Related Articles

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.

Objective

The present study was undertaken to analyze the impact of epigenetic alterations with a main focus on nuclear area, aneuploidy, hyperploidy, and proliferation in 70 ovarian cancer specimens.

Methods

Morphometric changes and somatic chromosomal ploidy status were assessed by Feulgen spectrophotometry. DNA-hypomethylation of LINE1 repeats was analyzed by means of MethyLight PCR, and methylation levels of satellite 2 (Sat2) and satellite alpha (Satα) DNA sequences in chromosome 1 were measured by Southern blot analysis. These parameters were analyzed with regard to correlations as well as to recurrence and survival.

Results

We identified a significant association between LINE1 DNA-hypomethylation and patient age (p = 0.029). Furthermore, LINE1 DNA-hypomethylation was positively correlated with the nuclear area (r = 0.47; p<0.001) and the proliferation index (r = 0.36; p<0.001). Univariate survival analysis showed that the nuclear area and LINE1 DNA-hypomethylation were prognostic factors for overall (p=0.015 and = 0.006, respectively) and progression-free survival (p=0.020 and p=0.001 respectively), the percentage of aneuploidy only for overall survival (p=0.031). Subgroup survival analyses revealed that the prognostic value of these factors is strictly confined to mucinous cancers. In serous cancers no prognostic value could be pointed out for any analyzed parameter. Multivariate analysis of the entire cohort showed that the percentage of hyperploidy was an independent prognostic parameter for overall survival (p=0.003) and LINE1 DNA-hypomethylation for progression-free survival (p=0.03). In mucinous cancers nuclear area and LINE1 DNA-hypomethylation were found to be independent predictors of progression-free and overall survival.

Conclusions

In this study we identified the correlations between early cancer-associated genome DNA-hypomethylation, nuclear morphometric changes, somatic chromosomal ploidy status and the proliferation index. Prognostic relevance of nuclear area and LINE1 DNA-hypomethylation was revealed exclusively in mucinous ovarian cancers.

Lower levels of global DNA methylation in white blood cell (WBC) DNA have been associated with adult cancers. It is unknown whether individuals with a family history of cancer also have lower levels of global DNA methylation early in life. We examined global DNA methylation in WBC (measured in three repetitive elements, LINE1, Sat2 and Alu, by MethyLight and in LINE1 by pyrosequencing) in 51 girls aged 6–17 years. Compared to girls without a family history of breast cancer, methylation levels were lower for all assays in girls with a family history of breast cancer and statistically significantly lower for Alu and LINE1 pyrosequencing. After adjusting for age, body mass index (BMI) and Tanner stage, only methylation in Alu was associated with family history of breast cancer. If these findings are replicated in larger studies, they suggest that lower levels of global WBC DNA methylation observed later in life in adults with cancer may also be present early in life in children with a family history of cancer.

DNA methylation measured in white blood cell DNA is increasingly being used in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran) and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources [WBC, Gran, mononuclear (MN) and lymphoblastoid cell lines (LCL)], we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA) and [3H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LCL, MN or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [3H]-methyl acceptance, LINE1 and Alu assays. Methylation in MN was correlated with methylation in WBC for the [3H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant correlations ranging from 0.3–0.7 for each cell type with one exception (Sat2 and Alu in MN). Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39–0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18–0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5’ gene region or promoter hypermethylation, satellite or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, 6 of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P<0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor cause of satellite and global DNA hypomethylation.

