Artificial receptors for the extraction of nucleoside metabolite 7-methylguanosine from aqueous media made by molecular imprinting

A series of imprinted polymers targeting nucleoside metabolites, prepared using a template analogue approach, are presented. These were prepared following selection of the optimum functional monomer by solution association studies using 1H-NMR titrations whereby methacrylic acid was shown to be the strongest receptor with and affinity constant of 621 ± 51 L mol-1 vs. 110 ± 16 L mol-1 for acrylamide. The best performing polymers were prepared using methanol as porogenic co-solvent and although average binding site affinities were marginally reduced, 2.3×104 L mol-1 vs. 2.7×104 L mol-1 measured for a polymer prepared in acetonitrile, these polymers contained the highest number of binding sites, 5.27 μmol g-1¬¬ vs. 1.64 μmol g-1, while they also exhibited enhanced selectivity for methylated guanosine derivatives. When applied as sorbents in the extraction of nucleoside derivative cancer biomarkers from synthetic urine samples, significant sample clean-up and recoveries of up to 90% for 7-methylguanosine were achieved.

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: a comprehensive profiling approach

BACKGROUND: Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC.

METHODS: The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations.

RESULTS: A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy.

CONCLUSIONS: High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

Distinct methylation patterns in genes that affect mitochondrial function are associated with kidney disease in blood-derived DNA from individuals with Type 1 diabetes

AIMS: Epigenetic modifications, such as DNA methylation, can influence the risk of developing kidney disease. We studied methylation profiles in genes related to mitochondrial function to assess whether differences in these epigenetic features were associated with diabetic kidney disease in people with Type 1 diabetes.

METHODS: A case-control association study was undertaken (n = 196 individuals with diabetic kidney disease vs. n = 246 individuals without renal disease). Participants were White and diagnosed with Type 1 diabetes before 31 years of age. Genes that encode mitochondrial proteins (n = 780) were downloaded from mitoproteome. org. DNA methylation profiles from blood-derived DNA were generated using the Illumina Infinium HumanMethylation450 (262 samples) and Illumina Infinium HumanMethylation27 (192 samples) arrays. Beta values (β) were calculated and quality control was conducted, including evaluating blind duplicate DNA samples.

RESULTS: Fifty-four Cytosine-phosphate-Guanine probes across 51 unique genes were significantly associated (P ≤ 10(-8) ) with diabetic kidney disease across both the 450K and the 27K methylation arrays. A subanalysis, employing the 450K array, identified 755 Cytosine-phosphate-Guanine probes in 374 genes that were significantly associated (P ≤ 10(-8) ) with end-stage renal disease. Forty-six of the top-ranked variants for diabetic kidney disease were also identified as being differentially methylated in individuals with end-stage renal disease. The largest change in methylation (Δβ = 0.2) was observed for cg03169527 in the TAMM41 gene, chromosome 3p25.2. Three genes, PMPCB, TSFM and AUH, were observed with differential methylation at multiple Cytosine-phosphate-Guanine sites each (P < 10(-12) ).

CONCLUSIONS: Differential methylation in genes that influence mitochondrial function are associated with kidney disease in individuals with Type 1 diabetes. 

Independent genomewide screens identify the tumor suppressor VTRNA2-1 as a human epiallele responsive to periconceptional environment

Background: Interindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants.

Results: First, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements.

Conclusions: The non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.

Arsenic-glutathione complexes - Their stability in solution and during separation by different HPLC modes

Complexes of arsenic compounds and glutathione are believed to play an essential part in the metabolism and transport of inorganic arsenic and its methylated species. Up to now, the evidence of their presence is mostly indirect. We studied the stability and Chromatographic behaviour of glutathione complexes with trivalent arsenic: i.e. As^III(GS)₃, MA ^III(GS)₂ and DMA^III(GS) under different conditions. Standard ion chromatography using PRP X-100 and carbonate or formic acid buffer disintegrated the complexes, while all three complexes are stable and separable by reversed phase chromatography (0.1% formic acid/acetonitrile gradient). As^III(GS)₃ and MA^III(GS)₂ were more stable than DMA^III(GS), which even under optimal conditions tended to degrade on the column at 25 °C. Chromatography at 6 °C can retain the integrity of the samples. These results shed more light on the interpretation of a vast number of previously published arsenic speciation studies, which have used Chromatographic separation techniques with the assumption that the integrity of the arsenic species is guaranteed. © The Royal Society of Chemistry 2004.

