Changes In DNA Methylation Patterns Research Articles

Myc-Mediated Lymphomagenesis Is Driven by DNA Methylation Changes Induced by DNMT3B7 Expression and Dnmt3b Heterozygosity

Abstract 225Cancer cells are characterized by abnormal DNA methylation, including overall genomic hypomethylation with concurrent region-specific hypo- and hyper-methylation, causing aberrant activation of some genes and the silencing of others. Three DNA methyltransferase (DNMT) enzymes catalyze DNA methylation in eukaryotic cells, DNMT1, DNMT3A, and DNMT3B. We discovered previously that cancer cells exhibit aberrant splicing of the DNMT3B gene, which produces transcripts containing premature stop codons that encode truncated proteins lacking the catalytic domain. When we bred transgenic mice expressing DNMT3B7, one of the aberrantly spliced DNMT3B isoforms found most commonly in cancer cells, with the Eμ-Myc mice, a mouse model for B cell lymphomas, we observed an acceleration of mediastinal lymphomagenesis along with changes in the expression of several genes involved in oncogenesis. The acceleration in tumorigenesis was associated with global DNA hypermethylation, and further analyses showed that these changes in DNA methylation were heterogeneous in tumors derived from Eμ-Myc/DNMT3B7 mice, a phenomenon reminiscent of human tumors. We hypothesized that DNMT3B7 altered DNA methylation by functioning as a dominant negative isoform of full-length endogenous mouse Dnmt3b, and therefore tested a second mouse model that has defects in DNA methylation.The introduction of Dnmt3b heterozygosity (Dnmt3b+/−) into the Eμ-Myc background accelerated mediastinal lymphomagenesis to an even greater extent, with more than 90% of the Eμ-Myc/Dnmt3b+/− mice developing mediastinal lymphomas within the first 120 days. This was also associated with an increase in global DNA methylation as measured by liquid chromatography-mass spectrometry, to a larger extent than in the Eμ-Myc/DNMT3B7 mice. Interestingly, the tumors from Eμ-Myc mice themselves showed global hypermethylation when compared to non-transformed cells from Eμ-Myc mice, suggesting that the transformation of cells that express Myc is a key aspect in the induction of global DNA hypermethylation.These observations led us to the hypothesis that Myc-mediated tumorigenesis is particularly sensitive to changes in DNA methylation. Brenner et al. demonstrated that Myc binds to Dnmt3a/b and recruits the methyltransferases to promoter regions of Myc targets, leading to DNA hypermethylation in these regions. We have also found previously that DNNMT3B7 binds with full-length DNMT3B, by co-immunoprecipitation studies. We hypothesize that either in the presence of DNMT3B7 or with Dnmt3b heterozygosity, Myc-Dnmt3a/b binding at promoters is enhanced, which leads to hypermethylation and repression of gene expression. Using Mycbp, a gene that was repressed in Eμ-Myc/DNMT3B7 tumors, we demonstrated that its promoter region was hypermethylated in both Eμ-Myc/DNMT3B7 and Eμ-Myc/Dnmt3b+/− tumors. The E-box, a conserved sequence located ∼100bp upstream of the transcription start site that Myc binds specifically, was hypomethylated in the Eμ-Myc/DNMT3B7 tumors, suggesting that there was an enrichment of Myc binding at this region. Chromatin immunoprecipitation analyses confirmed increased binding of Myc at the E-box of Mycbp in the Eμ-Myc/DNMT3B7 tumors. Furthermore, we also demonstrated that Myc expression induced all the three DNA methyltransferases, suggesting that Myc-mediated lymphomagenesis may occur using a feedback loop which enhances expression of the DNA methyltransferases to regulate particular genes involved in tumorigenesis. This study offers an insight into the mechanism behind Myc-mediated tumorigenesis and provides evidence for the central role played by changes in DNA methylation patterns in this process. Disclosures:No relevant conflicts of interest to declare.

Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

BackgroundThe human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood.ResultsIn this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments.ConclusionsThe identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes.

Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: Influence of cruciferous vegetables in an intervention study

Changes in DNA methylation patterns are a hallmark of tobacco-induced carcinogenesis. We have conducted a randomized 4-week intervention trial to investigate the effects of three dietary regimens to modify DNA methylation patterns in peripheral white blood cells of heavy smokers. A group of 88 smokers were randomly assigned to and distributed among three diets, including (1) normal isocaloric diet (balanced in fruits and vegetables), according to international guidelines; (2) a diet enriched in flavonoids and isothiocyanates (particularly cruciferous vegetables); (3) a regimen consisting of diet 1 supplemented with flavonoids (green tea and soy products). Methylation patterns were analyzed by pyrosequencing in LINE1 (Long Interspersed DNA Elements), RASSF1A, ARF and CDKN2a (tumor suppressor genes), MLH1 (mismatch DNA repair) and MTHFR (folate metabolism). Three distinct patterns of methylation were observed. In LINE1, methylation showed a small but reproducible increase with all three regimens. MTHFR was constitutively methylated with no significant modulation by diets. The four other loci showed low basal levels of methylation with no substantial change after intervention. These data suggest that the isocaloric diet may stabilize global epigenetic (LINE1 DNA methylation) patterns in peripheral white blood cells but does not provide evidence for methylation changes in specific genes associated with this short-term dietary intervention.

A putative “hepitype” in the ATM gene associated with chronic lymphocytic leukemia risk

Chronic lymphocytic leukemia (CLL) cells are characterized by several chromosomal lesions. Some of these aberrations imply chromosome breaks as a result of unrepaired double strand breaks (DSBs) in the DNA. The ATM (ataxia telangiectasia-mutated) protein is the principal integrator of cellular responses to DSBs. ATM deletion is also an adverse prognostic factor in CLL. Taking this into account, we evaluated if genetic and/or epigenetic variation in the ATM gene may modulate the individual susceptibility to develop CLL. Our case-control association study was performed in a large Spanish population of 1,503 individuals, including 742 patients with CLL and 761 controls. We identified one haplotype within the ATM gene that confers an increased risk of CLL development (OR = 1.33; 95% CI: 1.10-1.60). Two polymorphisms of this ATM haplotype eliminated one CpG site each in Introns 15 and 61, causing changes in DNA methylation pattern. These data provide the first evidence for the existence of a putative "hepitype" in the ATM gene associated with CLL risk.

Regulation of SNAIL1 and E-cadherin function by DNMT1 in a DNA methylation-independent context

Mammalian DNA methyltransferase 1 (DNMT1) is essential for maintaining DNA methylation patterns after cell division. Disruption of DNMT1 catalytic activity results in whole genome cytosine demethylation of CpG dinucleotides, promoting severe dysfunctions in somatic cells and during embryonic development. While these observations indicate that DNMT1-dependent DNA methylation is required for proper cell function, the possibility that DNMT1 has a role independent of its catalytic activity is a matter of controversy. Here, we provide evidence that DNMT1 can support cell functions that do not require the C-terminal catalytic domain. We report that PCNA and DMAP1 domains in the N-terminal region of DNMT1 are sufficient to modulate E-cadherin expression in the absence of noticeable changes in DNA methylation patterns in the gene promoters involved. Changes in E-cadherin expression are directly associated with regulation of β-catenin-dependent transcription. Present evidence suggests that the DNMT1 acts on E-cadherin expression through its direct interaction with the E-cadherin transcriptional repressor SNAIL1.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.

Dietary Patterns Are Associated with Levels of Global Genomic DNA Methylation in a Cancer-Free Population

Animal studies have provided direct evidence that dietary factors induce changes in DNA methylation patterns. In humans, studies on diet and DNA methylation have yielded inconsistent findings. Because humans tend to consume foods and nutrients that are highly interrelated, study of dietary patterns may have improved the power of detecting the effect of diet on DNA methylation. Using data collected from 149 participants aged 45–75 y in the North Texas Healthy Heart Study, we examined the relationship between dietary patterns and levels of genomic DNA methylation in peripheral blood leukocytes. Dietary data were collected from study participants using the Block FFQ. Genomic DNA methylation was measured using bisulfite conversion of DNA and real-time PCR (MethyLight) for LINE-1. Two dietary patterns were identified using factor analysis: a “prudent” dietary pattern characterized by a high intake of vegetables and fruits, and a “Western” dietary pattern characterized by a high intake of meats, grains, dairy, oils, and potatoes. The prudent dietary pattern was associated with a lower prevalence of DNA hypomethylation (Q4 vs. Q1; OR = 0.33, 95% CI: 0.12–0.92) and the association was dose dependent (P-trend = 0.04). There was no apparent association between the Western dietary pattern and global leukocyte DNA methylation (Q4 vs. Q1; OR = 1.28, 95% CI: 0.47–3.47; P-trend = 0.55). Thus, a dietary pattern characterized by a high intake of vegetables and fruits may protect against global DNA hypomethylation. Future studies with a larger sample size need to confirm that this association holds longitudinally.

