Genomic DNA Methylation Levels Research Articles

Role of DNA methylation in long-term low-dose γ-rays induced adaptive response in human B lymphoblast cells

Purpose: With widespread use of ionizing radiation, more attention has been attracted to low-dose radiation (LDR); however, the mechanisms of long-term LDR-induced bio-effects are unclear. Here, we applied human B lymphoblast cell line HMy2.CIR to monitor the effects of long-term LDR and the potential involvement of DNA methylation.Materials and methods: HMy2.CIR cells were irradiated with 0.032 Gy γ-rays three times per week for 1–4 weeks. Some of these primed cells were further challenged with 2 Gy γ-rays. Cell proliferation, micronuclei formation, gene expression of DNA methyltransferases (DNMT), levels of global genomic DNA methylation and protein expression of methyl CpG binding protein 2 (MeCP2) and heterochromatin protein-1 (HP1) were measured.Results: Long-term LDR enhanced cell proliferation and clonogenicity and triggered a cellular adaptive response (AR). Furthermore, global genomic DNA methylation was increased in HMy2.CIR cells after long-term LDR, accompanied with an increase of gene expression of DNMT1 and protein expression of MeCP2 and HP1. After treatment with 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, the long-term LDR-induced global genomic DNA hypermethylation was decreased and the AR was eliminated.Conclusion: Global genomic DNA hypermethylation accompanied with increases of DNMT1 and MeCP2 expression and heterochromatin formation might be involved in long-term LDR-induced adaptive response.

Long-term cadmium exposure leads to the enhancement of lymphocyte proliferation via down-regulating p16 by DNA hypermethylation

Cadmium (Cd) is a well-established carcinogen, however, the underlying mechanism, especially the role of epigenetics in it, is still poorly understood. Our previous work has disclosed that when rats were exposed to 0.5mg CdCl2 (kgd) for 8 and 12 weeks, the growth of peripheral white blood cells (WBC) was obviously stimulated but no over-proliferation of granulocyte–monocyte (GM) progenitor cells was observed in the bone marrow, suggesting that the over-proliferation of lymphocyte was promoted by Cd exposure. Is DNA-methylation involved in this Cd-stimulated cell proliferation? The present study found that when human B lymphoblast HMy2.CIR cells were exposed to Cd with a dose lower than 0.1μM for 3 months, both cell proliferation and mRNA expressions of DNA methyltransferases of DNMT1 and DNMT3b were increased, while the mRNA of tumor suppressor gene p16 was remarkably decreased. Furthermore, the level of genomic DNA methylation was increased and the CpG island in p16 promoter was hypermethylated in the Cd-exposed cells. A DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC), diminished Cd-stimulated cell proliferation associated with p16 overexpression. Our results suggested that the chronic exposure of low dose Cd could induce hypermethylation of p16 promoter and hence suppress p16 expression and then promote cell proliferation, which might contribute to Cd-induced carcinogenesis.

Pre-Diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women

BackgroundAbnormal one-carbon metabolism may lead to general genomic (global) hypomethylation, which may predispose an individual to the development of colorectal neoplasia.MethodsWe evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses’ Health Study (NHS). Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.ResultsOverall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45). Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05). Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05).ConclusionsWe found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.

Pre‐diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women

ObjectiveWe evaluated the association between pre‐diagnostic leukocyte genomic DNA methylation levels and colorectal cancer risk.MethodsWe conducted a nested case‐control study of 358 colorectal cancer cases and 661 matched controls within the Nurses’ Health Study of women. Among the controls, we further examined major plasma components in one‐carbon metabolism pathway in relation to genomic DNA methylation levels. Liquid chromatography electrospray ionization mass spectrometry method was used to measure DNA methylation. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.ResultsGenomic DNA methylation levels were not associated with colorectal cancer risk. Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile (p for trend, 0.45). We did not find associations of major plasma components of one‐carbon metabolism or several risk factors for colorectal cancer with genomic DNA methylation levels. The plasma components and risk factors for colorectal cancer did not modify the association between genomic DNA methylation and colorectal cancer risk.ConclusionsWe found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed in other populations including men.Funding source: This work was supported by research grants CA136950 and CA55075 from the National Institutes of Health

Димерные бисбензимидазолы: цитотоксичность и влияние на метилирование ДНК в нормальных и раковых клетках человека

Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a small extent stimulate the proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) display ability to penetrate into the nucleus of HeLa and F-977 cells and accumulate in various parts of the nuclei. DB(11) is not able to penetrate into the nuclei of these cells. The incubation of F-977 cells with 26 microM of DB(1) or DB(3) led to a decrease of the methylation of 18S rRNA gene, which is located in the region of DB(1) and DB(3) accumulation. A similar effect produces the same concentration of DB (3) in the F-977 cells. However, the overall level of genomic DNA methylation was not changed. These data suggest that DB(n) can be directed to act on specific genes demethylation and in the future may selectively inhibit the proliferation of cancer cells.

Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA

This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.

Effect of low temperature on genomic DNA methylation in Nile tilapia (Oreochromis niloticus)

In this study,using the methylation sensitive amplified polymorphism(MSAP) method,we globally assessed the effect of low temperature adaptation on the level and variations of cytosine methylation alterations at CCGG sites of genomic DNA in Nile tilapia(Oreochromis niloticus) strains with good coldresistance characters developed by the successive and directional selection breeding of multi-generations. Total of 849 fragments were identified by 18 pairs of se1ective primer combinations of which 411 and 438 were detected in cold-resistance tilapia strain and the control,respectively. Among these fragments,the methylation sites were 72 and 104,and the corresponding total-methylation levels w ere at 17.52% and 23.74%,respectively. The full methylation sites at the internal Cs were 37 and 65,and the corresponding full methylation levels were at 9. 00% and 14. 84%,respectively,and the hemi-methylation sites at the external Cs were 35 and 39,and the corresponding hemi-methylation levels were at 8.52% and 8.90% in coldresistance tilapia strain and the control,respectively. Further analysis of DNA methylation indicated that a statistically decrease in the overall level of total-methylation levels and both methylation types was detected in cold-resistance tilapia strain compared with the control(decrease at 6.22%,5.84% and 0.38%, respectively),and the changes of DNA methylation pattern were recognized mainly in the full methylation sites. The level of decrease in DNA methylation suggested that the levels of genomic DNA methylation were changed during the successive cold stress of multi-generations in tilapia,and it also indicated that DNA methylation alteration in tilapia treated with cold stress was mainly through de-methylation that occurred in some CCGG sites. All these results implied that the change of DNA methylation was closely associated with cold tolerance of tilapia. The present findings are valuable to further explore the application potential of DNA methylation alteration in tilapia genetic improvement and provide a new method and theoretical basis for fish stress resistance breeding.

DNA methylation status is associated with the formation of heterosis in Larix kaempferi intraspecific hybrids

Heterosis is prevalent in Larix kaempferi, but the molecular mechanism underlying heterosis in this species has not yet been elucidated. We have explored the relationships among heterosis and DNA sequence variations, changes in the DNA methylation status, and gene expression in a set of Larix kaempferi intraspecific parental lines and their reciprocal hybrids. The results indicate that the two intraspecific parental lines have highly similar genome structures and that the crosses did not produce substantial changes in the genomic DNA of the hybrid progeny. In contrast, there was a significant difference in the DNA methylation status between the two parental lines. The genomic DNA methylation level in the heterotic hybrids (26.47 %) was significantly lower than the midparent value (MPV) (33.80 %), indicating that significant extensive demethylation had occurred. However, the non-heterotic hybrids displayed a DNA methylation level (33.59 %) that was close to the MPV, and they contained similar numbers of methylation and demethylation events. The correlation analysis revealed that the increased gene expression in the heterotic hybrids was associated with its overall low genomic DNA methylation level and dynamic DNA methylation patterns. Twenty fragments with adjusted DNA methylation patterns were then cloned and the transcript level analyzed quantitatively. The results further indicated that the expressions of most but not all of the 12 possible genes were negatively correlated to their respective DNA methylation status. Based on these data, we suggest that adjustments in DNA methylation status may play critical roles in the formation of heterosis produced by the cross between two Larix kaempferi intraspecific parental lines.

Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures

KEY MESSAGE : The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities. In the aim of exploring the relationship between epigenetic stability and the long-term in vitro proliferation of plant tissues, we have studied changes in genomic DNA methylation levels in embryogenic suspensions of oil palm (Elaeis guineensis Jacq.). Five embryogenic callus lines were obtained from selected hybrid seeds and then proliferated as suspension cultures. Each clonal line obtained from a single genotype was subdivided into three independent subclonal lines. Once established, cultures proliferated for 12months and genomic DNA was sampled at 4months intervals for the estimation of global DNA methylation rates through high performance liquid chromatography (HPLC) quantitation of deoxynucleosides. Our results show that in vitro proliferation induces DNA hypermethylation in a time-dependent fashion. Moreover, this trend is statistically significant in several clonal lines and shared between subclonal lines originating from the same genotype. Interestingly, the only clonal line undergoing loss of genomic methylation in the course of proliferation has been found unable to generate somatic embryos. We discuss the possible implications of genome-wide DNA methylation changes in proliferating cells with a view to the maintenance of genomic and epigenomic stability.

The comprehensive effects of hyperlipidemia and hyperhomocysteinemia on pathogenesis of atherosclerosis and DNA hypomethylation in ApoE<sup>−/−</sup> mice

Atherosclerosis (AS) is a disease induced by multiple factors, including genetic and environmental elements. The aim of the present study is to investigate the comprehensive effects of high cholesterol, high methionine diet, and apolipoprotein E deficiency (ApoE(-/-)) on the pathogenesis of AS. ApoE(-/-) mice were fed with high cholesterol and methionine diet for 15 weeks to induce hyperlipidemia and hyperhomocysteinemia. The methylation levels of genomic DNA (gDNA) and B1 repetitive elements in aortic tissues were measured by both methylation-dependent restriction analysis and nested methylation-specific polymerase chain reaction (PCR). Methylation sequence-bias pattern was assayed by DNA methyl-accepting capacity with restriction endonuclease digestion. The mRNA expression of DNA methyltransferase-1, 3 (DNMT1, 3) was detected by real-time PCR. The concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were determined by high-performance liquid chromatography. The results showed hypomethylation of gDNA and B1 repetitive elements. The mRNA expression of DNMT1 was reduced. The levels of SAM, SAH, and SAM/SAH ratio were increased. The atherosclerotic lesion areas strongly correlated with the risk factors. The distribution of DNA demethylation was preferred to non-CpG islands, which may suggest the major impact of hypomethylation on DNA integrity and genomic instability. Overall, our data unequivocally showed that the comprehensive role of high cholesterol, high methionine diet, and ApoE(-/-) is not uniformly consistent with the role of a single risk factor. The DNA methylation pattern in AS is quite complex and depends on genetic background and many involved risk factors.

Genomic Methylation Changes Over Time in Peripheral Blood Mononuclear Cell DNA: Differences by Assay Type and Baseline Values

Lower levels of genomic DNA methylation in blood DNA has been associated with risk of different cancers and several cancer risk factors. To understand the use of genomic methylation measures as biomarkers of cancer risk, data are needed on within-individual changes over time. Using information from 77 subjects with blood collected at 2 visits on average 8 years apart, we examined whether levels of DNA methylation change with time and if so, whether selected cancer risk factors predict these changes. We measured DNA methylation levels in peripheral blood mononuclear cells (PBMC) using three assays that have been used in epidemiologic studies: (i) luminometric methylation assay (LUMA)(ii) LINE-1 by pyrosequencing, and (iii) Sat2 by MethyLight. Close to a third of all individuals had large changes over time (≥10%) in LUMA with 19.5% increasing and 13.0% decreasing. For Sat2, two-thirds of individuals had large changes with 40% increasing and 26% decreasing over time. In contrast, only 3.9% of individuals had large changes in LINE-1 over time. The degree of change in PBMC DNA methylation was statistically significantly inversely associated with methylation levels at baseline; greater decreases were observed in individuals with higher baseline values for each assay. These data, if replicated, suggest that changes in DNA methylation over time are highly associated with baseline values of the assay and vary by assay type. These findings suggest that assays that change more over time may warrant consideration for studies that measure later life exposures.

DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats

To explore the DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats. The primary macrophages and fibrocytes of SD rats were co-cultured directly and indirectly, which were exposed to SiO2 at the doses of 25, 50 and 100 g/ml. The transdifferentiation of cFb was identified with immunohistochemical assay. The genomic DNA methylation levels of cFb were detected with HPLC. Under the condition of indirect co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 19.9%, 26.9% and 30.3%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 22.0% (P < 0.05). Under the condition of ThinCert(TM) direct co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 22.2%, 30.2% and 36.7%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 20.6% (P < 0.05). Under the co-culture condition in vitro, SiO2 could reduce the genomic DNA methylation levels of cFb. The ThinCert(TM) direct co-culture can be used to study the silicosis fibrosis.

Expression Patterns of Non-Coding Spliced Transcripts from Human Endogenous Retrovirus HERV-H Elements in Colon Cancer

BackgroundUp-regulation of the most abundant H family human endogenous retrovirus (HERV-H), especially env-related transcripts, correlates with colon cancer. However, expression pattern of spliced non-coding transcripts of HERV-H is not clear.Methodology/Principal FindingsIn this study, expression of HERV-H spliced transcripts in colon cancer was investigated by a RT-PCR strategy using primers targeting the tRNAHis primer-binding site and the R region in the 3′ long terminal repeat (LTR), followed by cloning and sequencing of the amplicons. Sequences were then assigned to individual HERV-H loci by employing private nucleotide differences between loci. Different expression patterns of HERV-H spliced transcripts from distinct active elements were found in colon cancer cell lines HT29, LS 174T, RKO, SW480 and SW620. Furthermore, the expression patterns in SW480 and RKO were significantly changed by demethylation treatment. Interestingly, more HERV-H elements were found to be transcriptionally active in colon tumor tissues than in adjacent normal tissues (14 vs. 7).Conclusions/SignificanceThis is the first research to study the character of expression of non-coding spliced transcripts of HERV-H elements in colon cancer. Expression patterns of HERV-H spliced transcripts differed among colon cancer cell lines and could be affected by genomic DNA methylation levels. More importantly, besides the commonly accepted view of up-regulation of HERV-H expression in colon tumor tissues, we found more active HERV-H loci in colon tumor as compared with adjacent normal tissues.

Prenatal Smoke Exposure and Genomic DNA Methylation in a Multiethnic Birth Cohort

Exposure to prenatal tobacco smoke (PTS) has been associated with a number of health outcomes in the offspring, including some childhood cancers. Lower levels of genomic DNA methylation have also been associated with several types of cancers. We investigated whether PTS was associated with global DNA methylation levels in the offspring. Our sample was drawn from a birth cohort of women born between 1959 and 1963 in New York City (n = 90). We measured methylation of repetitive elements (Sat2, Alu, LINE-1) from peripheral blood granulocytes. We combined prospectively collected data on PTS with adult epidemiologic data and blood samples collected in 2001 to 2007 (mean age, 43 years). We used linear regression to assess the association between PTS and repetitive element methylation. Thirty-six percent of mothers smoked during pregnancy. We observed an inverse association between PTS and Sat2 methylation. This inverse association remained even after adjustment for potential mediators including child environmental tobacco smoke exposure, birth size, postnatal weight and height changes, and adult smoking status and alcohol intake (β = -0.22, 95% confidence interval = -0.40 to -0.03 for ever exposed to PTS vs. never exposed using models of log-transformed methylation levels). PTS exposure was not statistically significantly associated with LINE-1 or Alu methylation. PTS exposure, measured at the time of pregnancy and not retrospectively reported, was associated with a decrease in Sat2 methylation but not LINE-1 or Alu methylation. If replicated in larger studies, this study supports a persistent effect of PTS on DNA methylation levels, as measured by Sat2, in adulthood.

Quantitative measurement of genome-wide DNA methylation by a reliable and cost-efficient enzyme-linked immunosorbent assay technique

DNA methylation, the conversion of cytosine to 5-methylcytosine, is an important epigenetic modification involved in gene regulation. DNA methylation is essential for normal development whereas abnormal methylation has been implicated in pathological conditions including cancer. To evaluate the extent and variation of genome-wide DNA methylation and its changes during cellular differentiation and tumorgenesis as well as the interplay with histone modifications, accurate and reproducible quantification of the genomic DNA methylation level is required. These measurements have so far been achieved only by sophisticated and costly techniques. Here we report the generation of an enzyme-linked immunosorbent assay (methDNA-ELISA) for the accurate quantification of global DNA methylation levels. The linear region of this methDNA-ELISA ranges from 1 to 10%, making it highly suitable for the typical ranges from 2 to 6% in mammalian genomes. This method requires 10ng of isolated DNA per sample, thus permitting investigation with minimal amounts of DNA previously not applicable for global DNA methylation analysis, e.g., clinical biopsies or cells collected by microdissection.

