LINE-1 Repetitive Elements Research Articles

Birthweight, Maternal Weight Trajectories and Global DNA Methylation of LINE-1 Repetitive Elements

Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics.

Aberrant DNA methylation of cancer-associated genes in gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC–EURGAST)

Epigenetic events have emerged as key mechanisms in the regulation of critical biological processes and in the development of a wide variety of human malignancies, including gastric cancer (GC), however precise gene targets of aberrant DNA methylation in GC remain largely unknown. Here, we have combined pyrosequencing-based quantitative analysis of DNA methylation in 98 GC cases and 64 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and in cancer tissue and non-tumorigenic adjacent tissue of an independent series of GC samples. A panel of 10 cancer-associated genes ( CHRNA3, DOK1, MGMT, RASSF1A, p14ARF, CDH1, MLH1, ALDH2, GNMT and MTHFR) and LINE-1 repetitive elements were included in the analysis and their association with clinicopathological characteristics (sex, age at diagnosis, anatomical sub-site, histological sub-type) was examined. Three out of the 10 genes analyzed exhibited a marked hypermethylation, whereas two genes ( ALDH2 and MTHFR) showed significant hypomethylation, in gastric tumors. Among differentially methylated genes, we identified new genes ( CHRNA3 and DOK1) as targets of aberrant hypermethylation in GC, suggesting that epigenetic deregulation of these genes and their corresponding cellular pathways may promote the development and progression of GC. We also found that global demethylation of tumor cell genomes occurs in GC, consistent with the notion that abnormal hypermethylation of specific genes occurs concomitantly with genome-wide hypomethylation. Age and gender had no significant influence on methylation states, but an association was observed between LINE-1 and MLH1 methylation levels with histological sub-type and anatomical sub-site. This study identifies aberrant methylation patters in specific genes in GC thus providing information that could be exploited as novel biomarkers in clinics and molecular epidemiology of GC.

Renal kallikrein excretion and epigenetics in human acute kidney injury: Expression, mechanisms and consequences

BackgroundRenal kallikrein (KLK1) synthesis and urinary excretion are reportedly diminished during AKI (acute kidney injury) in animal models, and provision of kallikrein abrogates renal injury in this setting, but data in human AKI is limited. Therefore we first examined KLK1 renal excretion in human AKI, and then probed potential endocrine and epigenetic mechanisms for its alterations.MethodsKLK1 enzymatic activity excretion was evaluated in urine from patients with established or incipient AKI, versus healthy/non-hospital as well as ICU controls. Endocrine control of KLK1 excretion was then probed by catecholamine and aldosterone measurements in established AKI versus healthy controls. To examine epigenetic control of KLK1 synthesis, we tested blood and urine DNA for changes in promoter CpG methylation of the KLK1 gene, as well as LINE-1 elements, by bisulfite sequencing.ResultsPatients with early/incipient AKI displayed a modest reduction of KLK1 excretion, but unexpectedly, established AKI displayed substantially elevated urine KLK1 excretion, ~11-fold higher than healthy controls, and ~3-fold greater than ICU controls. We then probed potential mechanisms of the change. Established AKI patients had lower SBP, higher heart rate, and higher epinephrine excretion than healthy controls, though aldosterone excretion was not different. Promoter KLK1 CpG methylation was higher in blood than urine DNA, while KLK1 methylation in blood DNA was significantly higher in established AKI than healthy controls, though KLK1 methylation in urine tended to be higher in AKI, directionally consistent with earlier/incipient but not later/established changes in KLK1 excretion in AKI. On multivariate ANOVA, AKI displayed coordinate changes in KLK1 excretion and promoter methylation, though directionally opposite to expectation. Control (LINE-1 repetitive element) methylation in blood and urine DNA was similar between AKI and controls.ConclusionsUnexpectedly, increased KLK1 excretion in AKI patients was found; this increase is likely to be due in part to increments in adrenergic tone during BP depression. Epigenetic changes at KLK1 may also play a role in early changes of KLK1 expression and thus AKI susceptibility or recovery.