DNA methylation in cancer development, diagnosis and therapy--multiple opportunities for genotoxic agents to act as methylome disruptors or remediators

The role of DNA methylation and recently discovered hydroxymethylation in the function of the human epigenome is currently one of the hottest topics in the life sciences. Progress in this field of research has been further accelerated by the discovery that alterations in the methylome are not only associated with key functions of cells and organisms, such as development, differentiation and gene expression, but may underlie a number of human diseases, including cancer. This review describes both well established and more recent observations concerning alterations in the methylome, i.e. the global and local distribution of 5-methylcytosines, involved in its normal functions. Then, the changes in DNA methylation pattern seen in cancer cells are discussed in the context of their utilisation in cancer diagnostics and treatment. On this basis, comparisons are made between natural covalent DNA modification and that induced by genotoxic agents, chemical carcinogens and antitumour drugs as regards their impact on epigenetic mechanisms. The available data suggest that DNA damage by genotoxins can mimic epigenetic markers and in consequence disrupt the proper function of the epigenome. On the other hand, the same processes in cancer cells, e.g. DNA demethylation as a result of DNA methyltransferase blocking or the induction of DNA repair by DNA adducts, may restore the activity of hypermethylated anticancer genes. The observed multiple mechanisms by which genotoxic agents directly affect methylome function suggest that chemical carcinogens act primarily as epigenome disruptors, whereas mutations are secondary events that occur at later stages of cancer development when genome-protecting mechanisms have already been deregulated.

Abstract 85: Detection of promoter methylation of tumor supressor genes in serum DNA of breast cancer patients and controls

Abstract Background: Changes in DNA methylation patterns are a common feature of malignant cells. Methylation is an epigenetic change that affects gene expression. Methylation of a cytosine guanine dinucleotide-rich area (CpG island) of the promoter region of a gene that is normally unmethylated is correlated with silencing of that gene. Certain tumor suppressor genes are hypermethylated in breast tumor tissue and rarely methylated in normal breast tissue. Circulating serum DNA, presumably including DNA shed from the tumor, can be tested for promoter hypermethylation. Breast cancer detection with moderate sensitivity has been achieved in several small studies relying on a few genes and suggests that assaying methylation levels in a larger panel of genes might result in a useful serum marker for breast cancer detection. Methods: Bisulfite-modified serum DNA was evaluated for promoter methylation via pyrosequencing in 12 candidate tumor suppressor genes (GSTP1, SRFP1, RAR-beta, ER1, TWIST, HIN1, APC, ECAD, p16, BRAC1, CyclinD2, RASSF1). Study subjects included approximately 250 node-positive postmenopausal breast cancer cases, 75 node negative postmenopausal breast cancer cases and a comparison group of 250 postmenopausal women with benign breast disease types not strongly associated with breast cancer, all of whom donated blood at the time of diagnosis to the Mayo Serum Bank, a resource established in the 1970's to identify early markers of breast cancer. Results: As hypothesized, mean percent levels of serum DNA methylation were modestly but statistically higher in node-positive breast cancer cases than controls for TWIST (4.9% vs 3.9%; p-value =.002), CyclinD2 (3.2% vs 2.1%; p=.03), and HIN1 (3.9% vs 2.4%; p=.03). In the nine other genes, percent methylation levels were also moderately higher in node-positive cases than controls but did not reach statistical significance (e.g., SRFP1 [6.3% vs 4.3%; p=.06]). For TWIST, Cyclin D2, HIN1, mean percent levels were intermediate in node-negative patients compared to node-positive cases and controls. Additional analyses evaluating the predictive ability of multivariate sets of DNA methylation markers together with established breast cancer risk factors are underway. 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 85. doi:10.1158/1538-7445.AM2011-85

