Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzyme

We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed ‘helper’ oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called ‘helper-dependent chain reaction’. The process uses disabled primers called ‘drivers’ that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA.Funding for open access charge: Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. National Health & Medical Research Counci

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours’ biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription–quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes

Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation

The extent of variation in DNA methylation patterns in healthy individuals is not yet well documented. Identification of inter-individual epigenetic variation is important for understanding phenotypic variation and disease susceptibility. Using neutrophils from a cohort of healthy individuals, we generated base-resolution DNA methylation maps to document inter-individual epigenetic variation. We identified 12851 autosomal inter-individual variably methylated fragments (iVMFs). Gene promoters were the least variable, whereas gene body and upstream regions showed higher variation in DNA methylation. The iVMFs were relatively enriched in repetitive elements compared to non-iVMFs, and were associated with genome regulation and chromatin function elements. Further, variably methylated genes were disproportionately associated with regulation of transcription, responsive function and signal transduction pathways. Transcriptome analysis indicates that iVMF methylation at differentially expressed exons has a positive correlation and local effect on the inclusion of that exon in the mRNA transcript

GSK-3484862 Targets DNMT1 for Degradation in Cells

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner

A molecular portrait of high-grade ductal carcinoma in situ

Ductal carcinoma in situ (DCIS) is a noninvasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer whereas others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome, and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty-two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM, and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor-intrinsic subtypes, proliferative, immune scores, and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like, or ERBB2 + ) displayed signatures characteristic of activated Treg cells (CD4 + /CD25 + /FOXP3 + ) and CTLA4 + /CD86 + complexes indicative of a tumor-associated immunosuppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly, ncRNA and methylation profiles reproduce changes observed postinvasion. Among the most significant findings, we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a preinvasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer.Centro de Investigaciones Inmunológicas Básicas y Aplicada

GESTÃO DE RESÍDUOS

With the increase in demand for beauty products and services for the male market, there is also an increase in the offer of barber shops with a view to serving this audience. It is known that these barbershops generate contaminating residues, however, there is no information about the treatment of contaminating residues generated by barbershops. In the case of contaminating materials, treatment and disposal must meet specific criteria determined by law. Therefore, the question is: what is the way to dispose of contaminating waste generated by barber shops in Presidente Prudente / SP? In this sense, the present study is relevant, since it intends to analyze how the barber shops in Presidente Prudente / SP have treated and discarded the contaminating residues generated by them. To this end, the present research had a qualitative approach, exploratory and descriptive research, carried out through face-to-face interviews subsidized by a semi-structured interview script. For data analysis, the content analysis technique was used. It was observed that although the owners of the barber shops are not aware of the legislation that deals with the disposal of contaminating waste, they adopt practices that fit the requirements of the municipal health surveillance. It is hoped that the results of this study can support public policies aimed at waste management.Con el aumento de la demanda de productos y servicios de belleza para el mercado masculino, también aumenta la oferta de peluquerías con miras a atender a este público. Se sabe que estas barberías generan residuos contaminantes, sin embargo, no existe información sobre el tratamiento de residuos contaminantes generados por las barberías. En el caso de materiales contaminantes, el tratamiento y la eliminación deben cumplir con criterios específicos determinados por la ley. Por tanto, la pregunta es: ¿cuál es la forma de disponer de los residuos contaminantes generados por las peluquerías en Presidente Prudente / SP? En este sentido, el presente estudio es relevante, ya que pretende analizar cómo las peluquerías de Presidente Prudente / SP han tratado y descartado los residuos contaminantes generados por ellas. Para ello, esta investigación se basó en un enfoque cualitativo, una investigación exploratoria y descriptiva, realizada a través de entrevistas presenciales subvencionadas por un guión de entrevista semiestructurado. Para el análisis de datos se utilizó la técnica de análisis de contenido. Se observó que si bien los dueños de las peluquerías desconocen la legislación que se ocupa de la disposición de residuos contaminantes, adoptan prácticas que se ajustan a los requisitos de la vigilancia sanitaria municipal. Se espera que los resultados de este estudio puedan apoyar las políticas públicas dirigidas a la gestión de residuos.Com aumento na demanda por produtos e serviços de beleza para o mercado masculino, se observa também o aumento na oferta de barbearias com vista a atender esse público. Sabe-se que essas barbearias geram resíduos contaminantes, no entanto, não se tem informações a respeito da forma de tratamento dos resíduos contaminantes gerados pelas barbearias. Tratando–se de materiais contaminantes o tratamento e descarte deve atender a critérios específicos determinados por lei. Sendo assim, questiona-se: qual a forma de descarte dos resíduos contaminantes, gerados pelas barbearias em Presidente Prudente/SP? Nesse sentido o presente estudo é relevante, uma vez que pretende analisar como as barbearias de Presidente Prudente/SP tem tratado e descartado os resíduos contaminantes gerados por elas. Para tanto, a presente pesquisa contou com uma abordagem qualitativa, pesquisa exploratória e descritiva, realizada por meio de entrevista face a face subsidiada por roteiro de entrevista semiestruturado. Para análise de dados empregou-se a técnica de análise de conteúdo. Observou-se que embora os proprietários das barbearias não tenham conhecimento da legislação que trata do descarte de resíduos contaminantes, adotam práticas que se enquadram as exigências da vigilância sanitária municipal. Espera-se que os resultados deste estudo possam subsidiar políticas públicas direcionadas a gestão de resíduos