Polycomb group genes are targets of aberrant DNA methylation in renal cell carcinoma

The combined effects of genetic and epigenetic aberrations are well recognized as causal in tumorigenesis. Here, we defined profiles of DNA methylation in primary renal cell carcinomas (RCC) and assessed the association of these profiles with the expression of genes required for the establishment and maintenance of epigenetic marks. A bead-based methylation array platform was used to measure methylation of 1,413 CpG loci in ~800 cancer-associated genes and three methylation classes were derived by unsupervised clustering of tumors using recursively partitioned mixture modeling (RPMM). Quantitative RT-PCR was performed on all tumor samples to determine the expression of DNMT1, DNMT3B, VEZF1 and EZH2. Additionally, methylation at LINE-1 and AluYb8 repetitive elements was measured using bisulfite pyrosequencing. Associations between methylation class and tumor stage (p = 0.05), LINE-1 (p < 0.0001) and AluYb8 (p < 0.0001) methylation, as well as EZH2 expression (p < 0.0001) were noted following univariate analyses. A multinomial logistic regression model controlling for potential confounders revealed that AluYb8 (p < 0.003) methylation and EZH2 expression (p < 0.008) were significantly associated with methylation class membership. Because EZH2 is a member of the Polycomb repressive complex 2 (PRC2), we next analyzed the distribution of Polycomb group (PcG) targets among methylation classes derived by clustering the 1,413 array CpG loci using RPMM. PcG target genes were significantly enriched (p < 0.0001) in methylation classes with greater differential methylation between RCC and non-diseased kidney tissue. This work contributes to our understanding of how repressive marks on DNA and chromatin are dysregulated in carcinogenesis, knowledge that might aid the development of therapies or preventive strategies for human malignancies.

Abstract 3754: DNA methylation in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer death in the world. In the last three decades, HCC incidence has been increasing throughout the western world and research to uncover molecular mechanisms and markers of the disease has expanded. DNA methylation is a relevant molecular alteration in liver carcinogenesis. We carried out a retrospective study of 31 HCC cases to better understand the role of promoter methylation and global methylation changes in association with viral status, smoking exposure and other clinicopathological characteristics of HCC. Levels of DNA methylation were determined by pyrosequencing in DNA extracted from tumor and non-tumor adjacent tissue. We selected a panel of genes previously found to be implicated in epigenetic changes during liver carcinogenesis. The promoter regions of P16, APC, RASSF1A, CHGA and TP73 were investigated and the promoter region of the interspersed repetitive element LINE1 was used as a marker of global DNA methylation. We found statistically significant differences between the levels of DNA methylation in tumor and non-adjacent tumor tissue for P16, APC and TP73. Elevated methylation percentages were found for all markers in tumor tissue when compared to the corresponding non-tumor tissue DNA (P16tumor =35.9% vs. P16normal =4.9%, p&amp;lt;0.0001; APCtumor =42.1% vs. APCnormal =12.5%, p&amp;lt;0.0001; TP73tumor =37.7% vs. TP73normal =23.6%, p=0.007). Global methylation levels as measured by LINE1, were significantly lower in tumor tissue than in non-tumor adjacent tissue DNA (LINE1tumor =55.9% vs. LINE1normal =72.7%, p&amp;lt;0.0001). Statistically significant differences were also found between smokers and non-smokers in the levels of global DNA methylation of tumor tissue DNA (LINE1non-smokers = 60.30% vs. LINE1smokers=69.04%, p=0.05). Additional research into the association between DNA methylation and viral hepatitis status revealed no statistically significant differences between HCV carriers and non-carriers for any of the methylation markers studied. Clinicopathological characteristics of these patients were also analyzed in association with DNA methylation. Differences in tumor stage and survival were not associated with a statistically significant difference in DNA methylation percentages of tumor tissue DNA for these markers. However, CHGA promoter methylation was statistically significantly higher in low grade tumors than high grade tumors (CHGAgrade &amp;gt;2 = 19.83% vs. CHGAgrade ≤2 = 22.11%, p=0.04). Our results suggest that smoking might affect epigenetic mechanisms involved in liver carcinogenesis, and revealed potentially new prognostic epigenetic markers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3754. doi:10.1158/1538-7445.AM2011-3754

Methylation Status of Imprinted Genes and Repetitive Elements in Sperm DNA from Infertile Males

