAIL and will evaluate its response by Annexin/PI dual staining and western blot analysis. Our results demonstrated that TMSI gene is methylated in colorectal cancer cells and repression of methylation by 5-Aza-2′-deoxycytidine restored the expression of TMS1 gene. In conclusion, methylation of TMS1 may prove to be a useful prognostic marker and/or predictor of patient survival in colorectal and can be utilized for targeted therapy. 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 4813. doi:10.1158/1538-7445.AM2011-4813

Long interspersed nuclear element-1 hypomethylation in cancer: biology and clinical applications

Epigenetic changes in long interspersed nuclear element-1s (LINE-1s or L1s) occur early during the process of carcinogenesis. A lower methylation level (hypomethylation) of LINE-1 is common in most cancers, and the methylation level is further decreased in more advanced cancers. Consequently, several previous studies have suggested the use of LINE-1 hypomethylation levels in cancer screening, risk assessment, tumor staging, and prognostic prediction. Epigenomic changes are complex, and global hypomethylation influences LINE-1s in a generalized fashion. However, the methylation levels of some loci are dependent on their locations. The consequences of LINE-1 hypomethylation are genomic instability and alteration of gene expression. There are several mechanisms that promote both of these consequences in cis. Therefore, the methylation levels of different sets of LINE-1s may represent certain phenotypes. Furthermore, the methylation levels of specific sets of LINE-1s may indicate carcinogenesis-dependent hypomethylation. LINE-1 methylation pattern analysis can classify LINE-1s into one of three classes based on the number of methylated CpG dinucleotides. These classes include hypermethylation, partial methylation, and hypomethylation. The number of partial and hypermethylated loci, but not hypomethylated LINE-1s, is different among normal cell types. Consequently, the number of hypomethylated loci is a more promising marker than methylation level in the detection of cancer DNA. Further genome-wide studies to measure the methylation level of each LINE-1 locus may improve PCR-based methylation analysis to allow for a more specific and sensitive detection of cancer DNA or for an analysis of certain cancer phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1007/s13148-011-0032-8) contains supplementary material, which is available to authorized users.

Hypermethylation-modulated Downregulation of RASSF1A Expression Is Associated with the Progression of Esophageal Cancer

Chromosome 3p21 is an important locus harboring critical tumor suppressor genes (TSGs) implicated in the pathogenesis of multiple tumors including esophageal carcinoma (EC). Aberrant promoter methylation is a fundamental mechanism of inactivation of TSGs in cancer. RASSF1A, a candidate tumor suppressor gene, recently cloned from the lung tumor locus at 3p21.3, is frequently inactivated by hypermethylation of its promoter region in a number of malignancies. We undertook this study to investigate the methylation status of RASSF1A and its significance in esophageal squamous cell carcinoma (ESCC). Real-time RT-PCR and real-time methylation-specific PCR (real-time MSP) were used to detect RASSF1A expression and the methylation status of the RASSF1A promoter, respectively, in 124 primary ESCC tissues. Hypermethylation, partial methylation and unmethylation of the promoter region of RASSF1A were detected in 56 (45.2%), 23 (18.6%) and 45 (36.2%) of 124 ESCC samples, respectively. Unmethylation of the promoter region of RASSF1A was detected in 119 (96%) of the 124 corresponding noncancerous tissues. Five (4.0%) of 124 noncancerous tissues showed partial methylation. The presence of hypermethylation was statistically associated with loss of RASSF1A mRNA expression in primary ESCC (p <0.05). There were statistically significant correlations between the presence of hypermethylation and regional lymph node involvement (p=0.000), histological differentiation (p=0.009) and tumor stage (p=0.000). Our results suggest that RASSF1A may be one of the ESCC-related TSGs located at 3p21, and hypermethylation of the CpG island promoter of the RASSF1A is associated with the progression of ESCC.

Expansion of CpG methylation in the SFRP2 promoter region during colorectal tumorigenesis.

