scholarly journals Predicting Carcinogenic Mechanisms of Non-Genotoxic Carcinogens via Combined Analysis of Global DNA Methylation and In Vitro Cell Transformation

2020 ◽  
Vol 21 (15) ◽  
pp. 5387 ◽  
Author(s):  
Sung-Hee Hwang ◽  
Hojin Yeom ◽  
Byeal-I Han ◽  
Byung-Joo Ham ◽  
Yong-Moon Lee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Death ◽  
Cell Transformation ◽  
Integrated Approach ◽  
Global Dna Methylation ◽  
Promoter Regions ◽  
Combined System ◽  
Relationship Analysis ◽  
Genotoxic Carcinogens

An in vitro cell transformation assay (CTA) is useful for the detection of non-genotoxic carcinogens (NGTXCs); however, it does not provide information on their modes of action. In this study, to pursue a mechanism-based approach in the risk assessment of NGTXCs, we aimed to develop an integrated strategy comprising an in vitro Bhas 42 CTA and global DNA methylation analysis. For this purpose, 10 NGTXCs, which were also predicted to be negative through Derek/Sarah structure–activity relationship analysis, were first tested for transforming activity in Bhas 42 cells. Methylation profiles using reduced representation bisulfite sequencing were generated for seven NGTXCs that were positive in CTAs. In general, the differentially methylated regions (DMRs) within promoter regions showed slightly more bias toward hypermethylation than the DMRs across the whole genome. We also identified 13 genes associated with overlapping DMRs within the promoter regions in four NGTXCs, of which seven were hypermethylated and six were hypomethylated. Using ingenuity pathway analysis, the genes with DMRs at the CpG sites were found to be enriched in cancer-related categories, including “cell-to-cell signaling and interaction” as well as “cell death and survival”. Moreover, the networks related to “cell death and survival”, which were considered to be associated with carcinogenesis, were identified in six NGTXCs. These results suggest that epigenetic changes supporting cell transformation processes occur during non-genotoxic carcinogenesis. Taken together, our combined system can become an attractive component for an integrated approach for the testing and assessment of NGTXCs.

scholarly journals Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells

2004 ◽  
Vol 24 (3) ◽  
pp. 1270-1278 ◽  
Author(s):  
Jonathan C. Cheng ◽  
Daniel J. Weisenberger ◽  
Felicidad A. Gonzales ◽  
Gangning Liang ◽  
Guo-Liang Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Promoter Regions ◽  
Bladder Cancer Cells ◽  
Continuous Application ◽  
Aberrant Dna Methylation ◽  
Human Bladder Cancer Cells ◽  
Partial Depletion

ABSTRACT During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(β-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5′ region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2′-deoxycytidine followed by zebularine hindered the remethylation of the p16 5′ region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.

scholarly journals Global DNA methylation: role, status and genome-wide approaches to study epigenetic mark in cloned embryos

2020 ◽  
pp. 41-59
Author(s):  
Shivani Malpotra ◽  
Ahmad Hussain
Keyword(s):  
Dna Methylation ◽  
Developmental Stages ◽  
Gene Expression Pattern ◽  
Methylation Profile ◽  
Global Dna Methylation ◽  
Epigenetic Mark ◽  
Dna Methylation Profile ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) technique has been proving its worth for more than two decades now as over 20 different species have been successfully cloned. SCNT protocol for cloning is well established but efficiency in terms of live birth rate is still low. Epigenetic abnormality following nuclear reprogramming is considered as the main culprit behind its low efficiency. DNA methylation is one of the most important epigenetic modifications that directly or indirectly regulate gene expression pattern, development and genome stability. Embryos produced through SCNT are found to express abnormal DNA methylation profile in comparison with in vivo or in vitro produced embryos. In order to improve DNA methylation profile in cloned embryos, a complete database of whole genome is required to find out specific faulty targets. Many techniques including low throughput and high throughput approach has been used to profile DNA methylation pattern in bovine embryos throughout the developmental stages. In the present review, we have compiled the overall status of global DNA methylation, the effect of aberrant DNA methylation on development and evolution in methodologies used for profiling global DNA methylome in cloned embryos.

scholarly journals Blood‐Based Epigenetic Markers of FKBP5 Gene Methylation in Patients With Dilated Cardiomyopathy

2021 ◽  
Author(s):  
Kento Wada ◽  
Tomofumi Misaka ◽  
Tetsuro Yokokawa ◽  
Yusuke Kimishima ◽  
Takashi Kaneshiro ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dilated Cardiomyopathy ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Epigenetic Modification ◽  
Quantitative Polymerase Chain Reaction ◽  
Pressure Overload ◽  
Cpg Methylation ◽  
Promoter Regions