Abstract A84: Reduced LINE-1 methylation in peripheral blood mononuclear cells is associated with risk of melanoma in families segregating CDKN2A germline mutations

Abstract Background: CDKN2A is a major susceptibility gene for familial melanoma, but its incomplete penetrance suggests that other factors may modify the effect of this gene in melanoma-prone families. Epigenetic changes involving reduced levels of global DNA methylation in blood have been associated with genomic instability and cancer risk. In addition, genetic background has the potential to influence epigenetic processes through cis- or trans-regulatory variation. Therefore, we hypothesize that the CDKN2A carrier mutation state may be an important factor influencing global DNA methylation in predisposed individuals and that aberrant methylation may be an indicator of modified risk in individuals from melanoma-prone families segregating CDKN2A germline mutations. Methods: We measured the overall level of genomic DNA methylation using bisulfite pyrosequencing at four CpG sites (CpG1, CpG2, CpG3 and CpG4) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in peripheral blood mononuclear cells (PBMCs) from 42 CDKN2A mutation positive cases (20 males and 22 females) and 73 controls (29 CDKN2A carrier controls [9 males and 20 females] and 44 non-carrier controls [19 males and 25 females]) in 26 families with CDKN2A mutations. LINE-1 methylation was reported as the average (of all 4 CpGs) and as site-specific percentage methylation. Odds' ratios (OR) and P-values were obtained by comparing melanoma cases to controls using unconditional logistic regression with adjustment for family correlation (using the generalized estimating equations [GEE] approach), age, sex, number of moles and with melanoma status as the outcome variable. Percentage LINE-1 methylation was categorized using tertiles with the highest tertile as the reference. Results: Male gender was significantly associated with higher average LINE-1 methylation (P=&amp;lt;0.001) and methylation at all CpG sites (P=&amp;lt;0.03). In contrast, age, solar injury, dysplastic nevi (DN) and number of moles were not associated with average LINE-1 methylation. Although mean levels of LINE-1 methylation were similar in melanoma cases, carrier controls, and non-carrier controls, after adjusting for family correlation, age, sex and number of moles, low average LINE-1 methylation (tertile 1) was significantly associated with increased risk of melanoma (OR=3.87, 95% confidence interval [CI]: 1.11–13.5, P=0.033, compared to all controls; OR=1.83, 95% CI: 1.09–11.7, P=0.035, compared to carrier controls; and OR=3.43, 95% CI: 1.17–10.0, P= 0.024, compared to non-carrier controls). This association was observed for most CpG sites with the most significant association seen for site 3 and risk of melanoma compared to all controls (OR=5.18, 95% CI: 1.54–17.4, P=0.008). We observed no significant association between average or CpG site-specific LINE-1 methylation and CDKN2A mutation status among controls although the comparison was limited by small sample size. Conclusions: Melanoma cases from families segregating CDKN2A germline mutations have significantly lower LINE-1 global methylation in their PBMCs compared to controls. Changes in LINE-1 methylation in PBMCs from CDKN2A mutation positive cases may reflect a potential genetic background influence on global DNA methylation in these individuals. Further studies are needed to clarify these preliminary observations. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A84.

Abnormal DNA methylation in CD4 + T cells from people with latent autoimmune diabetes in adults

Aberrant DNA methylation in T cells has been linked to pathogenesis of autoimmune diseases. To investigate genomic and gene-specific DNA methylation levels in CD4 + T cells from patients with latent autoimmune diabetes in adults (LADA), and to investigate changes in the expression of genes that regulate methylation as well as the autoimmune-related gene FOXP3 in these patients. Global CD4 + T cell DNA methylation was measured in 15 LADA patients and 11 healthy controls using a methylation quantification kit. mRNA levels of DNA methytransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and FOXP3 were measured by real time PCR. Methylation of a FOXP3 regulatory element region was determined by bisulphite genomic sequencing. Genomic DNA methylation in CD4 + T cells from LADA patients was significantly increased compared to controls. DNMT3b mRNA levels were higher in CD4 + T cells from LADA patients than in controls. DNMT3b expression positively correlated with global DNA methylation in LADA CD4 + T cells. FOXP3 expression was decreased, and the FOXP3 promoter region was hypermethylated in CD4 + T cells from LADA patients compared with controls. DNA methylation levels are altered in CD4 + T cells from LADA patients, which may contribute to disease onset and progression by affecting the expression of autoimmune-related genes.

