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1.  Global DNA methylation in a population with aflatoxin B1 exposure 
Epigenetics  2013;8(9):962-969.
We previously reported that global DNA hypomethylation, measured as Sat2 methylation in white blood cells (WBC), and aflatoxin B1 (AFB1) exposure were associated with increased hepatocellular carcinoma risk. In this study, we assessed the association between AFB1 exposure and global DNA methylation. We measured LINE-1 and Sat2 methylation in WBC DNA samples from 1140 cancer free participants of the Cancer Screening Program (CSP) cohort. Blood and urine samples were used to determine the level of AFB1-albumin (AFB1-Alb) adducts and urinary AFB1 metabolites. In continuous models, we found reverse associations of urinary AFB1 with LINE-1 and Sat2 methylation. The odds ratio (OR) per 1 unit decrease were 1.12 (95%CI = 1.03–1.22) for LINE-1 and 1.48 (95%CI = 1.10–2.00) for Sat2 methylation. When compared with subjects in the highest quartile of LINE-1, we found that individuals in the 2nd and 3rd quartiles were less likely to have detectable AFB1-Alb adducts, with ORs (95%CI) of 0.61 (0.40–0.93), 0.61 (0.40-.94), and 1.09 (0.69–1.72), respectively. The OR for detectable AFB1-Alb was 1.81 (95%CI = 1.15–2.85) for subjects in the lowest quartile of Sat2 methylation. The OR for detection of urinary AFB1 for those with LINE-1 methylation in the lowest quartile compared with those in the highest quartile was 1.87 (95%CI = 1.15–3.04). The corresponding OR was 1.75 (95%CI = 1.08–2.82) for subjects in the lowest quartile of Sat2 methylation. The association between AFB1 exposure and global DNA methylation may have implications for the epigenetic effect of AFB1 on hepatocellular carcinoma development and also suggests that changes in DNA methylation may represent an epigenetic biomarker of dietary AFB1 exposure.
PMCID: PMC3883773  PMID: 23867725
DNA methylation; Hepatitis B virus; LINE-1; Sat2; aflatoxin B1; global DNA methylation; white blood cell
2.  Aberrant Promoter Hypermethylation and Genomic Hypomethylation in Tumor, Adjacent Normal Tissues and Blood from Breast Cancer Patients 
Anticancer research  2010;30(7):2489-2496.
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.
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).
These results suggest that further studies of WBC methylation, including prospective studies, may provide biomarkers of breast cancer risk.
PMCID: PMC3568974  PMID: 20682973
Breast cancer; promoter hypermethylation; genomic methylation; tumor suppressor genes; repetitive elements; WBC DNA
3.  Global DNA methylation levels in white blood cells as a biomarker for hepatocellular carcinoma risk: a nested case–control study 
Carcinogenesis  2012;33(7):1340-1345.
Global DNA hypomethylation is associated with genomic instability and human cancer and blood DNAs collected at the time of cancer diagnosis have been used to examine the relationship between global methylation and cancer risk. To test the hypothesis that global hypomethylation is associated with increased risk of hepatocellular carcinoma (HCC), we conducted a prospective case–control study nested within a community-based cohort with 16 years of follow-up. We measured methylation levels in Satellite 2 (Sat2) by MethyLight and LINE-1 by pyrosequencing using baseline white blood cell DNA from 305 HCC cases and 1254 matched controls. We found that Sat2 hypomethylation was associated with HCC risk [odds ratio (OR) per unit decrease in natural log Sat2 methylation = 1.77, 95% confidence interval (CI) = 1.06–2.95]. The association was significant among individuals diagnosed with HCC before age 62 (OR per unit decrease in natural log Sat2 methylation = 2.47, 95% CI = 1.06–5.73) but not after (OR = 1.67, 95% CI = 0.84–3.32). We did not observe an association of LINE-1 with HCC overall risk by age at diagnosis. Among carriers of hepatitis B virus surface antigen (HBsAg), with each 1U decrease in natural log Sat2 methylation level, the OR for HCC increased by 2.19 (95% CI = 1.00–4.89). LINE-1 hypomethylation was associated with about a 2-fold increased risk of HCC, with ORs (95% CI) of 2.39 (1.06–5.39), 2.09 (0.91–4.77) and 2.28 (0.95–5.51, P trend = 0.14) for HBsAg carriers in the third, second and lowest quartile of LINE-1 methylation, respectively compared with carriers in the fourth. These results suggest that global hypomethylation may be a useful biomarker of HCC susceptibility.