Background Blood‐based DNA methylation patterns are linked to types of diseases. FKBP prolyl isomerase 5 (FKBP5), a protein cochaperone, is known to be associated with the inflammatory response, but the regulatory mechanisms by leukocyte FKBP5 DNA methylation in patients with dilated cardiomyopathy (DCM) remain unclear. Methods and Results The present study enrolled patients with DCM (n=31) and age‐matched and sex‐matched control participants (n=43). We assessed FKBP5 CpG (cytosine‐phosphate‐guanine) methylation of CpG islands at the 5′ side as well as putative promoter regions by methylation‐specific quantitative polymerase chain reaction using leukocyte DNA isolated from the peripheral blood. FKBP5 CpG methylation levels at the CpG island of the gene body and the promoter regions were significantly decreased in patients with DCM. Leukocyte FKBP5 and IL‐1β (interleukin 1β) mRNA expression levels were significantly higher in patients with DCM than in controls. The protein expressions of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) in leukocytes were significantly reduced in patients with DCM. In vitro methylation assay revealed that FKBP5 promoter activity was inhibited at the methylated conditions in response to immune stimulation, suggesting that the decreased FKBP5 CpG methylation was functionally associated with elevation of FKBP5 mRNA expressions. Histological analysis using a mouse model with pressure overload showed that FKBP5‐expressing cells were substantially infiltrated in the myocardial interstitium in the failing hearts, indicating a possible role of FKBP5 expressions of immune cells in the cardiac remodeling. Conclusions Our findings demonstrate a link between specific CpG hypomethylation of leukocyte FKBP5 and DCM. Blood‐based epigenetic modification in FKBP5 may be a novel molecular mechanism that contributes to the pathogenesis of DCM.

scholarly journals O6D.2 Evidence of dna methylation changes by carbon nanotubes in a translational study design

2019 ◽  
Vol 76 (Suppl 1) ◽  
pp. A57.2-A57
Author(s):  
Manosij Ghosh ◽  
Deniz Öner ◽  
Lode Godderis ◽  
Peter Hoet
Keyword(s):  
Dna Methylation ◽  
Cross Sectional Study ◽  
Methylation Pattern ◽  
Global Dna Methylation ◽  
Limited Information ◽  
Cross Sectional ◽  
Translational Study ◽  
Exposed Workers

IntroductionWhile studies have addressed genotoxic effects of CNT, only limited information are available on epigenetic effects. We designed a study to investigate DNA methylation alterations in vitro, in vivo and in occupationally exposed workers.Material and methodsIn vitro studies were performed in 16-HBE and THP-1 cells. For the in vivo study, BALB/c mice were administered intratracheally with single-wall CNT (SWCNTs) and multi-wall (MWCNTs) at high (2.5 mg/kg) and low (0.25 mg/kg) doses. For the cross sectional study, 24 workers exposed to aggregates of MWCNT of 500 nm–100 µm with concentrations of 4.6–42.6 µg/m3 and 43 unexposed referents were recruited. Global DNA methylation and demethylation patterns were analysed by LC-MS/MS. Methylation of specific genes was measured by Pyromark 24® (Qiagen). Genome-wide assessment of DNA methylation was performed with Infinium HumanMethylation450 BeadChip Array.ResultsIn general, we did not find global DNA methylation alteration for both CNTs. In 16-HBE cells, differentially methylated and expressed genes (MWCNTs>SWCNTs) from p53 signalling, DNA damage repair and cell cycle pathways were observed. In THP-1 cells, CNTs induced promoter-specific methylation of genes involved in several signaling cascade, vascular endothelial growth factor and platelet activation pathways. In lungs of BALB/c mice CNTs affected methylation of ATM gene. Finally, analysis of gene-specific DNA methylation in exposed workers revealed significant changes for DNMT1, ATM, SKI, and HDAC4 promoter CpGs.ConclusionsEpigenetic changes seem to occur at sub cyto-genotoxic concentrations in vitro. Alteration in DNA methylation pattern could be a natural reaction of cells but could also silence critical genes and reprogram cellular functions.

scholarly journals Hypo-methylation mediates chromosomal instability in pancreatic NET

2017 ◽  
Vol 24 (3) ◽  
pp. 137-146 ◽  
Author(s):  
I Marinoni ◽  
A Wiederkeher ◽  
T Wiedmer ◽  
S Pantasis ◽  
A Di Domenico ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromosomal Instability ◽  
Global Dna Methylation ◽  
Epigenetic Changes ◽  
Knock Down ◽  
Alternative Lengthening ◽  
Increased Risk ◽  
Pancreatic Net ◽  
G0 Phase

DAXX and or ATRX loss occur in 40% of pancreatic neuroendocrine tumors (PanNETs). PanNETs negative for DAXX or ATRX show an increased risk of relapse. The tumor-associated pathways activated upon DAXX or ATRX loss and how this event may induce chromosomal instability (CIN) and alternative lengthening telomeres (ALT) are still unknown. Both DAXX and ATRX are involved in DNA methylation regulation. DNA methylation of heterochromatin and of non-coding sequences is extremely important for the maintenance of genomic stability. We analyzed the association of DAXX and/or ATRX loss and CIN with global DNA methylation in human PanNET samples and the effect of DAXX knock-down on methylation and cell proliferation. We assessedLINE1as well as global DNA methylation in 167 PanNETs, and we found that DAXX and or ATRX-negative tumors and tumors with CIN were hypomethylated. DAXX knock-down in PanNET cell lines blocked cells in G1/G0 phase and seemed to increase CIN in QGP-1 cells. However, no direct changes in DNA methylation were observed after DAXX knock-downin vitro. In conclusion, our data indicate that epigenetic changes are crucial steps in the progression of PanNETs loss and suggest that DNA methylation is the mechanism via which CIN is induced, allowing clonal expansion and selection.

