Long Interspersed Nuclear Element-1 Hypomethylation Research Articles

LINE-1 hypomethylation, increased retrotransposition and tumor-specific insertion in upper gastrointestinal cancer.

The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.

LINE-1 Methylation Status in Canine Splenic Hemangiosarcoma Tissue and Cell-Free DNA.

Splenic hemangiosarcoma is one of the most common malignant tumors in dogs, and early diagnosis is of great importance for achieving a good prognosis. DNA methylation plays an important role in cancer development. Long interspersed nuclear element 1 (LINE-1) is the most abundant repetitive element in the genome. LINE-1 hypomethylation has been shown to be related to carcinogenesis in humans, and it has been used as a novel cancer biomarker. This study aimed to evaluate the methylation status of LINE-1 in tumor tissue and circulating cell-free DNA and assess its clinical significance in canine splenic hemangiosarcoma. Genomic DNA was isolated from splenic masses of 13 dogs with hemangiosarcoma, 11 with other malignant tumors, and 15 with benign lesions. LINE-1 methylation was quantified using methylation-sensitive and -insensitive restriction enzyme digestion followed by real-time polymerase chain reaction. Additionally, blood samples were collected from eight patients to isolate cell-free DNA to determine LINE-1 methylation status changes during the treatment course. LINE-1 methylation in tumor samples was significantly lower in patients with hemangiosarcoma than in those with other malignant tumors and benign lesions. Non-significant but similar results were observed for the cell-free DNA samples. Our results demonstrate that LINE-1 methylation status is a potential biomarker for splenic hemangiosarcoma.

What Is Driving Early-Onset Colorectal Cancer?

What Is Driving Early-Onset Colorectal Cancer?

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content

BackgroundHypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect.MethodsLINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules’ in situ level (n = 40).ResultsSignificantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05).ConclusionOur results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

1221Baseline Global DNA Hypermethylation Increase the Risk of Cerebrovascular Disease Mortality in Japanese Population

Abstract Background DNA methylation plays an important role in progression of cardiovascular disease (CVD). The methylation status of long interspersed nuclear element-1 (LINE-1) reflects the global DNA methylation level and is associated with lipid profiles and glycemia. We conducted a longitudinal study to examine the association between LINE-1 DNA methylation and CVD mortality in a Japanese elderly population. Methods We targeted for 357 subjects (143 men and 214 women) who were more than 60 years, participated in the health checkup in 1990, and had no clinical history of cancer, stroke, or ischemic heart disease. During 29 years of follow-up period, total 69 subjects were died from CVD, and died of cerebrovascular disease (CBD) and ischemic heart disease (IHD) were 25 and 13, respectively. LINE-1 DNA methylation was measured using PBMCs. We defined the hypermethylation group as greater than median of the LINE-1 DNA methylation levels and hypomethylation group as others. Multivariable Hazard ratios (HRs) for each disease mortality were calculated by the Cox proportional hazard model. We adjusted for age, sex, smoking habits, and alcohol consumption. Results Significantly higher HRs for CVD and CBD mortality were observed in the hypermethylation group compared to the hypomethylation group (CVD: HR 1.67 [95%CI 1.02-2.73], CBD: HR 3.29 [95%CI 1.29-8.40]). However, IHD mortality did not associated with LINE-1 DNA methylation. Conclusions We found that LINE-1 DNA hypermethylation in PBMCs was associated with high CVD mortality, especially CBD mortality. Key messages Higher levels of LINE-1 methylation in PBMCs can be a predictive marker for CBD risk

LINE-1 Hypomethylation Is Associated With Malignant Traits and Cell Proliferation in Lung Adenocarcinoma.

Long interspersed nuclear element-1 (LINE-1) methylation status is a marker for global DNA methylation. However, the relationship between LINE-1 methylation and the biology of lung adenocarcinoma remains unclear. Here, we aimed to examine the role of LINE-1 in lung cancer. LINE-1 methylation levels were quantified by bisulfite pyrosequencing of resected tumor specimens from 162 patients with lung adenocarcinoma. The relationships of LINE-1 methylation with clinicopathological factors, gene mutations, and Ki-67 immunoreactivity were investigated. LINE-1 hypomethylation was associated with tumor invasion and advanced stage. TP53 mutations were more frequently detected in the LINE-1 hypomethylation group than in the hypermethylation group. LINE-1 hypomethylation was associated with poor recurrence-free survival, high maximum standardized uptake value in positron-emission tomography, and high Ki-67 expression in tumors. LINE-1 hypomethylation was associated with high-grade malignancy and poor prognosis in lung adenocarcinoma, but was not related to driver mutations.