Obesity-Associated Alterations in Inflammation, Epigenetics, and Mammary Tumor Growth Persist in Formerly Obese Mice

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable to control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL-6 levels, expression of pro-inflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice, and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus non-obese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression

Investigations on DNA damage and frequency of micronuclei in occupational exposure to electromagnetic fields (EMFs) emitted from video display terminals (VDTs)

The potential effect of electromagnetic fields (EMFs) emitted from video display terminals (VDTs) to elicit biological response is a major concern for the public. The software professionals are subjected to cumulative EMFs in their occupational environments. This study was undertaken to evaluate DNA damage and incidences of micronuclei in such professionals. To the best of our knowledge, the present study is the first attempt to carry out cytogenetic investigations on assessing bioeffects in personal computer users. The study subjects (n = 138) included software professionals using VDTs for more than 2 years with age, gender, socioeconomic status matched controls (n = 151). DNA damage and frequency of micronuclei were evaluated using alkaline comet assay and cytochalasin blocked micronucleus assay respectively. Overall DNA damage and incidence of micronuclei showed no significant differences between the exposed and control subjects. With exposure characteristics, such as total duration (years) and frequency of use (minutes/day) sub-groups were assessed for such parameters. Although cumulative frequency of use showed no significant changes in the DNA integrity of the classified sub-groups, the long-term users (> 10 years) showed higher induction of DNA damage and increased frequency of micronuclei and micro nucleated cells

Genome-wide DNA methylation profiles in both precancerous conditions and clear cell renal cell carcinomas are correlated with malignant potential and patient outcome

To clarify genome-wide DNA methylation profiles during multistage renal carcinogenesis, bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA) was performed. Non-cancerous renal cortex tissue obtained from patients with clear cell renal cell carcinomas (RCCs) (N) was at the precancerous stage where DNA hypomethylation and DNA hypermethylation on multiple bacterial artificial chromosome (BAC) clones were observed. By unsupervised hierarchical clustering analysis based on BAMCA data for their N, 51 patients with clear cell RCCs were clustered into two subclasses, Clusters AN (n = 46) and BN (n = 5). Clinicopathologically aggressive clear cell RCCs were accumulated in Cluster BN, and the overall survival rate of patients in Cluster BN was significantly lower than that of patients in Cluster AN. By unsupervised hierarchical clustering analysis based on BAMCA data for their RCCs, 51 patients were clustered into two subclasses, Clusters AT (n = 43) and BT (n = 8). Clinicopathologically aggressive clear cell RCCs were accumulated in Cluster BT, and the overall survival rate of patients in Cluster BT was significantly lower than that of patients in Cluster AT. Multivariate analysis revealed that belonging to Cluster BT was an independent predictor of recurrence. Cluster BN was completely included in Cluster BT, and the majority of the BAC clones that significantly discriminated Cluster BN from Cluster AN also discriminated Cluster BT from Cluster AT. In individual patients, DNA methylation status in N was basically inherited by the corresponding clear cell RCC. DNA methylation alterations in the precancerous stage may generate more malignant clear cell RCCs and determine patient outcome