Parental ages and levels of DNA methylation in the newborn are correlated

BackgroundChanges in DNA methylation patterns with age frequently have been observed and implicated in the normal aging process and its associated increasing risk of disease, particularly cancer. Additionally, the offspring of older parents are at significantly increased risk of cancer, diabetes, and neurodevelopmental disorders. Only a proportion of these increased risks among the children of older parents can be attributed to nondisjunction and chromosomal rearrangements.ResultsUsing a genome-wide survey of 27,578 CpG dinucleotides in a cohort of 168 newborns, we examined the relationship between DNA methylation in newborns and a variety of parental and newborn traits. We found that methylation levels of 144 CpGs belonging to 142 genes were significantly correlated with maternal age. A weaker correlation was observed with paternal age. Among these genes, processes related to cancer were over-represented, as were functions related to neurological regulation, glucose/carbohydrate metabolism, nucleocytoplasmic transport, and transcriptional regulation. CpGs exhibiting gender differences in methylation were overwhelmingly located on the X chromosome, although a small subset of autosomal CpGs were found in genes previously shown to exhibit gender-specific differences in methylation levels.ConclusionsThese results indicate that there are differences in CpG methylation levels at birth that are related to parental age and that could influence disease risk in childhood and throughout life.

DNA methylation restricts spontaneous multi-lineage differentiation of mesenchymal progenitor cells, but is stable during growth factor-induced terminal differentiation

The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells.

DNA Methylation in Genomes of Several Annual Herbaceous and Woody Perennial Plants of Varying Ploidy as Detected by MSAP

Polyploidization is known to accompany altered DNA methylation in higher plants, which plays an important role in gene expression regulation and maintaining genome stability. While the characteristics of DNA methylation in different polyploid plants are still to be elucidated; here, status of genomic DNA methylation in a series of diploid, triploid, and tetraploid annual herbaceous plants (watermelon and Salvia) and woody perennials (pear, Poplar, and loquat) were explored by methylation-specific amplified polymorphism analysis. The results indicated that levels of DNA methylation in triploid watermelon and Salvia were lower than their diploid parents. In triploid Poplar and pear, higher levels of DNA methylation were detected, and no significant difference was observed between triploid and tetraploid in all tested materials. Further data analysis suggested that about half of the total detected sites underwent changes of DNA methylation patterns in triploid watermelons and Salvia, as well as an obvious trend towards demethylation. However, the changes of DNA methylation patterns in three triploid woody perennials were only 17.54–33.40%. This implied that the characteristics of DNA methylation are significantly different during the polyploidization of different plant species. Furthermore, the results suggested that the level of DNA methylation was nonlinearly related to the ploidy level, and triploid plants displayed more interesting DNA methylation status. The characteristics and possible functions of DNA methylation in different ploidy series are further discussed.

Cytosine Methyltransferases as Tumor Markers

Changes in DNA methylation patterns is a prominent characteristic of human tumors. Tumor cells display reduced levels of genomic DNA methylation and site-specific CpG island hypermethylation. Methylation of CpG dinucleotides is catalyzed by the enzyme family of DNA methyltransferases (DNMTs). In this review, the role of DNA methylation and DNMTs as key determinants of carcinogenesis is further elucidated. The chromatin modifying proteins that are known to interact with DNMTs are also described. Finally, the role of DNMTs as potential therapeutic targets is addressed.

Immunomodulatory Effects of Azacitidine Treatment In High Risk Myelodysplastic Syndrome