PMCID: PMC3499052  PMID: 22581841
4.  Analysis of repetitive element DNA methylation by MethyLight 
Nucleic Acids Research  2005;33(21):6823-6836.
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.
PMCID: PMC1301596  PMID: 16326863
5.  LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype (CIMP) in colorectal cancer 
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.
PMCID: PMC2630175  PMID: 18366060
colon cancer; methylation; epigenomics; CIMP; LINE-1
6.  Unique Association between Global DNA Hypomethylation and Chromosomal Alterations in Human Hepatocellular Carcinoma 
PLoS ONE  2013;8(9):e72312.
Global DNA hypomethylation is a characteristic feature of cancer cells that closely associates with chromosomal instability (CIN). However, the association between these characteristics during hepatocarcinogenesis remains unclear. Herein, we determined the relationship between hypomethylation and CIN in human hepatocellular carcinoma (HCC) by analyzing 179 HCCs, 178 matched non-tumor livers and 23 normal liver tissues. Hypomethylation at three different repetitive DNA (rDNA) sequences and hypermethylation of 12 CpG loci, including 11 tumor suppressor gene (TSG) promoters, were quantified using MethyLight or combined bisulfite restriction analysis. Fractional allelic loss (FAL) was used as a marker for CIN, calculated by analyzing 400 microsatellite markers. Gains and losses at each chromosome were also determined using semi-quantitative microsatellite analysis. The associations between rDNA hypomethylation and FAL, as well as between TSG hypermethylation and FAL were investigated. Significantly more hypomethylation was observed in HCC tissues than in normal liver samples. Progression of hypomethylation during carcinogenesis was more prominent in hepatitis C virus (HCV)-negative cases, which was in contrast to our previous reports of significantly increased TSG methylation levels in HCV-positive tumors. Absence of liver cirrhosis and higher FAL scores were identified as independent contributors to significant hypomethylation of rDNA in HCC. Among the chromosomal alterations frequently observed in HCC, loss of 8p, which was unique in the earliest stages of hepatocarcinogenesis, was significantly associated with hypomethylation of rDNA by multivariable analysis (p = 0.0153). rDNA hypomethylation was also associated with a high FAL score regardless of tumor differentiation (p = 0.0011, well-differentiated; p = 0.0089, moderately/poorly-differentiated HCCs). We conclude that DNA hypomethylation is an important cause of CIN in the earliest step of HCC, especially in a background of non-cirrhotic liver.
PMCID: PMC3759381  PMID: 24023736
7.  Global methylation profiles in DNA from different blood cell types 
Epigenetics  2011;6(1):76-85.
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.
PMCID: PMC3052916  PMID: 20890131
[3H]-methyl acceptance assay; Alu; DNA demethylation; epigenetics; LINE1; LUMA; methylight; Sat2
8.  Genome-Wide Hypomethylation in Head and Neck Cancer Is More Pronounced in HPV-Negative Tumors and Is Associated with Genomic Instability 
PLoS ONE  2009;4(3):e4941.
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.
PMCID: PMC2654169  PMID: 19293934
9.  Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids 
AIM: To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers.