Global DNA hypomethylation has been associated with increased risk for cancers of the colorectum, bladder, breast, head and neck, and testicular germ cells. The aim of this study was to examine whether global hypomethylation in blood leukocyte DNA is associated with the risk of hepatocellular carcinoma (HCC). A total of 315 HCC cases and 356 age-, sex- and HBsAg status-matched controls were included. Global methylation in blood leukocyte DNA was estimated by analyzing long interspersed element-1 (LINE-1) repeats using bisulfite-polymerase chain reaction (PCR) and pyrosequencing. We observed that the median methylation level in HCC cases (percentage of 5-methylcytosine (5mC)=77.7%) was significantly lower than that in controls (79.5% 5mC) (P=0.004, Wilcoxon rank-sum test). The odds ratios (ORs) of HCC for individuals in the third, second, and first (lowest) quartiles of LINE-1 methylation were 1.1 (95% confidence interval (CI) 0.7–1.8), 1.4 (95% CI 0.8–2.2), and 2.6 (95% CI 1.7–4.1) (P for trend <0.001), respectively, compared to individuals in the fourth (highest) quartile. A 1.9-fold (95% CI 1.4–2.6) increased risk of HCC was observed among individuals with LINE-1 methylation below the median compared to individuals with higher (>median) LINE-1 methylation. Our results demonstrate for the first time that individuals with global hypomethylation measured in LINE-1 repeats in blood leukocyte DNA have an increased risk for HCC. Our data provide the evidence that global hypomethylation detected in the easily obtainable DNA source of blood leukocytes may help identify individuals at risk of HCC.

Background

Global loss of methylated cytosines in DNA, thought to predispose to chromosomal instability and aneuploidy, has been associated with an increased risk of colorectal neoplasia. Little is known about the relationships between global hypomethylation and lifestyle, demographics, dietary measures and genetic factors.

Methods

Our data were collected as part of a randomized clinical trial testing the efficacy of aspirin and folic acid for the prevention of colorectal adenomas. At a surveillance colonoscopy approximately three years after the qualifying exam, we obtained two biopsies of the normal-appearing mucosa from the right colon and two from the left colon. Specimens were assayed for global hypomethylation using a pyrosequencing assay for LINE-1 (long interspersed nucleotide elements) repeats.

Results

The analysis included data from 388 subjects. There was relatively little variability in LINE methylation overall. Mean LINE-1 methylation levels in normal mucosa from the right bowel were significantly lower than those on the left side (p<0.0001). No significant associations were found between LINE-1 methylation and folate treatment, age, sex, body-mass-index, smoking status, alcohol use, dietary intake or circulating levels of B-vitamins, homocysteine, or selected genotypes. Race, dietary folic acid and plasma B6 showed associations with global methylation that differed between the right and left bowel. The effect of folic acid on risk of adenomas did not differ according to extent of LINE-1 methylation and we found no association between LINE-1 methylation and risk of adenomas.

Conclusions

LINE-1 methylation is not influenced by folic acid supplementation, but differs by colon subsite.

Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.

Background

Promoter hypermethylation and global hypomethylation in the human genome are hallmarks of most cancers. Detection of aberrant methylation in white blood cells (WBC) has been suggested as a marker for cancer development, but has not been extensively investigated. This study was carried out to determine whether aberrant methylation in WBC DNA can be used as a surrogate biomarker for breast cancer risk.

Patients and Methods

Promoter hypermethylation of 8 tumor suppressor genes (RASSF1A, APC, HIN1, BRCA1, cyclinD1, RARβ, CDH1 and TWIST1) and DNA methylation for three repetitive elements (LINE1, Sat2M1 and AluM2) were analyzed in invasive ductal carcinoma of the breast, paired adjacent normal tissue and WBC from 40 breast cancer patients by the MethyLight assay. Methylation in WBC from 40 controls was also analyzed.

Results

Tumor and adjacent tissues showed frequent hypermethylation for all genes tested, while WBC DNA was rarely hypermethylated. For HIN1, RASSF1A, APC and TWIST1 there was agreement between hypermethylation in tumor and adjacent tissues (P=0.04, P=0.02, P=0.005 and P<0.0001, respectively). DNA methylation for the three repetitive elements was lower in tumor compared to adjacent tissue and WBC DNA. Significant correlations in the methylation of Sat2M1 between tumor and adjacent tissues and WBC DNA were found (P<0.0001 and P=0.046, respectively). There was also a significant difference in methylation of Sat2M1 between cases and controls (P=0.01).