Identification of arsenic species in sheep-wool extracts by different chromatographic methods

Sheep on the island of North Ronaldsay (Orkney, UK) feed mostly on seaweed, which contains high concentrations of dimethylated arsenoribosides. Wool of these sheep contains dimethylated, monomethylated and inorganic arsenic, in addition to unidentified arsenic species in unbound and complexed form. Chromatographic techniques using different separation mechanisms and detectors enabled us to identify five arsenic species in water extracts of wool. The wool contained 5.2 ± 2.3 μg arsenic per gram wool. About 80% of the arsenic in wool was extracted by boiling the wool with water. The main species is dimethylarsenic, which accounted for about 75 to 85%, monomethylated arsenic at about 5% and the rest is inorganic arsenic. Depending on the separation method and condition, the chromatographic recovery of arsenic species was between 45% for the anion exchange column, 68% for the size exclusion chromatography (SEC) and 82% for the cation exchange column. The SEC revealed the occurrence of two unknown arsenic compounds, of which one was probably a high molecular mass species. Since chromatographic recovery can be improved by either treating the extract with CuCl/HCl (CAT: 90%) or longer storage of the sample (CAT: 105%), in particular for methylated arsenic species, it can be assumed that labile arsenic -protein-like coordination species occur in the extract, which cannot be speciated with conventional chromatographic methods. It is clear from our study of sheep wool that there can be different kinds of 'hidden' arsenic in biological matrices, depending on the extraction, separation and detection methods used. Hidden species can be defined as species that are not recordable by the detection system, not extractable or do not elute from chromatographic columns. Copyright © 2003 John Wiley & Sons, Ltd.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.

MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.

RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.

CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.

RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.

CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility

Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p < 0.01). Merging gene expression and DNA methylation data revealed CHN2 as consistently hypermethylated and downregulated in this disease (Spearman -0.71, p < 0.001). Mutations in TP53 which were found in more than half of the cohort (15/28) and Kazald1 hypomethylation were both were indicative of poor survival (p = 0.03, HR = 3.2 and p = 0.01, HR = 4.9 respectively). By integrating high-throughput mutational, gene expression and DNA methylation data, this study reveals for the first time the distinct molecular profile of small bowel adenocarcinoma and highlights potential clinically exploitable markers.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

Spatial Variations of the Methanogenic Communities in the Sediments of Tropical Mangroves

Methane production by methanogens in mangrove sediments is known to contribute significantly to global warming, but studies on the shift of methanogenic community in response to anthropogenic contaminations were still limited. In this study, the effect of anthropogenic activities in the mangrove sediments along the north and south coastlines of Singapore were investigated by pyrosequencing of the mcrA gene. Our results showed that hydrogenotrophic, acetoclastic and methylotrophic methanogens coexist in the sediments. The predominance of the methylotrophic Methanosarcinales reflects the potential for high methane production as well as the possible availability of low acetate and high methylated C-1 compounds as substrates. A decline in the number of acetoclastic/methylotrophic methanogens in favor of hydrogenotrophic methanogens was observed along a vertical profile in Sungei Changi, which was contaminated by heavy metals. The diversity of methanogens in the various contaminated stations was significantly different from that in a pristine St. John’s Island. The spatial variation in the methanogenic communities among the different stations was more distinct than those along the vertical profiles at each station. We suggest that the overall heterogeneity of the methanogenic communities residing in the tropical mangrove sediments might be due to the accumulated effects of temperature and concentrations of nitrate, cobalt, and nickel.