METHODS: DNA was extracted from undiluted pancreatic and biliary fluids. As a surrogate for a genome-wide hypomethylation assay, levels of long interspersed nuclear element-1 (LINE-1) methylation were analyzed using bisulfite pyrosequencing. CpG island hypermethylation of 10 tumor-associated genes, aryl-hydrocarbon receptor repressor, adenomatous polyposis coli, calcium channel, voltage dependent, T type α1G subunit, insulin-like growth factor 2, O-6-methyl-guanine-DNA methyltransferase, neurogenin 1, CDKN2A, runt-related transcription factor 3 (RUNX3), secreted frizzled-related protein 1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), was analyzed using MethyLight. To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1, human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 μmol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h. After the treatment, UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction.
RESULTS: Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease (58.7% ± 4.3% vs 61.7% ± 2.2%, P = 0.027; 53.8% ± 6.6% vs 57.5% ± 1.7%, P = 0.007); however, LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids (45.4% ± 5.5% vs 58.7% ± 4.3%, P < 0.001). CpG island hypermethylation of tumor-associated genes was detected at various frequencies, but it was not correlated with LINE-1 hypomethylation. Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic (67%) or biliary (70%) fluids from patients with pancreatobiliary cancer. As a single marker, hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancers. Treatment with a demethylating agent, 5-AZA-2’-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines.
CONCLUSION: Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.
PMCID: PMC3607748  PMID: 23555160
Pancreatobiliary cancers; DNA methylation; Pancreatobiliary fluids; Ubiquitin carboxyl-terminal esterase L1; Runt-related transcription factor 3
10.  LINE-1 Hypomethylation in Cancer Is Highly Variable and Inversely Correlated with Microsatellite Instability 
PLoS ONE  2007;2(5):e399.
Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer.
Methodology/Principal Findings
We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process.
Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer.
PMCID: PMC1851990  PMID: 17476321
11.  Adult global DNA methylation in relation to pre-natal nutrition 
Background Exposure to a pre-natal famine environment has been associated with a persistent decrease in DNA methylation of the IGF2 gene, although study findings on other loci have been highly variable. There have been no studies to date of the relation between pre-natal famine and overall global DNA methylation in adulthood.
Methods Our study population includes 350 births with pre-natal exposure to the Dutch famine of 1944–45 selected from three birth clinics, 290 births from these clinics born before or after the famine as unexposed time controls and 307 same-sex siblings of either birth group as unexposed family controls. All study subjects were interviewed and underwent a medical examination at a mean age of 58 years when blood samples were also collected. As measures of genomic DNA methylation, we analysed two repetitive elements, LINE-1 (long interspersed nucleotide element 1) and Sat2 (Satellite 2 DNA sequence) by pyrosequencing and MethyLight, respectively, and overall genomic DNA methylation using the Luminometric methylation assay (LUMA).
Results Mean DNA methylation by LUMA was 75.2% [standard deviation (SD) 4.7], by LINE-1 was 77.1% (SD 2.5) and by Sat2 was 122.2 (SD 56.2). Pre-natal famine exposure was associated with negligible changes in all three assays {LUMA: −0.16% [95% confidence interval (95% CI) −0.49 to 0.81], P = 0.63; LINE-1: −0.05 % (95% CI −0.33 to 0.22), P = 0.70; and Sat2: −0.51% (95% CI −7.38 to 6.36), P = 0.88} relative to unexposed controls, adjusting for age at examination and within family clustering.
Conclusion Our results show no relation between overall global DNA methylation in adults and pre-natal famine exposure. Further work should focus on selected regions in the genome that may be differentially methylated in response to changes in early life exposures and predict adult health outcomes.
PMCID: PMC3304521  PMID: 22422450
Pre-natal exposure delayed effects; Dutch famine; The Netherlands; World War II; DNA methylation; LINE-1; Sat2; LUMA
12.  DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer 
Gynecologic oncology  2011;121(1):24-31.
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.
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.
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.
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.
PMCID: PMC3062717  PMID: 21251700
DNA ploidy; Nuclear area; Proliferation index; DNA-hypomethylation; Ovarian cancer
13.  LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer 
BMC Cancer  2012;12:574.
Long interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed.
Formalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples.
LINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC.
LINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.