AbstractAbstract 1860Immune editing is recognized as being important in the pathogenesis of myelodysplastic syndrome (MDS). In low risk MDS, inappropriate immune reactions are thought to contribute to the initiation and progression of the disease. IL-17 producing CD4+ T-helper cells have been associated with various autoimmune diseases and are found in increased frequencies in low-risk MDS. These increased Th17 frequencies are accompanied by a reduced FoxP3+ T-regulatory (Treg) frequency. On the other hand, in high-risk MDS, the elevated number of bone marrow blasts was associated with increased numbers of Treg. These increased Treg may inhibit immune responses directed against the dysplastic blasts and as such permit progression of the disease. The demethylating agents '5-aza-2'-deoxycytidine and '5-azacitidine (Aza) have shown significant clinical benefits in the treatment of MDS. Although aberrant DNA methylation patterns appear to play a role in the pathogenesis of MDS, clinical responses to DNA demethylating agents could not be related to changes in DNA methylation patterns. Cytokine production and FoxP3 expression are also regulated by epigenetic mechanisms, and FoxP3 expression is increased upon in vitro treatment with Aza. Since cytokines and FoxP3+ Treg may play a role in the pathogenesis of MDS, we set out to investigate the effects of Aza (Vidaza®) treatment on CD4+ T-helper subset frequencies in high-risk MDS patients. Nine patients treated with 75 mg/m2/day subcutaneously for 7 consecutive days in a 28 day cycle were included in this study. Peripheral blood samples were drawn before, 15 days after start of treatment and at the end of the third cycle. There was no change in absolute or relative CD3+ and CD3+CD4+ T-cell numbers during treatment. The proportion of Treg was slightly but not significantly increased (paired student's T-test, p=0.08) on day 15 (11.9 % of CD4+) compared to levels before treatment (9.6 % of CD4+) while numbers dropped to pretreatment levels after the third cycle(9.6 % of CD4+). Cytokine producing cells were enumerated after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin in the presence of Brefeldin A. No significant changes in IFNγ (23.8%, 22.5% and 17.9% of CD4+ cells pretreatment, 15d post treatment and after the third cycle respectively) TNFα (38.3, 35.9 and 39.3% of CD4+ cells) or IL-4 (0.6, 1.0 and 0.6% of CD4+ cells) producing cells were observed. However, the proportion of IL-17 producing cells progressively decreased during treatment (1.1, 0.7 and 0.6% of CD4+ cells; ANOVA, p=0.006). In conclusion, Aza may modulate the immune response and in particular reduce IL-17 responses. This effect of Aza may provide a rationale for expanding the group of patients eligible for Aza treatment with low risk MDS patients, because low risk MDS has been associated with elevated, potential pathogenic, Th17 cells. Disclosures:Ossenkoppele:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Wheat plant DNA methylation pattern changes at chronic seed γ irradiation

Alterations of DNA methylation patterns of two wheat sorts Al’batros odesskii and Donetskaya-48 whose seeds were irradiated with a low dose rate (3 × 10−7 Gy/s) for 4 months have been studied. Six restriction endonucleases were used in the experiments. Primary distinction in DNA methylation patterns of the studied sorts has been demonstrated. Chronic irradiation resulted in an increase of the methylation level on the recognion site for GluI and Sou3AI and in a decrease of this index in recognion sites for endonucleases GlaI and HpaII. The alterations of the methylation level in recognition sites for restrictases MboI and MspI were not found. The considerable increase of chromosome aberration level at the same dose of chronic irradiation has been shown. The role of DNA methylation pattern changes in development of irradiation damage and organism protective reactions is discussed.

Association of P-mobile element activity and DNA methylation pattern changes in conditions of Drosophila Melanogaster prolonged irradiation

Association of the radiosensitivity and epigenetic pattern DNA changes at the conditions of prolonged irradiation was investigated. Two laboratory Drosophila melanogaster strains (Canton-S and ri) irradiated for 20 generations to low doses rate (1.2 x 10(-1), 0.8 x 10(-8) and 0.12 x 10(-8) Gy/s) were used as experimental objects. DNA for the analysis was extracted separately for the flies of males and females. Restriction endonucleases Glul, Glal were used. Restriction analysis has shown that there are different DNA methylated patterns for males and females as for control and exposed variants. At the chronic irradiation there was the decline of methylation level at the enzymes Glul, Glal sites recognition.