Secreted frizzled-related protein 2, (SFRP2) is a Wnt inhibitor whose promoter CpGs were recently found to be methylated at high frequency in colorectal cancers (CRCs). We hypothesized that the pattern of SFRP2 methylation may differ throughout the promoter during progressive tumorigenesis. Using combined bisulfite restriction analysis (COBRA), two methylation-sensitive regions (Regions A and B) of the SFRP2 promoter were investigated in 569 specimens of colorectal tissue:222 CRCs, 103 adenomatous polyps (APs), 208 normal colonic mucosa from CRC patients (N-Cs), and 36 normal colonic mucosa from subjects with no evidence of colorectal neoplasia at colonoscopy (N-Ns). Extensive (including both Regions A and B) and partial (either Region A or B) SFRP2 methylation levels were found in 61.7% and 24.8% of CRCs, 8.7% and 37.9% of APs, 3.9% and 39.9% of N-Cs, and 0% and 30.6% of N-Ns, respectively. Extensive methylation of the SFRP2 promoter was present primarily in CRCs, while partial methylation was common in APs. Whereas APs with the KRAS mutant showed no correlation to any pattern of SFRP2 methylation, extensive methylation of the SFRP2 promoter was significantly associated with KRAS mutant CRCs (p＜.0001), suggesting that genetic alteration in the RAS-RAF pathway might precede the spread of CpG methylation through the SFRP2 promoter, which is observed in over 60% of advanced colorectal tumors.

Methylation pattern of IFN-γ and IL-10 genes in periodontal tissues

DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands. Unmethylated CpGs are related to transcriptionally active structure, whereas methylated CpG recruits methyl-binding proteins that promote chromatin compaction. DNA methylation can influence the expression of cytokines and affect the development of periodontal disease. ObjectivesThe purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-γ) and interleukin-10 (IL-10) genes in periodontal tissues. DesignMethylation-specific polymerase chain reaction (MSP) and DNA sequencing analysis were used to verify the DNA methylation status of the IFN-γ and IL-10 genes, respectively, in samples from subjects without periodontitis and individuals with chronic periodontitis. Histological sections stained by hematoxylin–eosin were used for histopathological evaluation of samples. ResultsThe methylation status of the IFN-γ and IL-10 genes was similar among the groups. Most of the samples were positive for IFN-γ methylation. Only 11% of the periodontitis group showed unmethylated DNA. Considering the IL-10 gene, no unmethylated sample was observed. The profile of total or partial methylation was detected in CpGs evaluated. ConclusionsThe results showed evidence that methylation of IFN-γ and IL-10 genes is a usual feature on periodontal tissues. Further studies are needed to determine the functional relevance of these alterations.

Epigenetic investigation of variably X chromosome inactivated genes in monozygotic female twins discordant for primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune chronic cholestatic liver disease with a strong genetic susceptibility due to the high concordance in monozygotic (MZ) twins and a striking female predominance. Women with PBC manifest an enhanced X monosomy rate in peripheral lymphocytes and we thus hypothesized an X chromosome epigenetic component to explain PBC female prevalence. While most genes on the female inactive X chromosome are silenced by promoter methylation following X chromosome inactivation (XCI), approximately 10% of X- linked genes exhibit variable escape from XCI in healthy females. This study was designed to test the hypothesis that susceptibility to PBC is modified by one or more X-linked gene with variable XCI status. Peripheral blood mRNA and DNA samples were obtained from a unique cohort of MZ twin sets discordant and concordant for PBC. Transcript levels of the 125 variable XCI status genes was determined by quantitative RT-PCR analysis and two genes (CLIC2 and PIN4) were identified as consistently downregulated in the affected twin of discordant pairs. Both CLIC2 and PIN4 demonstrated partial and variable methylation of CpG sites within 300 bp of the transcription start site that did not predict the XCI status. Promoter methylation of CLIC2 manifested no significant difference between samples and no significant correlation with transcript levels. PIN4 methylation showed a positive trend with transcription in all samples but no differential methylation was observed between discordant twins. A genetic polymorphism affecting the number of CpG sites in the PIN4 promoter did not impact methylation or transcript levels in a heterozygous twin pair and showed a similar frequency in independent series of unrelated PBC cases and controls. Our results suggest that epigenetic factors influencing PBC onset are more complex than methylation differences at X-linked promoters and variably 3 inactivated X-linked genes may be characterized by partial promoter methylation and biallelic transcription.