Conclusion

These results suggest that further studies of WBC methylation, including prospective studies, may provide biomarkers of breast cancer risk.

Increased and decreased methylation at specific sequences (hypermethylation and hypomethylation, respectively) is characteristic of tumor DNA compared to normal DNA and promotes carcinogenesis in multiple ways including genomic instability. Long interspersed element (LINE), an abundant class of retrotransposons, provides a surrogate marker for global hypomethylation. We developed methylation-specific multiplex ligation-dependent probe amplification assays to study LINE-1 methylation in cases of colorectal, gastric, and endometrial cancer (N = 276), stratified by patient category [sporadic; Lynch syndrome (LS); familial colorectal cancer type X (FCCX)] and microsatellite instability status. Within each patient group, LINE-1 showed lower methylation in tumor DNA relative to paired normal DNA and hypomethylation was statistically significant in most cases. Interestingly, normal colorectal mucosa samples from different patient groups displayed differences in LINE-1 methylation that mirrored differences between the respective tumor tissues, with a decreasing trend for LINE-1 methylation from patients with sporadic colorectal cancer to LS to FCCX. Despite the fact that the degree of LINE-1 methylation is generally tissue specific, normal colorectal mucosa, gastric mucosa, and endometrium from LS patients showed similar levels of LINE-1 methylation. Our results suggest that the degree of LINE-1 methylation may constitute a “field defect” that may predispose normal tissues for cancer development.

Electronic supplementary material

The online version of this article (doi:10.1007/s00109-011-0854-z) contains supplementary material, which is available to authorized users.

Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.

Genome-wide DNA hypomethylation plays has an important role in genomic instability and colorectal carcinogenesis. However, the relationship between cellular DNA methylation level and patient outcome remains uncertain. Using 643 colon cancers in two independent prospective cohorts, we quantified DNA methylation in repetitive long interspersed nucleotide element-1 (LINE-1) elements using pyrosequencing, which is a good indicator of global DNA methylation level. We used Cox proportional hazard models to calculate hazard ratios (HRs) of colon cancer–specific and overall mortality, adjusting for patient and tumoral features, including CpG island methylator phenotype (CIMP). Statistical tests were two-sided. LINE-1 hypomethylation was linearly associated with a statistically significant increase in colon cancer–specific mortality (for a 30% decrease in LINE-1 methylation: multivariable HR = 2.37, 95% confidence interval [CI] = 1.42 to 3.94; Ptrend < .001) and overall mortality (multivariable HR = 1.85, 95% CI = 1.25 to 2.75; Ptrend = .002). The association was consistent across the two independent cohorts and strata of clinical and molecular characteristics, including sex, age, tumor location, stage, and CIMP, microsatellite instability, KRAS, BRAF, p53, and chromosomal instability status. In conclusion, tumoral LINE-1 hypomethylation is independently associated with shorter survival among colon cancer patients.

The CpG island methylator phenotype (CIMP) with widespread promoter CpG island methylation is a phenotype in colorectal cancer, associated with microsatellite instability (MSI) and BRAF mutation. Genome-wide hypomethylation may also play an important role in genomic instability. However, the relation between global DNA methylation level and methylation in individual CpG islands remains uncertain. Utilizing 869 population-based colorectal cancers, we measured LINE-1 (long interspersed nucleotide element-1) methylation level by Pyrosequencing, which correlates with global DNA methylation level. We quantified DNA methylation in 8 CIMP-specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) by real-time PCR (MethyLight technology). LINE-1 methylation levels in tumors were approximately normally distributed (mean 61.4%, median 62.3%, standard deviation 9.6%). Among the 869 tumors, 128 (15%) were classified as CIMP-high (≥6/8 methylated promoters). The mean LINE-1 methylation level was higher in CIMP-high tumors (65.1%, p<0.0001) than non-CIMP-high tumors (60.7%), and higher in MSI-high tumors (64.7%, p<0.0001) than non-MSI-high tumors (60.7%). When tumors were stratified by MSI/CIMP status, compared to non-MSI-high non-CIMP-high tumors (mean LINE-1 methylation level 60.4%), the mean LINE-1 methylation level was higher in MSI-high CIMP-high (64.8%, p<0.0001), MSI-high non-CIMP-high (64.6%, p=0.03) and non-MSI-high CIMP-high tumors (66.1%, p=0.0003). In addition, 18q loss of heterozygosity in non-MSI-high tumors was correlated with LINE-1 hypomethylation (p=0.004). In conclusion, both CIMP-high and MSI-high are inversely associated with LINE-1 hypomethylation, suggesting that CIMP/MSI and genomic hypomethylation may represent different pathways to colorectal cancer. Our data also support a possible link between global hypomethylation and chromosomal instability.