268 LIQUID BIOPSY IN ESOPHAGEAL ADENOCARCINOMA: ROLE OF LINE-1 HYPOMETHYLATION

Abstract Esophageal Adenocarcinoma (EADC) prognosis is still poor. Clinical staging doesn’t allow accurate prediction of response to neoadjuvant treatment and survival. Molecular characterization of EADC is an expanding field aimed to predict treatment response and prognosis. Hypomethylation of Long Interspersed Nuclear Element 1 (LINE-1) sequences recently emerged as a frequent epigenetic alteration in EADC. Aim of the study was to evaluate methylation patterns of LINE-1 that is considered a good surrogate of global DNA methylation. Methods We investigated cell free DNA (cfDNA) of 21 patients, 19 with EADC and 2 with Barrett’s esophagus (BE). To verify the possibility to use LINE-1 methylation status to predict tumor behaviour, we extended the analysis to a few longitudinal cases, one EADC and two BE. One BE patient progressed to low-grade dysplasia (LGD) and high-grade dysplasia (HGD)/EADC during surveillance, while the other remains stable. LINE-1 promoter methylation was analyzed using restriction enzymes and DNA amplification. Results Using this approach, we were able to detect the presence of hypomethylated LINE-1 sequences in EADC cfDNA. The difference in methylation level between cfDNA and constitutive DNA was statistically significant (p = 0.0001). In one EADC patient, treated with esophagectomy, LINE-1 hypomethylation was found at lower level after resection and at higher level at recurrence. In another patient affected by BE, LINE-1 hypomethylation increased during the progression from metaplastic epithelium to EADC, switching back to the level of constitutive DNA after endoscopic resection. The third patient, with stable BE during 4 years follow-up, showed non-significant variations in methylation status of LINE-1. Conclusion Our study demonstrated the feasibility of our methodological approach to detect hypomethylation events in EADC patient cfDNA. Longitudinal preliminary analysis suggested a correlation between methylation status of LINE-1 and tumor progression. Further studies on larger population are needed to confirm these encouraging data. LINE-1 methylation analysis could be useful as a new molecular assay to integrate patient monitoring.

The prevalence and persistence of aberrant promoter DNA methylation in benzene-exposed Chinese workers

Aberrant DNA methylation patterns are common in cancers and environmental pollutant exposed subjects. Up to date, few studies have examined the aberrant DNA methylation patterns in benzene exposed workers. We recruited 141 benzene-exposed workers, including 83 benzene-exposed workers from a shoe factory in Wenzhou and 58 workers from a painting workshop in Wuhu, 35 workers in Wuhu were followed from 2009 to 2013, and 48 indoor workers as controls from Wenzhou. We used high-resolution melting (HRM) to quantitate human samples of DNA methylation in long interspersed nuclear element-1 (LINE-1), (6)-methylguanine-DNA methyltransferase (MGMT), and DNA mismatch repair gene human mutator L homologue 1 (hMLH1). AML-5 cells were treated with benzoquinone (BQ) and hydroquinone (HQ), and the promoter methylation of MGMT and hMLH1 was detected using the bisulfite sequencing PCR method. The degree of LINE-1 methylation in benzene-exposed workers was significantly lower than that of the controls (p<0.001), and the degree of MGMT (p<0.001) and hMLH1 (p = 0.01) methylation was significantly higher than that of the controls. The in vitro study validated the aberrant hypermethylation of hMLH1 after treatment with BQ. Among the cohort workers who were followed from 2009 to 2013, the LINE1 methylation elevated in 2013 than 2009 (p = 0.004), and premotor methylation in hMLH1 reduced in 2013 than 2009 (p = 0.045) with the reduction of the benzene exposure. This study provides evidence that benzene exposure can induce LINE-1 hypomethylation and DNA repair gene hypermethylation.