PMCID: PMC3534591  PMID: 23216958
LINE-1; DNA methylation; Colorectal cancer; Laser microdissection
14.  DNA Methylation Changes in Atypical Adenomatous Hyperplasia, Adenocarcinoma In Situ, and Lung Adenocarcinoma 
PLoS ONE  2011;6(6):e21443.
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.
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.
PMCID: PMC3121768  PMID: 21731750
15.  Coordinated cancer germline antigen promoter and global DNA hypomethylation in ovarian cancer: association with the BORIS/CTCF expression ratio and advanced stage 
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.
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.
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.
PMCID: PMC3079045  PMID: 21296871
DNA methylation; DNA hypomethylation; cancer germline antigens; cancer testis antigens; ovarian cancer
16.  Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors 
Oncogene  2006;25(18):2636-2645.
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.
PMCID: PMC1449872  PMID: 16532039
DNA hypomethylation; DNA hypermethylation; DNA methyltransferases; ovarian tumors
17.  Association of hypomethylation of LINE-1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma 
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.
PMCID: PMC3190095  PMID: 21960343
Cancer risk; Epigenetics; Global hypomethylation; Hepatocellular carcinoma; LINE-1 repetitive element
18.  Inactive X chromosome-specific reduction in placental DNA methylation 
Human Molecular Genetics  2009;18(19):3544-3552.
Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P = 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for ∼20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta.
PMCID: PMC2742397  PMID: 19586922
19.  LINE-1 Hypomethylation in a Choline-Deficiency-Induced Liver Cancer in Rats: Dependence on Feeding Period 
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.
PMCID: PMC1479888  PMID: 16877811
20.  Prenatal maternal immune activation causes epigenetic differences in adolescent mouse brain 
Translational Psychiatry  2014;4(9):e434-.
Epigenetic processes such as DNA methylation have been implicated in the pathophysiology of neurodevelopmental disorders including schizophrenia and autism. Epigenetic changes can be induced by environmental exposures such as inflammation. Here we tested the hypothesis that prenatal inflammation, a recognized risk factor for schizophrenia and related neurodevelopmental conditions, alters DNA methylation in key brain regions linked to schizophrenia, namely the dopamine rich striatum and endocrine regulatory centre, the hypothalamus. DNA methylation across highly repetitive elements (long interspersed element 1 (LINE1) and intracisternal A-particles (IAPs)) were used to proxy global DNA methylation. We also investigated the Mecp2 gene because it regulates transcription of LINE1 and has a known association with neurodevelopmental disorders. Brain tissue was harvested from 6 week old offspring of mice exposed to the viral analog PolyI:C or saline on gestation day 9. We used Sequenom EpiTYPER assay to quantitatively analyze differences in DNA methylation at IAPs, LINE1 elements and the promoter region of Mecp2. In the hypothalamus, prenatal exposure to PolyI:C caused significant global DNA hypomethylation (t=2.44, P=0.019, PolyI:C mean 69.67%, saline mean 70.19%), especially in females, and significant hypomethylation of the promoter region of Mecp2, (t=3.32, P=0.002; PolyI:C mean 26.57%, saline mean 34.63%). IAP methylation was unaltered. DNA methylation in the striatum was not significantly altered. This study provides the first experimental evidence that exposure to inflammation during prenatal life is associated with epigenetic changes, including Mecp2 promoter hypomethylation. This suggests that environmental and genetic risk factors associated with neurodevelopmental disorders may act upon similar pathways. This is important because epigenetic changes are potentially modifiable and their investigation may open new avenues for treatment.
PMCID: PMC4203009  PMID: 25180573
21.  Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues 
PLoS ONE  2013;8(11):e79044.
DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.
PMCID: PMC3832524  PMID: 24260150
22.  LINE-1 hypomethylation in familial and sporadic cancer 
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.