RUNX3 expression and its methylation of 5'-CpG island in salivary gland adenoid cystic carcinoma cell lines ACC-2, ACC-3 and ACC-M

To investigate the methylation of 5'-CpG island and expression of RUNX3 in salivary gland adenoid cystic carcinoma cell lines. RT-PCR (reverse transcription-polymerase chain reaction), laser scanning confocal microscope (LSCM) and Western blot were used to detect the expression of RUNX3 gene and protein in salivary gland adenoid cystic carcinoma cell lines, ACC-2, ACC-3, and ACC-M, before/after a treatment of 5-Aza-dc respectively. A weak expression of RUNX3 was found in ACC-2 and ACC-3. And no expression of RUNX3 was found in ACC-3 cell line. After a treatment of 300 nmol/L 5-Aza-dc for 72 hours, the expression of RUNX3 in ACC-2 and ACC-3 cells was enhanced, and in ACC-M was restored. LSCM results showed that the RUNX3 protein was located mainly in the cytoplasm of ACC cell lines. After a treatment of 300 nmol/L 5-Aza-dc for 72 h, both nuclear and cytoplasmic location of RUNX3 positive signals were found in the ACC-2 and ACC-3 cells. However, a weak positive signal was still only found in the cytoplasm of ACC-M cells. Partial methylation in promoter 5'-CpG island of RUNX3 gene was found in all three cell lines. And the methylation degree of CpG island was 50%, 75% and 33% in ACC-2, ACC-M and ACC-3 respectively. After a treatment of 5-Aza-dc, the RUNX3 gene showed unmethylated status in all three cell lines. The methylation of RUNX3 plays an important role in the silencing of RUNX3 expression in ACC cell lines. The cytoplasmic mislocalization of RUNX3 may be correlated with the inhibition of its function in ACC cells.

Effect oftrans-resveratrol and ascorbigens on the fire blight pathogenErwinia amylovorain the BioArena system

On the basis of our earlier observations, preliminary experiments were conducted with the fire blight pathogen Erwinia amylovora with the BioArena system using the potential antibacterial substances 1′-methylascorbigen (MeAG) as formaldehyde (HCHO) precursor and trans-resveratrol (RES) as HCHO mobilizer, capturer, and scavenger. Results showed characteristic, strong, dose-dependent antibacterial effects of both MeAG and RES present as chromatographic spots on the adsorbent layer. It is supposed that the weak antibacterial effect of AG may have originated from MeAG formed in situ on the layer by partial enzymatic methylation of AG. Addition of HCHO capture compounds (l-arginine, glutathione, glucosamine) to the culture medium before inoculation partially or totally reduced the antibacterial effect of both molecules, that is, this simplest aliphatic aldehyde, the endogenous HCHO molecule, participates in the antibacterial activity of these compounds. It follows from these results that there is a possibility...

Frequent Epigenetic Inactivation of MSX2 In Acute Myeloid Leukemia

AbstractAbstract 4645DNA hypermethylation has been implicated in the tumorigenesis and prognosis in acute myeloid leukemia (AML). To identify and validate relevant methylated genes in AML, we have compared expression levels and methylation status of 26 candidate genes. One of the interesting candidates identified in our study is MSX2. MSX2 is a member of muscle segment homeobox gene family. MSX2 plays a role in promoting cell growth under certain conditions and may be an important target for RAS signaling pathways. However, the mechanism of transcriptional regulation and functional role of MSX2 in hematological malignancies, especially AML, are poorly understood. In our study, we determined the methylation status, and analyzed the expression levels of MSX2 in AML cell lines and primary AML cells using RT-PCR and/or Taqman real-time PCR. MSX2 mRNA expression was robust in the normal granulocytes and blasts of human bone-marrow, but was either absent or significantly diminished in 6 of 9 (66.7%) AML cell lines. The expression levels of MSX2 in those 6 AML cell lines were restored after treatment of 5-aza 2′-deoxycytidine. In addition, COBRA (Combined Bisulfite Restriction) analysis demonstrated hypermethylation of MSX2 in those AML cell lines (6 of 9, 66.7%), and partial methylation in 3 of 9 AML cell lines. The methylation status was inversely correlated with the mRNA expression levels of MSX2 in those cell lines. Furthermore, the expression levels and methylation status of MSX2 in human primary AML cells were evaluated. COBRA analysis demonstrated frequent hypermethylation of MSX2 in primary AML patient samples (19 of 32, 59.3%). Importantly, the mRNA expression levels of MSX2 as shown by Taqman real-time PCR in those 19 primary AML patient samples were inversely correlated with the methylation status of MSX2. These findings confirmed the role of frequent DNA hypermethylation in silencing MSX2 in AML. We are in the process of determining the functional role of MSX2 in the pathogenesis of AML. In addition, diagnostic and prognostic values of MSX2 in AML are being pursued. Disclosures:No relevant conflicts of interest to declare.