Chronic exposure to arsenic, LINE-1 hypomethylation, and blood pressure: a cross-sectional study in Bangladesh

BackgroundChronic exposure to arsenic is associated with cancer and hypertension. Growing evidence suggests that altered methylation in long interspersed nuclear element-1 (LINE-1) is involved in many types of disorders, including cardiovascular disease. Here we evaluated the association between arsenic exposure and LINE-1 methylation levels, especially in relation to blood pressure (BP).MethodsA total of 236 subjects (175 from arsenic-endemic areas and 61 from a non-endemic area) in rural Bangladesh were recruited. The subjects’ arsenic exposure levels (i.e., drinking water, hair and nail arsenic concentrations) were measured by inductively coupled plasma mass spectroscopy. The subjects’ LINE-1 methylation levels were determined by pyrosequencing.ResultsThe average LINE-1 methylation levels of the subjects living in the arsenic-endemic areas were significantly (p < 0.01) lower than those of the subjects living in the non-endemic area. In a sex-stratified analysis, the arsenic exposure levels in female but not male subjects showed a significant inverse association with LINE-1 methylation levels before (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) and after (water arsenic: p < 0.01, hair arsenic: p < 0.05, nail arsenic: p < 0.001) adjustment for age, body mass index and smoking. Analyses examining interactions among arsenic levels, BP and LINE-1 methylation showed that arsenic-related elevated levels of BP were associated with LINE-1 hypomethylation.ConclusionsOur findings demonstrated that chronic exposure to arsenic was inversely associated with LINE-1 methylation levels in blood leukocyte DNA and this was more pronounced in females than males; in addition, the decreased levels of LINE-1 methylation might be involved in the arsenic-induced elevation of BP.

LINE-1 hypomethylation is associated to specific clinico-pathological features in Stage I non-small cell lung cancer

ObjectivesWe hypothesize that selected genetic and/or epigenetic changes associated with advanced tumours may help identifying early non-small cell lung cancers (NSCLCs) that recur after resection. Among epigenetic changes, long interspersed nuclear element-1 (LINE-1) hypomethylation is seen early during carcinogenesis and may act in concert with genetic alterations to cancer progression. LINE-1 hypomethylation and gene mutations frequently involved in lung cancer, were analysed to evaluate their prognostic role in resected stage I NSCLC. MethodsGene mutations and LINE-1 methylation were analysed in 167 Caucasian patients with stage I NSCLC, namely 100 adenocarcinomas (ADC) and 67 squamous-cell carcinomas (SqCC), using mass-spectrometry and pyrosequencing. We evaluated the correlation between molecular results and clinico-pathological data: age, gender, smoking status, period of surgery, histology, grading, pathological stage, p53 expression, LINE-1 hypomethylation. These variables have been assessed as possible predictors of cancer related survival by regression analysis. ResultsFrequency and spectrum of gene mutations were significantly different in ADCs compared with SqCCs. p53 positivity was more common in SqCC, while EGFR or KRAS mutations were mainly detected in ADC. LINE1 hypomethylation was associated with SqCC histology, p53 immunoreactivity and smoking habit. Stage IB, LINE-1 hypomethylation and PIK3CA mutation independently predicted a worse cancer-related survival. When combined into a scoring system, their prognostic power was strengthened. ConclusionsIn many stage I NSCLC a mutation pattern of advanced disease was observed. Stage IB, LINE-1 hypomethylation and PIK3CA mutation were associated to poor prognosis. Genetic and epigenetic events occurring in early carcinogenesis may help identifying stage I NSCLC patients who deserve adjuvant therapy.

LINE-1 hypomethylation status of circulating cell-free DNA in plasma as a biomarker for colorectal cancer.

Colorectal cancer (CRC) is a serious public health problem and non-invasive biomarkers improving diagnosis or therapy are strongly required. Circulating cell-free DNA (cfDNA) has been a promising target for this purpose. In this study, we evaluated the potential of long interspersed nuclear element-1 (LINE-1) hypomethylation as a blood biomarker for CRC. LINE-1 hypomethylation level in plasma cfDNA in 114 CRC patients was retrospectively examined by absolute quantitative analysis of methylated alleles real-time PCR, and was expressed using LINE-1 hypomethylation index (LHI) [unmethylated copy number/ (methylated copy number + unmethylated copy number)]. Greater LHI values indicated enhanced hypomethylation. In our clinicopathological analysis, CRC patients with large tumors (≥6.0 cm), advanced N stage (≥2), and distant metastasis (M1) had statistically significantly higher cfDNA LHI than other CRC patients, suggesting cfDNA LHI as a disease progression biomarker for CRC. Furthermore, early stage I/II (n = 57) as well as advanced stage III/IV (n =57) CRC patients had significantly higher cfDNA LHI than healthy donors (n=53) [stage I/II: median 0.369 (95% confidence interval, 0.360–0.380) vs. 0.332 (0.325–0.339), P < 0.0001; stage III/IV: 0.372 (0.365–0.388) vs. 0.332 (0.325–0.339), P < 0.0001]. The receiver operating characteristic analysis showed that cfDNA LHI had the detection capacity of CRC with area under the curve(AUC) of 0.79 and 0.83 in stage I/II and stage III/IV CRC patients, respectively. The present study demonstrated for the first time the potential of plasma cfDNA LHI as a novel biomarker for CRC, particularly for early stage detection.