DNA methylation profile of liver of mice conceived by in vitro fertilization

2021 ◽  
pp. 1-9
Author(s):  
Saúl Lira-Albarrán ◽  
Xiaowei Liu ◽  
Seok Hee Lee ◽  
Paolo Rinaudo
Keyword(s):  
Dna Methylation ◽  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Functional Enrichment ◽  
Global Dna Methylation ◽  
Analysis Tool ◽  
Gene Promoters ◽  
Vitro Fertilization ◽  
Natural Mating

Abstract Offspring generated by in vitro fertilization (IVF) are believed to be healthy but display a possible predisposition to chronic diseases, like hypertension and glucose intolerance. Since epigenetic changes are believed to underlie such phenotype, this study aimed at describing global DNA methylation changes in the liver of adult mice generated by natural mating (FB group) or by IVF. Embryos were generated by IVF or natural mating. At 30 weeks of age, mice were sacrificed. The liver was removed, and global DNA methylation was assessed using whole-genome bisulfite sequencing (WGBS). Genomic Regions for Enrichment Analysis Tool (GREAT) and G:Profilerβ were used to identify differentially methylated regions (DMRs) and for functional enrichment analysis. Overrepresented gene ontology terms were summarized with REVIGO, while canonical pathways (CPs) were identified with Ingenuity® Pathway Analysis. Overall, 2692 DMRs (4.91%) were different between the groups. The majority of DMRs (84.92%) were hypomethylated in the IVF group. Surprisingly, only 0.16% of CpG islands were differentially methylated and only a few DMRs were located on known gene promoters (n = 283) or enhancers (n = 190). Notably, the long-interspersed element (LINE), short-interspersed element (SINE), and long terminal repeat (LTR1) transposable elements showed reduced methylation (P < 0.05) in IVF livers. Cellular metabolic process, hepatic fibrosis, and insulin receptor signaling were some of the principal biological processes and CPs modified by IVF. In summary, IVF modifies the DNA methylation signature in the adult liver, resulting in hypomethylation of genes involved in metabolism and gene transcription regulation. These findings may shed light on the mechanisms underlying the developmental origin of health and disease.

scholarly journals Heat stress and immune response phenotype affect DNA methylation in blood mononuclear cells from Holstein dairy cows

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. M. Livernois ◽  
B. A. Mallard ◽  
S. L. Cartwright ◽  
A. Cánovas
Keyword(s):  
Dna Methylation ◽  
Immune Response ◽  
Heat Stress ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Heat Waves ◽  
Promoter Regions ◽  
Blood Mononuclear Cells

AbstractHeat stress negatively affects health and production in cows. Examining the cellular response to heat stress could reveal underlying protective molecular mechanisms associated with superior resilience and ultimately enable selection for more resilient cattle. This type of investigation is increasingly important as future predictions for the patterns of heat waves point to increases in frequency, severity, and duration. Cows identified as high immune responders based on High Immune Response technology (HIR) have lower disease occurrence compared to their average and low immune responder herd-mates. In this study, our goal was to identify epigenetic differences between high and low immune responder cows in response to heat stress. We examined genome-wide DNA methylation of blood mononuclear cells (BMCs) isolated from high and low cows, before and after in vitro heat stress. We identified differential methylation of promoter regions associated with a variety of biological processes including immune function, stress response, apoptosis, and cell signalling. The specific differentially methylated promoter regions differed between samples from high and low cows, and results revealed pathways associated with cellular protection during heat stress.

Exposure to high levels of fumarate and succinate leads to apoptotic cytotoxicity and altered global DNA methylation profiles in vitro

Biochimie ◽  
2017 ◽  
Vol 135 ◽  
pp. 28-34 ◽  
Author(s):  
Johannes F. Wentzel ◽  
Angélique Lewies ◽  
Abel J. Bronkhorst ◽  
Etresia van Dyk ◽  
Lissinda H. du Plessis ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Global Dna Methylation

Cell Transformation Assays: Are we Barking up the Wrong Tree?

2012 ◽  
Vol 40 (2) ◽  
pp. 115-130 ◽  
Author(s):  
Robert D. Combes
Keyword(s):  
Validation Studies ◽  
Cell Transformation ◽  
Metabolic Activation ◽  
Morphological Transformation ◽  
Genotoxic Carcinogens ◽  
Principal Finding ◽  
High Level ◽  
Transformation Systems

There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed.

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