SOCS1 and SOCS3 Genes Are Not Methylated In Waldenstrom Macroglobulinemia

AbstractAbstract 2481 Background.The suppressor of cytokine signalling (SOCS) 1 and 3 are inhibitors of interleukin 6 (IL6) signalling pathway and demonstrated tumour-suppressor activity. The methylation of SOCS family, resulting in transcriptional silencing, has been suggested to play an important role in development of several hematological disorders, including multiple myeloma. Waldenstrom's macroglobulinemia (WM) is a lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM immunoglobulin. Gene expression profiling has demonstrated an up-regulation in IL6 signalling in WM. IL6 also plays a critical role in cell proliferation and survival of WM cells and in the interaction between the tumour cells and their bone marrow microenvironment. We hypothesized that aberrant SOCS1 and SOCS3 expression may play a role in the pathogenesis of MW. Our study aimed to explore genetic and epigenetic aberrations of SOCS1 and SOCS3 genes. Methods.32 patients (pts) diagnosed with WM were included. Diagnosis was done following guidelines, and pts were not treated at time of BM collection. Approval of this protocol was obtained from the CHRU of Lille and was in accordance with the declaration of Helsinki. We studied RL and MEC1, cell lines that secrete IgM, and BCWM1 developed from a pt with WM. DNA and RNA were extracted following negative CD19 B cells selection. The levels of mRNA of SOCS1 and SOCS3 were evaluated by real-time PCR. Samples were examined at steady state and following PMA stimulation. The CpG island methylation status of SOCS1 and SOCS3 genes in the promoter region was investigated by methylation-specific (MSP) PCR. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both loss of heterozygosity (LOH) and copy number alteration (CNA). Paired DNA samples (tumor cells / normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Results.SOCS1 and SOCS3 genes were expressed in BCWM1, RL and MEC1 cell lines but the baseline expression in cell lines was heterogeneous. PMA induced SOCS1 and SOCS3 expression in all cell lines. No methylation of SOCS1 gene was observed in these cell lines. In contrast, partial methylation of SOCS3 promoter gene was observed in RL and BCMW1. SOCS1 and SOCS3 genes were constitutively expressed at heterogeneous levels in WM pts. The expression varied for SOCS1 from 0.31 to 43 and for SOCS3 from 1.35 to 73. SOCS1 and SOCS3 expressions were enhanced by PMA treatment (n=3 pts). In order to explore genetic aberration on SOCS1 and SOCS3 genes in WM patients, SNP arrays were performed in 17 WM pts. No deletion of SOCS 1 and 3 genes were observed. In one case (5.8%), SNP array karyotyping identified an acquired uniparental disomy (UPD), a LOH without variation of copy number changes, at the SOCS3 locus (17q25). Gain of SOCS1 gene (16p13) was observed in another pt with high mRNA SOCS1 expression. Gene methylation in the promoter was studied in 25 WM pts. No methylation of CpG islands in the SOCS1 and SOCS3 gene promoters was detected in the WM pts. Conclusion.SOCS1 and SOCS3 genes are expressed in WM cells. Copy number aberration, UPD and epigenetic inactivation of SOCS1 and SOCS3 genes seem to be rare events in WM. Other mechanisms might explain for the heterogeneous expression levels of SOCS1 and SOCS3 genes that may contribute for IL6 signalling deregulation in WM. Disclosures:Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Improved measurement of LINE-1 sequence methylation for cancer detection

Background Methylation of long interspersed nuclear element-1 (LINE-1) sequences varies among normal cells and it is often decreased in cancer genomes and white blood cells (WBC) of cancer patients. Current measurement techniques of genome-wide level are inadequate because LINE-1 methylation is distinctive at each locus. Here, we improved the detection of cancer by combining information of LINE-1 methylation pattern and level. Methods Combined bisulfite restriction analysis (COBRA) of LINE-1, COBRA LINE-1, was used to test cancer cell lines, two oral rinse cohorts, and WBC from normal and cancer patients. COBRA LINE-1 separated LINE-1 sequences into 4 products depending on the methylation statuses of 2 CpG dinucleotides, as follows: 2 unmethylated CpGs ( uC uC), partial methylation ( mC uC), 1 methylated CpG ( mC), and 1 unmethylated CpG ( uC). Results The association between mC uC and uC uC was directly correlated in normal cells (r = 0.4895, p = 0.0009) but inversely correlated in cancer (r = − 0.8979, p = 0.0002). Oral rinse AUC values of uC uC were 0.763 and 0.926 and methylation levels were 0.707 and 0.621, respectively. uC uC, but not overall methylation level, differentiated cancer WBC from normal (p = 0.0082 and p = 0.4830, respectively). Conclusion LINE-1 partial methylation represents hypomethylation in normal cells but hypermethylation in cancer cells. This information improves LINE-1 methylation detection in cancer.