Gene-specific DNA methylation profiles and LINE-1 hypomethylation are associated with myocardial infarction risk.

BackgroundDNA methylation profiles are responsive to environmental stimuli and metabolic shifts. This makes DNA methylation a potential biomarker of environmental-related and lifestyle-driven diseases of adulthood. Therefore, we investigated if white blood cells’ (WBCs) DNA methylation profiles are associated with myocardial infarction (MI) occurrence.Whole-genome DNA methylation was investigated by microarray analysis in 292 MI cases and 292 matched controls from the large prospective Italian European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (EPICOR study). Significant signals (false discovery rate (FDR) adjusted P < 0.05) were replicated by mass spectrometry in 317 MI cases and 262 controls from the Dutch EPIC cohort (EPIC-NL). Long interspersed nuclear element-1 (LINE-1) methylation profiles were also evaluated in both groups.ResultsA differentially methylated region (DMR) within the zinc finger and BTB domain-containing protein 12 (ZBTB12) gene body and LINE-1 hypomethylation were identified in EPICOR MI cases and replicated in the EPIC-NL sample (ZBTB12-DMR meta-analysis: effect size ± se = −0.016 ± 0.003, 95 % CI = −0.021;−0.011, P = 7.54 × 10−10; LINE-1 methylation meta-analysis: effect size ± se = −0.161 ± 0.040, 95 % CI = −0.239;−0.082, P = 6.01 × 10−5).Moreover, cases with shorter time to disease had more pronounced ZBTB12-DMR hypomethylation (meta-analysis, men: effect size ± se = −0.0059 ± 0.0017, PTREND = 5.0 × 10−4; women: effect size ± se = −0.0053 ± 0.0019, PTREND = 6.5 × 10−3) and LINE-1 hypomethylation (meta-analysis, men: effect size ± se = −0.0010 ± 0.0003, PTREND = 1.6 × 10−3; women: effect size ± se = −0.0008 ± 0.0004, PTREND = 0.026) than MI cases with longer time to disease.In the EPIC-NL replication panel, DNA methylation profiles improved case-control discrimination and reclassification when compared with traditional MI risk factors only (net reclassification improvement (95 % CI) between 0.23 (0.02–0.43), P = 0.034, and 0.89 (0.64–1.14), P < 1 × 10−5).ConclusionsOur data suggest that specific methylation profiles can be detected in WBCs, in a preclinical condition, several years before the occurrence of MI, providing an independent signature of cardiovascular risk. We showed that prediction accuracy can be improved when DNA methylation is taken into account together with traditional MI risk factors, although further confirmation on a larger sample is warranted. Our findings support the potential use of DNA methylation patterns in peripheral blood white cells as promising early biomarkers of MI.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0164-3) contains supplementary material, which is available to authorized users.

LINE-1 hypomethylation is neither present in rectal aberrant crypt foci nor associated with field defect in sporadic colorectal neoplasia.

BackgroundAberrant crypt foci (ACF) are considered the first identifiable preneoplastic lesion in colorectal cancer (CRC), and have been proposed as a potential biomarker for CRC risk. Global DNA hypomethylation is an early event in colorectal carcinogenesis, and long interspersed nuclear element-1 (LINE-1) methylation status is a well-known surrogate marker for genome-wide DNA methylation levels. Despite the gradual increase in DNA hypomethylation in the adenoma–carcinoma sequence, LINE-1 methylation in ACF has never been studied. Moreover, recent studies have reported a field defect for LINE-1 hypomethylation, suggesting that LINE-1 methylation status in normal mucosa could be used to stratify CRC risk and tailor preventive strategies. Thus, we assessed LINE-1 status by pyrosequencing in rectal ACF and paired normal colorectal mucosa from individuals with sporadic colon cancer (CC) (n = 35) or adenoma (n = 42), and from healthy controls (n = 70).FindingsCompared with normal mucosa, LINE-1 in ACF were hypermethylated across all groups (P < 0.0001). Furthermore, LINE-1 methylation status in normal colorectal mucosa was independent of the presence of adenoma or CC (P = 0.1072), and did not differ depending on the distance to the adenoma or CC. Interestingly, when we compared the LINE-1 methylation status in normal mucosa from different segments of the colorectum, we found higher hypomethylation in the rectum compared with the descending colon (P < 0.0001).ConclusionsOverall, our results suggest that global hypomethylation is not present in rectal ACF and argues against the existence of LINE-1 methylation field defect in sporadic colon cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1868-7083-6-24) contains supplementary material, which is available to authorized users.