Epigenetic changes in long interspersed nuclear element-1s (LINE-1s or L1s) occur early during the process of carcinogenesis. A lower methylation level (hypomethylation) of LINE-1 is common in most cancers, and the methylation level is further decreased in more advanced cancers. Consequently, several previous studies have suggested the use of LINE-1 hypomethylation levels in cancer screening, risk assessment, tumor staging, and prognostic prediction. Epigenomic changes are complex, and global hypomethylation influences LINE-1s in a generalized fashion. However, the methylation levels of some loci are dependent on their locations. The consequences of LINE-1 hypomethylation are genomic instability and alteration of gene expression. There are several mechanisms that promote both of these consequences in cis. Therefore, the methylation levels of different sets of LINE-1s may represent certain phenotypes. Furthermore, the methylation levels of specific sets of LINE-1s may indicate carcinogenesis-dependent hypomethylation. LINE-1 methylation pattern analysis can classify LINE-1s into one of three classes based on the number of methylated CpG dinucleotides. These classes include hypermethylation, partial methylation, and hypomethylation. The number of partial and hypermethylated loci, but not hypomethylated LINE-1s, is different among normal cell types. Consequently, the number of hypomethylated loci is a more promising marker than methylation level in the detection of cancer DNA. Further genome-wide studies to measure the methylation level of each LINE-1 locus may improve PCR-based methylation analysis to allow for a more specific and sensitive detection of cancer DNA or for an analysis of certain cancer phenotypes.

Electronic supplementary material

The online version of this article (doi:10.1007/s13148-011-0032-8) contains supplementary material, which is available to authorized users.

Background:

The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and BRAF mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis.

Methods:

Utilizing MethyLight technology (real-time PCR), we quantified DNA methylation in 8 CIMP-specific promoters {CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487) and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with ≥ 2 markers showing LOH; and 138 LOH-negative tumors with ≥ 3 informative markers and no LOH).

Results:

CIMP-0 (0/8 methylated promoters) was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002) than 18q LOH-negative tumors (44% = 61/138), while CIMP-low/high (1/8–8/8 methylated promoters) was significantly more common (56%) in 18q LOH-negative tumors than 18q LOH-positive tumors (41%). These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry), or KRAS/BRAF mutation.

Conclusion:

18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in colorectal cancer.

Background

Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation.

Methodology/Principal Findings

We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer.

Conclusions/Significance

The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions.

Background: DNA methylation is a potential pathway linking environmental exposures to disease. Exposure to particulate air pollution has been associated with increased cardiovascular morbidity and mortality, and lower blood DNA methylation has been found in processes related to cardiovascular morbidity.

Objective: We hypothesized that prolonged exposure to particulate pollution would be associated with hypomethylation of repetitive DNA elements and that this association would be modified by genes involved in glutathione metabolism and other host characteristics.

Methods: DNA methylation of the long interspersed nucleotide element–1 (LINE-1) and the short interspersed nucleotide element Alu were measured by quantitative polymerase chain reaction pyrosequencing in 1,406 blood samples from 706 elderly participants in the Normative Aging Study. We estimated changes in repetitive element DNA methylation associated with ambient particles (particulate matter ≤ 2.5 µm in aerodynamic diameter), black carbon (BC), and sulfates (SO4), with mixed models. We examined multiple exposure windows (1–6 months) before DNA methylation measurement. We investigated whether this association was modified by genotype and phenotype.