Down-Regulation of Serum/Glucocorticoid Regulated Kinase 1 in Colorectal Tumours Is Largely Independent of Promoter Hypermethylation

BackgroundWe have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples.Methodology/Principal FindingsWe investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG.Conclusions/SignificanceOur results show that even though partial methylation of the promoter region of SGK1 is present, this does not account for the different expression levels seen between normal and tumour tissue.

Gene methylation of SFRP2, P16, DAPK1, HIC1, and MGMT and KRAS mutations in sporadic colorectal cancer

The aim of this study was to investigate the methylation of the SFRP2, P16, DAPK1, HIC1, and MGMT genes, as well as the mutation of amino acid codons 12 and 13 of the KRAS gene in normal and tumor tissue DNA of patients diagnosed with sporadic colorectal cancer (SCRC). The methylation of gene regions and the KRAS mutations of normal (N) and tumor tissue (T) DNA obtained from 17 patients diagnosed with SCRC and 20 healthy controls were investigated using the polymerase chain reaction and reverse-hybridization methods. There was an Asp mutation in four patients, an Asp and Ser mutations in one patient in codon 12 of the KRAS gene, and an Asp mutation in codon 13 in eight patients. Overall promoter methylation (OPM) in the SFRP2 gene was observed in one N and four T, whereas partial promoter methylation (PPM) was observed in two N and five T. OPM in the P16 gene was present in one T. In the DAPK1 gene, OPM existed in seven T and five N, while PPM was present in two N. In the HIC1 gene, OPM was demonstrated in three T, while PPM was noted in two N; however, no methylation existed in N. In the MGMT gene, OPM occurred in five T and two N, and PPM was present in one T. KRAS mutations in Turkish patients with SCRC are similar to those of other population groups. Methylations in the genes, which underwent methylation analysis, were higher in T in comparison with N, and it has been suggested that significant results would be obtained by making a study with a larger population.

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K 2HPO 4–KH 2PO 4–H 2O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy ( 1H, 13C). These results showed AAPS-2 is a polysaccharide with a backbone of (1 → 3)-linked-β- d-glucopyranosyl and (1 → 3, 6)-linked-β- d-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β- d-glucopyranosyl at the O-6 position of (1→3, 6)-linked-β- d-glucopyranosyl of the main chain.

Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.

Frequent promoter hypermethylation of TGFBI in epithelial ovarian cancer

Objectives Using pharmacologic unmasking and genome-wide differential methylation analysis, we identified a novel methylated gene in ovarian cancers. Methods Two ovarian cancer cells (OVCAR-3, ES-2) that showed synergistic growth inhibition by 5-aza-dC and cisplatin were selected. After treatment with 5-aza-dC, differential expression profiles were compared using microarray that contained 38,500 genes. Reactivation of candidate genes and their promoter methylation were validated by real-time RT-PCR, MS-PCR and bisulfite sequencing. Methylation status was tested by MS-PCR in 56 patients with epithelial ovarian cancer and compared to the 38 normal ovarian tissues. Results We identified 103 candidate genes that were reactivated by 5-aza-dC treatment. Among those, SFN and TGFBI were commonly reactivated in both cells. Since SFN is a well known methylated marker, we selected TGFBI for further validation. Bisulfite sequencing revealed complete promoter methylation in ES-2 and partial methylation in OVCAR-3. In addition, silencing of TGFBI at the transcription level was reversed by 5-aza-dC treatment. TGFBI methylation was observed in 23 out of 38 (60.5%) cases of ovarian cancer, in no normal ovarian tissues (0 of 38, P = 0.001), and in 5 out of 18 (27.8%) borderline tumors ( P = 0.044). In our cohort, we did not observe any association between methylation of TGFBI and clinicopathologic variables or clinical outcomes. Conclusion Our results confirm that TGFBI is frequently methylated in ovarian cancer. Its methylation can be used as a novel epigenetic biomarker in discriminating ovarian cancer from non-cancer or borderline tumors.