Association between LINE-1 hypomethylation and cancer risk: a systematic review and meta-analysis

Background DNA methylation, a major epigenetic mechanism, is involved in various biological processes including cancer. Long Interspersed Nuclear Element-1 (LINE-1) hypomethylation, as biomarker of global methylation, was observed in several types of cancer and was associated with a poor prognosis. In the present systematic review, global DNA methylation was evaluated as a biomarker for cancer risk. Methods A literature search, using the PubMED database, was performed to select studies, published up to March …

Hypomethylation of long interspersed nuclear element-1 (LINE-1) is associated with poor prognosis via activation of c-MET in hepatocellular carcinoma.

Long interspersed nuclear element-1 (LINE-1) methylation status, representing global DNA methylation levels, is associated with patient prognosis in several types of cancer. This study was designed to examine the prognostic significance of LINE-1 methylation in patients with hepatocellular carcinoma (HCC) and the possible mechanisms related to oncogene activation. Seventy-five HCC patients who underwent hepatectomy between 2006 and 2012 were enrolled in this study. Quantitative pyrosequencing was performed to quantify the methylation level of three CpG sites in the LINE-1 promoter. Clinicopathological variables and prognosis were compared between LINE-1 hypo- and hypermethylation groups. LINE-1-inserted c-MET (L1-MET) gene expression and its correlation with LINE-1 methylation levels also were analyzed. LINE-1 was significantly hypomethylated in tumor tissues compared with nontumor tissues (48.3 ± 12.2 % vs. 68.2 ± 2.0 %, respectively, p < 0.0001). LINE-1 hypomethylation was not associated with any clinicopathological factors in HCC patients, except sex (p < 0.05). However, patients with LINE-1 hypomethylation exhibited significantly poorer outcome, and multivariate analysis revealed that LINE-1 hypomethylation was an independent risk factor for overall survival (hazard ratio (HR) = 6.1, p = 0.031) and disease-free survival (HR = 2.34, p = 0.045). L1-MET expression was significantly higher in tumor tissues (p < 0.01). L1-MET expression levels were inversely correlated with LINE-1 methylation levels, and positively correlated with c-MET expression (p < 0.05). Furthermore, higher c-MET protein expression was observed in the LINE-1 hypomethylated tumor tissues compared with hypermethylated tumor tissues (p = 0.032). LINE-1 hypomethylation is significantly associated with poor prognosis in patients with HCC, possibly due to activation of c-MET expression.

Chromosomal instability associated with global DNA hypomethylation is associated with the initiation and progression of esophageal squamous cell carcinoma.

Global DNA hypomethylation is associated with increased chromosomal instability and plays an important role in tumorigenesis. The methylation status of the long interspersed nuclear element-1 (LINE-1) element is a useful surrogate marker for global DNA methylation. Although LINE-1 hypomethylation is recognized as a poor prognostic marker, the correlation of LINE-1 methylation level with tumor suppressor gene mutation, chromosomal instability, and clinical significance in esophageal squamous cell carcinoma (ESCC) remains unclear. Using resected tumor tissues and the corresponding normal esophageal mucosa from 105 patients with ESCC, bisulfite pyrosequencing analysis was performed to quantify the LINE-1 methylation levels. p53 mutations in exons two to ten were detected by polymerase chain reaction direct sequencing. Chromosomal instability was assessed by single nucleotide polymorphism array comparative genomic hybridization analysis. The LINE-1 methylation level of ESCC was significantly lower than matched normal mucosa. LINE-1 methylation levels of normal mucosa from the esophagus had a significant inverse correlation with both cigarette smoking and alcohol consumption of the study subjects. LINE-1 hypomethylation of ESCC was significantly associated with lymph node metastasis, lymphovascular invasion, the frequency of p53 mutation and poor survivability. The LINE-1 methylation levels in ESCC had a significant inverse association with the percentage of copy number alterations in the whole genome, mirroring chromosomal instability. Our results suggested that whole genome hypomethylation caused by chronic inflammation could initiate carcinogenesis of esophageal squamous cells through chromosomal instability. In addition, chromosomal instability associated with the global hypomethylation might correlate highly with the progression of ESCC.

Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis

ObjectiveHypomethylation of LINE-1 elements has emerged as a distinguishing feature in human cancers. Limited evidence indicates that some LINE-1 elements encode an additional internal antisense promoter, and increased hypomethylation of...

Global and MGMT promoter hypomethylation independently associated with genomic instability of lymphocytes in subjects exposed to high-dose polycyclic aromatic hydrocarbon

Global hypomethylation, gene-specific methylation, and genome instability are common events in tumorigenesis. To date, few studies have examined the aberrant DNA methylation patterns in coke oven workers, who are highly at risk of lung cancer by occupational exposure to polycyclic aromatic hydrocarbons (PAHs). We recruited 82 PAH-exposed workers and 62 unexposed controls, assessed exposure levels by urinary 1-hydroxypyrene, and measured genetic damages by comet assay, bleomycin sensitivity, and micronucleus assay. The PAHs in coke oven emissions (COE) were estimated based on toxic equivalency factors. We used bisulfite-PCR pyrosequencing to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and O(6)-methylguanine-DNA methyltransferase (MGMT). Further, the methylation alteration was also investigated in COE-treated human bronchial epithelial (16HBE) cells. We found there are higher levels of PAHs in COE. Among PAH-exposed workers, LINE-1 and MGMT methylation levels (with CpG site specificity) were significantly lowered. LINE-1, MGMT, and its hot CpG site-specific methylation were negatively correlated with urinary 1-hydroxypyrene levels (r = -0.329, p < 0.001; r = -0.164, p = 0.049 and r = -0.176, p = 0.034, respectively). In addition, LINE-1 methylation was inversely associated with comet tail moment and micronucleus frequency, and a significant increase of micronucleus in low MGMT methylation group. In vitro study revealed that treatment of COE in 16HBE cells resulted in higher production of BPDE-DNA adducts, LINE-1 hypomethylation, hypomethylation, and suppression of MGMT expression. These findings suggest hypomethylation of LINE-1 and MGMT promoter could be used as markers for PAHs exposure and merit further investigation.

Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids

To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers. 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. 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. Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.

Long Interspersed Nuclear Element-1 Hypomethylation and Oxidative Stress: Correlation and Bladder Cancer Diagnostic Potential

Although, increased oxidative stress and hypomethylation of long interspersed nuclear element-1 (LINE-1) associate with bladder cancer (BCa) development, the relationship between these alterations is unknown. We evaluated the oxidative stress and hypomethylation of the LINE-1 in 61 BCa patients and 45 normal individuals. To measure the methylation levels and to differentiate the LINE-1 loci into hypermethylated, partially methylated and hypomethylated, peripheral blood cells, urinary exfoliated cells and cancerous tissues were evaluated by combined bisulfite restriction analysis PCR. The urinary total antioxidant status (TAS) and plasma protein carbonyl content were determined. The LINE-1 methylation levels and patterns, especially hypomethylated loci, in the blood and urine cells of the BCa patients were different from the levels and patterns in the healthy controls. The urinary TAS was decreased, whereas the plasma protein carbonyl content was increased in the BCa patients relative to the controls. A positive correlation between the methylation of LINE-1 in the blood-derived DNA and urinary TAS was found in both the BCa and control groups. The urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content provided the best diagnostic potential for BCa prediction. Based on post-diagnostic samples, the combination test improved the diagnostic power to a sensitivity of 96% and a specificity of 96%. In conclusion, decreased LINE-1 methylation is associated with increased oxidative stress both in healthy and BCa subjects across the various tissue types, implying a dose-response association. Increases in the LINE-1 hypomethylation levels and the number of hypomethylated loci in both the blood- and urine-derived cells and increase in the oxidative stress were found in the BCa patients. The combination test of the urinary hypomethylated LINE-1 loci and the plasma protein carbonyl content may be useful for BCa screening and monitoring of treatment.