Results: An interquartile range (IQR) increase in BC over a 90-day period was associated with a decrease of 0.31% 5-methylcytosine (5mC) (95% confidence interval, 0.12–0.50%) in Alu. An IQR increase in SO4 over a 90-day period was associated with a decrease of 0.27% 5mC (0.02–0.52%) in LINE-1. The glutathione S-transferase mu-1–null genotype strengthened the association between BC and Alu hypomethylation.

Conclusion: Prolonged exposure to BC and SO4 particles was associated with hypomethylation of two types of repetitive elements.

Purpose

Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer.

Experimental Design

LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation.

Results

Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer (95% CI, 1.12-2.90) in models controlling for gender, age and smoking, and the association was stronger in women than in men (ORs = 2.48, 95% CI 1.19-5.17 in women and 1.47, 95% CI 0.79-2.74 in men). Amongst controls, women were more likely to have lower LINE1 methylation than men (p-value 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (p-value 0.04).

Conclusions

LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially amongst women.

Summary

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methyl cytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and to date no base-resolution maps exist to support and refine it. Here, we generated genome-scale DNA methylation maps in mouse gametes and through post-implantation embryogenesis. We find that the oocyte already exhibits global hypomethylation, most prominently at specific families of long interspersed element-1 and long terminal repeat retro-elements, which are disparate between gametes and resolve to lower methylation values in zygote. Surprisingly, the oocyte contributes a unique set of Differentially Methylated Regions (DMRs), including many CpG Island promoter regions, that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and resolve to hypermethylation after the blastocyst stage. Our data provide a complete genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.

Hepatocellular carcinoma (HCC) is known to be associated with both HBV and HCV and HVC. While epigenetic changes have been previously reported to be associated with hepatocellular carcinoma (HCC), whether the epigenetic profile of HBC associated HCC differs from that of HCV associated HCC is unclear. We analyzed DNA methylation of ten genes (APC, CCND2, CDKN2A, GSTP1, HOXA9, RARB, RASSF1, RUNX, SFRP1, and TWIST1) using MethyLight assays on 65 archived liver tissue blocks. Three genes (APC, CCND2, and GSTP1) were frequently methylated in normal liver tissues. Five genes (APC, CDKN2A, HOXA9, RASSF1, and RUNX) were significantly more frequently methylated in malignant liver tissues than normal liver tissues. Among HCC cases, HOXA9, RASSF1 and SFRP1 were methylated more frequently in HBV positive HCC cases, while CDKN2A were significantly more frequently methylated in HCV positive HCC cases. Our data support the hypothesis that HCC resulting from different viral etiologies are associated with different epigenetic changes.

Purpose

Cancer germline (CG) antigens are frequently expressed and hypomethylated in epithelial ovarian cancer (EOC), but the relationship of this phenomenon to global DNA hypomethylation is unknown. In addition, the potential mechanisms leading to DNA hypomethylation, and its clinicopathological significance in EOC, have not been determined.

Experimental Design

We used quantitative mRNA expression and DNA methylation analyses to determine the relationship between expression and methylation of X-linked (MAGE-A1, NY-ESO-1, XAGE-1) and autosomal (BORIS, SOHLH2) CG genes, global DNA methylation (5mdC levels, LINE-1, Alu, and Sat-α methylation), and clinicopathology, using 75 EOC samples. In addition, we examined the association between these parameters and a number of mechanisms proposed to contribute to DNA hypomethylation in cancer.

Results

CG genes were coordinately expressed in EOC and this was associated with promoter DNA hypomethylation. Hypomethylation of CG promoters was highly correlated and strongly associated with LINE-1 and Alu methylation, moderately with 5mdC levels, and rarely with Sat-α methylation. BORIS and LINE-1 hypomethylation, and BORIS expression, were associated with advanced stage. GADD45A expression, MTHFR genotype, DNMT3B isoform expression, and BORIS mRNA expression did not associate with methylation parameters. In contrast, the BORIS/CTCF expression ratio was associated with DNA hypomethylation, and furthermore correlated with advanced stage and decreased survival.