Evaluating the Global CpG Methylation Status of Native DNA Utilizing a Bipartite Split-Luciferase Sensor

Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys(2)-His(2) zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor.

Effects of long-term low-dose formaldehyde exposure on global genomic hypomethylation in 16HBE cells

Formaldehyde (FA), a volatile organic compound, is a ubiquitous air pollutant that is classified as ‘Carcinogenic to humans (Group 1)’ by IARC (2006). As a well-recognized human carcinogen, its carcinogenic mechanisms are still poorly understood. Previous studies have emphasized on genetic changes. However, little is known about the epigenetic mechanisms of FA exposure. In this study, We not only characterized the epigenomic response to long-term low-dose FA exposure in 16HBE cells, but also examined the expression of DNA methyltransferases (DNMTs) and the methyl-CpG-binding protein DNA-binding domain protein 2 (MBD2). Each week the 16HBE cells were treated with 10 μM FA for 24 h (h). After 24 weeks (W) of exposure to FA, the level of genomic DNA methylation gradually decreased in a time-related manner. Moreover, our results showed that FA exposure down-regulated the expression of DNMT3a and DNMT3b at both mRNA and protein level, and up-regulated the levels of DNMT1 and MBD2 at both mRNA and protein level. Our study indicated that long-term FA exposure could disrupt genomic DNA methylation, which may be one of the possible underlying carcinogenic mechanisms of FA.

Hydroxychloroquine reverses ultraviolet ray-induced genomic DNA hypomethylation in CD4+ T cells from patients with systemic lupus erythematosus

Objective To explore the effect of hydroxychloroquine on ultraviolet ray-induced genomic DNA hypomethylation in CD4+ T cells from patients with systemic lupus erythematosus (SLE). Methods Thirty patients with SLE and 10 normal human controls were enrolled in the study. CD4+ T cells were isolated from these subjects by using magnetic beads, and cultured. Hydroxychloroquine of 50 mg/L was added to the culture medium of CD4+ T cells before or after the exposure to narrow band ultraviolet B (NB-UVB) 311 nm.After additional culture, the levels of genomic DNA methylation in CD4+ T cells were determined with the Imprint Methylated DNA Quantification kit. Results The levels of DNA methylation was lower in SLE patients than in the normal controls [(3.922 ±2.215)% vs. (10.210 ± 5.573)%, t= 3.450, P = 0.026]. After exposure to UVB at 45 and 100 mJ/cm2, the DNA methylation level in patients with active SLE decreased from (7.235 ±3.846)% to (1.784 ± 1.033)% and (1.932 ± 1.844)% respectively (t= 3.000, 4.118, both P< 0.05). Decreased DNA methylation level was observed in CD4+ T cells from patients with active SLE compared with those from patients with stable SLE and normal human controls [(1.932 ± 1.844)% vs. (7.235 ± 3.846)% and (5.472 ±5.573)%, t = 2.648, 3.000, both P< 0.05] after irradiation with UVB of 100 mj/cm2. A significant increase in the methylation level was observed in active SLE patient-derived CD4+ T cells treated with hydroxychloroquine following the irradiation with UVB of 45 (4.698% ± 1.948%) and 100 mJ/cm2(8.698% ± 3.151%) compared with those only treated with UVB irradiation (t = 4.827, 3.184, both P< 0.05), as well as in those treated with hydroxychloroquine before and after the irradiation with UVB of 45 mJ/cm2 compared with those receiving irradiation alone [(5.404 ± 2.308)% vs. (1.784 ± 1.033)%, t = 4.827, P< 0.01]. Conclusion Hydroxychloroquine can reverse the UVB-induced genomic DNA hypomethylation in CD4+ T cells from patients with SLE,especially in those from patients with active SLE. Key words: Lupus erythematosus,sytemic; Ultraviolet rays; Hydroxychloroquine; DNA methylatio