Hypomethylation and induction of retinoic acid receptor beta 2 by concurrent action of adenosine analogues and natural compounds in breast cancer cells

DNA methylation is considered as a potential cause of aberrations in regulation of gene expression during carcinogenesis. Therefore, changes in DNA methylation patterns may be targets for chemoprevention. In the present study, we investigated effects of all- trans retinoic acid (ATRA), vitamin D 3, and resveratrol alone and in combination with adenosine analogues: 2-chloro-2′-deoxyadenosine (2CdA) and 9-beta- d-arabinosyl-2-fluoroadenine (F-ara-A), on methylation and expression of retinoic acid receptor beta 2 ( RARbeta2) in MCF-7 and MDA-MB-231 breast cancer cell lines. Alterations in methylation and expression levels after treatment of cells with the tested compounds were evaluated by methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. RARbeta2 promoter in the tested fragment was partially methylated in MCF-7 cells and non-methylated in MDA-MB-231 cells. In MCF-7 cells, all compounds, except for resveratrol, inhibited promoter methylation and increased expression of RARbeta2. All natural compounds improved the action of 2CdA and F-ara-A on RARbeta2 methylation and/or expression. Combination of ATRA or vitamin D 3 with 2CdA was the most effective. In MDA-MB-231 cells, only 2CdA, F-ara-A, and ATRA induced RARbeta2 expression without any notable effects in combined treatment. Our results demonstrate that both natural compounds and adenosine analogues are able to reduce promoter methylation and/or induce expression of RARbeta2 in non-invasive MCF-7 cells. Furthermore, the natural compounds improve effects of adenosine analogues, however only at early non-invasive stages of carcinogenesis.

Abstract 152: Methylated-DNA enrichment and PCR array technology: A qPCR-based method to rapidly screen for changes in promoter DNA methylation patterns

Abstract As the only known enzymatic modification of DNA in mammals, methylation of cytosines at CpG dinucleotides is recognized as a “third pathway” in Knudson's two-hit hypothesis for cancer which suggests that at least two DNA insults (hits) are required for cancer progression. This epigenetic event alters gene expression patterns associated with normal development and disease progression without changing the actual DNA sequence. In cancer, hypermethylation of tumor suppressor gene promoters abolishes their transcription, while hypomethylation of proto-oncogene promoters increases their transcription, effectively driving tumorigenesis in either case. Given the importance of this event for tumorgenesis, various techniques have been developed to identify DNA methylation based diagnostic biomarkers. Unfortunately, these methods often fall short in terms of throughput capacity, cost, or equipment requirements. This work presents a novel combination of technologies to efficiently screen for changes in DNA methylation patterns within a panel of genes simultaneously. Using lung cancer as an experimental model, this study establishes that methylated DNA enrichment combined with real-time PCR (qPCR) technology offers great potential to identify putative biomarkers. This one-day assay is quite simple, and with very little hands-on time it can be easily adapted for high-throughput screening. A genomic DNA sample of interest is sheared, denatured, and incubated with a highly specific anti-5-methylcytosine antibody immobilized to a resin. The methylated DNA is then released, purified over a column, and applied to a pathway-focused qPCR array. The sensitivity of the method is very high; even one methylated CpG dinucleotide is enough to selectively enrich for a methylated target of interest, and the assay detection limit is well below 2.5% of a target in a mixed genomic DNA background. Importantly, data derived from this method has been validated with data derived from the Illumina® Infinium® Methylation Assay platform, further attesting to the potential for discovering methylation biomarkers with this system. In conclusion, the combination of methylated-DNA enrichment and qPCR array technology has been extensively optimized to discriminate methylated-CpG dinucleotides with low background. With this method, researchers will be able to quickly determine whether changes in gene expression (upregulation or downregulation) are associated with promoter methylation changes. This combination of methylated-DNA enrichment with qPCR array technology offers great potential as a methylated CpG dinucleotide screening tool, without the high cost. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 152.

Hypomethylation of Repeated Dna Sequences in Cancer

An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine. Hypomethylation of the genome can affect both single-copy genes, repeat DNA sequences and transposable elements, and is highly variable among and within cancer types. Here, we review our current understanding of genome hypomethylation in cancer, with a particular focus on hypomethylation of the different classes and families of repeat sequences. The emerging data provide insights into the importance of methylation of different repeat families in the maintenance of chromosome structural integrity and the fidelity of normal transcriptional regulation. We also consider the events underlying cancer-associated hypomethylation and the potential for the clinical use of characteristic DNA methylation changes in diagnosis, prognosis or classification of tumors.

DNA methylation pattern changes upon long‐term culture and aging of human mesenchymal stromal cells

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo. This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.