Conclusions

DNA hypomethylation coordinately affects CG antigen gene promoters and specific repetitive DNA elements in EOC, and correlates with advanced stage disease. The BORIS/CTCF mRNA expression ratio is closely associated with DNA hypomethylation and confers poor prognosis in EOC.

Background

The product of CDKN2A, p16 is an essential regulator of the cell cycle controlling the entry into the S-phase. Herein, we evaluated CDKN2A promoter methylation and p16 protein expression for the differentiation of hepatocellular carcinoma (HCC) from other liver tumors.

Methods

Tumor and corresponding non-tumor liver tissue samples were obtained from 85 patients with liver tumors. CDKN2A promoter methylation was studied using MethyLight technique and methylation-specific PCR (MSP). In the MethyLight analysis, samples with ≥ 4% of PMR (percentage of methylated reference) were regarded as hypermethylated. p16 expression was evaluated by immunohistochemistry in tissue sections (n = 148) obtained from 81 patients using an immunoreactivity score (IRS) ranging from 0 (no expression) to 6 (strong expression).

Results

Hypermethylation of the CDKN2A promoter was found in 23 HCCs (69.7%; mean PMR = 42.34 ± 27.8%), six (20.7%; mean PMR = 31.85 ± 18%) liver metastases and in the extralesional tissue of only one patient. Using MSP, 32% of the non-tumor (n = 85), 70% of the HCCs, 40% of the CCCs and 24% of the liver metastases were hypermethylated. Correspondingly, nuclear p16 expression was found immunohistochemically in five (10.9%, mean IRS = 0.5) HCCs, 23 (92%; mean IRS = 4.9) metastases and only occasionally in hepatocytes of non-lesional liver tissues (mean IRS = 1.2). The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively).

Conclusion

Promoter methylation of CDKN2A gene and lack of p16 expression characterize patients with HCC.

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, accounting for an estimated 600,000 deaths annually. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients. We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further, selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly, several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression.

BACKGROUND AND AIMS

Although critical for methylation reactions, how dietary folate and B vitamins affect global DNA methylation level in colon cancers is currently unknown. Long interspersed nucleotide element-1 (LINE-1) is an emerging indicator of genome-wide DNA methylation level that has previously been linked to colon cancer survival.

METHODS

We examined the association between dietary intake of folate, alcohol, and B vitamins and LINE-1 hypomethylation in 609 incident colon cancers, utilizing the database of two independent prospective cohort studies.

RESULTS

Participants with ≥400µg folate intake per day were significantly less likely to develop LINE-1 hypomethylated colon cancers than those reporting <200µg of folate intake per day (Relative risk (RR)=0.57, 95% confidence interval (CI)=0.36–0.91) for <55% LINE-1 methylated colon tumors; RR=0.74, 95% CI=0.51–1.06 for 55–64% LINE-1 methylated colon tumors; and RR=1.08, 95% CI=0.66–1.75 for ≥65% LINE-1 methylated tumors; Pinteraction=0.01). By contrast, high alcohol consumption conferred a higher risk of LINE-1 hypomethylated cancers (≥15g alcohol per day versus none, RR=1.67, 95% CI=1.04–2.67 for <55% LINE1 methylated tumors; and RR=1.55, 95% CI=1.10–2.18 for 55–64% LINE-1 methylated tumors) but had no association with ≥65% LINE-1 methylated tumors (RR=1.06, 95% CI=0.69–1.62). High intakes of vitamin B6, B12, or methionine were not significantly associated with colon cancers, regardless of LINE-1 methylation level.

CONCLUSION

The influence of dietary folate intake and alcohol consumption on colon cancer risk differs significantly according to tumoral LINE-1 methylation level.