PMCID: PMC3383956  PMID: 22228215
LINE-1; DNA methylation; Hereditary cancer; Gastrointestinal cancer; Lynch syndrome
23.  Changes in DNA methylation of tandem DNA repeats are different from interspersed repeats in cancer 
Hypomethylation of DNA repetitive elements is a common finding in cancer, but very little is known about the DNA methylation changes of different types of DNA repetitive elements, such as interspersed repeats (LINE1 and Alu Yb8) and tandem repeats (Sat-α, NBL-2 and D4Z4). We used bisulfite-PCR Pyrosequencing to quantitatively measure the DNA methylation of 5 different DNA repetitive elements in normal tissue and cancer. In all we studied 10 different tissues from 4 individuals undergoing autopsy, 34 paired normal and tumor tissues from patients with bladder cancer, 58 patients with chronic myelogenous leukemia and 23 patients with acute promyelocytic leukemia. We found that the DNA methylation of interspersed repeats (LINE1 and Alu Yb8) was very consistent from person to person and tissue to tissue while tandem DNA repeats appeared more variable in normal tissues. In bladder cancer we found clear hypomethylation of LINE1, Alu Yb8, Sat-α, and NBL-2. Conversely, we found an increase in the DNA methylation levels of D4Z4 from normal to cancer. In contrast leukemia showed no significant changes in the DNA methylation of LINE1 and Alu Yb8, but DNA methylation increases in NBL-2 and D4Z4 tandem repeats. Our findings show that the changes in DNA methylation levels of individual DNA repetitive elements are unique for each repetitive element, which may reflect distinct epigenetic factors and may have important implications in the use of DNA methylation of repetitive elements as global DNA methylation biomarkers. Keywords: DNA methylation, DNA repetitive elements, bladder cancer, leukemia
PMCID: PMC4086885  PMID: 19437537
DNA methylation; DNA repetitive elements; bladder cancer; leukemia
24.  Epigenetic Effects of the Continuous Exposure to Peroxisome Proliferator WY-14,643 in Mouse Liver are Dependent upon Peroxisome Proliferator Activated Receptor α 
Mutation research  2007;625(1-2):62-71.
Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129 mice were fed a control or WY-14,643-containing (1000 ppm) diet for 1 wk, 5 wks or 5 months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at 5 months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparα-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of PPARα.
PMCID: PMC2111058  PMID: 17586532
25.  Global Hypomethylation Identifies Loci Targeted for Hypermethylation in Head and Neck Cancer 
The human epigenome is profoundly altered in cancers, with a characteristic loss of methylation in repetitive regions and concomitant accumulation of gene-promoter methylation. The degree to which these processes are coordinated is unclear so we investigated both in head and neck squamous cell carcinomas.
Experimental Design
Global methylation was measured using the luminometric methylation assay (LUMA), and pyrosequencing of LINE-1Hs and AluYb8 repetitive elements in a series of 138 tumors. We also measured methylation of over 27,000 CpG loci with the Illumina HumanMethylation27 microarray (n=91).
LINE-1 methylation was significantly associated with LUMA and Infinium loci methylation (Spearman’s rho=0.52/rho=0.56, both p<0.001), but not that of AluYb8. Methylation of LINE-1, AluYb8, and Infinium loci differed by tumor site (each Kruskal-Wallis p<0.05). Also, LINE-1 and LUMA methylation were associated with HPV16 E6 serology (each Mann-Whitney p<0.05). Comparing LINE-1 methylation to gene-associated methylation, we identified a distinct subset of CpG loci with significant hypermethylation associated with LINE-1 hypomethylation. An investigation of sequence features for these CpG loci revealed that they were significantly less likely to reside in repetitive elements (GSEA p<0.02), enriched in CpG islands (p<0.001), and were proximal to transcription factor binding sites (p<0.05). We validated the top CpG loci that had significant hypermethylation associated with LINE-1 hypomethylation (at EVI2A, IFRD1, KLHL6, and PTPRCAP) by pyrosequencing independent tumors.
These data indicate that global hypomethylation and gene-specific methylation processes are associated in a sequence-dependent manner, and that clinical characteristics and exposures leading to HNSCC may be influencing these processes.
PMCID: PMC3107873  PMID: 21505061

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