Stochastic, environmentally and/or genetically induced disturbances in the genome-wide epigenetic reprogramming processes during male germ-cell development may contribute to male infertility. To test this hypothesis, we have studied the methylation levels of 2 paternally (H19 and GTL2) and 5 maternally methylated (LIT1, MEST, NESPAS, PEG3, and SNRPN) imprinted genes, as well as of ALU and LINE1 repetitive elements in 141 sperm samples, which were used for assisted reproductive technologies (ART), including 106 couples with strictly male-factor or combined male and female infertility and 28 couples with strictly female-factor infertility. Aberrant methylation imprints showed a significant association with abnormal semen parameters, but did not seem to influence ART outcome. Repeat methylation also differed significantly between sperm samples from infertile and presumably fertile males. However, in contrast to imprinted genes, ALU methylation had a significant impact on pregnancy and live-birth rate in couples with male-factor or combined infertility. ALU methylation was significantly higher in sperm samples leading to pregnancy and live-birth than in those that did not. Sperm samples leading to abortions showed significantly lower ALU methylation levels than those leading to the birth of a baby.

A phase I biological study of azacitidine (VidazaTM) to determine the optimal dose to inhibit DNA methylation

This study aims to determine the optimal dosing and administration route for azacitidine to reduce global DNA methylation levels in the peripheral blood of patients with hematologic malignancies. Seventeen patients were enrolled into one of five dose level treatment groups (3 at 25mg/m2, 4 at 50mg/m2, 4 at 75mg/m2, 3 at 100mg/m2, and 3 at 150mg/m2) and received IV azacitidine at their respective dose on days 1-5 of cycle one. All patients received 75mg/m2/day IV on days 1-5 of cycle 2. Subcutaneous dosing of 75mg/m2/day on days 1-5 was used in cycle 3. Peripheral blood was collected on days 1, 3, and 5 of each cycle, and global DNA methylation was measured using bisulfite-PCR pyrosequencing of the DNA repetitive element LINE-1. 14 patients were evaluable for response with 2 CR, 2 PR, 7 SD and 3 PD reported. LINE-1 DNA methylation decreased by 1.37, 2.29, 4.81, 1.94 and 4.05% on day 5 for the 25mg/m2, 50mg/m2, 75mg/m2, 100mg/m2 and 150mg/m2 cycle one dose levels respectively. Mean decrease in LINE-1 DNA methylation was 3.7% with 75mg/m2 IV and 3.4% by subcutaneous adminstration. The data indicates that 75mg/m2 azacitidine given IV or SC effectively leads to global DNA methylation reduction by LINE-1 analysis.

A phase 1 and pharmacodynamic study of decitabine in combination with carboplatin in patients with recurrent, platinum‐resistant, epithelial ovarian cancer

Aberrant DNA methylation is a hallmark of cancer, and DNA methyltransferase inhibitors have demonstrated clinical efficacy in hematologic malignancies. On the basis of preclinical studies indicating that hypomethylating agents can reverse platinum resistance in ovarian cancer cells, the authors conducted a phase 1 trial of low-dose decitabine combined with carboplatin in patients with recurrent, platinum-resistant ovarian cancer. Decitabine was administered intravenously daily for 5 days, before carboplatin (area under the curve, 5) on Day 8 of a 28-day cycle. By using a standard 3 + 3 dose escalation, decitabine was tested at 2 dose levels: 10 mg/m(2) (7 patients) or 20 mg/m(2) (3 patients). Peripheral blood mononuclear cells (PBMCs) and plasma collected on Days 1 (pretreatment), 5, 8, and 15 were used to assess global (LINE-1 repetitive element) and gene-specific DNA methylation. Dose-limiting toxicity (DLT) at the 20-mg/m(2) dose was grade 4 neutropenia (2 patients), and no DLTs were observed at 10 mg/m(2). The most common toxicities were nausea, allergic reactions, neutropenia, fatigue, anorexia, vomiting, and abdominal pain, the majority being grades 1-2. One complete response was observed, and 3 additional patients had stable disease for >/=6 months. LINE-1 hypomethylation on Days 8 and 15 was detected in DNA from PBMCs. Of 5 ovarian cancer-associated methylated genes, HOXA11 and BRCA1 were demethylated in plasma on Days 8 and 15. Repetitive low-dose decitabine is tolerated when combined with carboplatin in ovarian cancer patients, and demonstrates biological (ie, DNA-hypomethylating) activity, justifying further testing for clinical efficacy. Cancer 2010. (c) 2010 American Cancer Society.

As we bring demethylating drugs to the clinic, we better know the DICE being cast

In this issue, Weber and coworkers report that DNA-demethylating drugs alter the transcriptional expression of the cMet proto-oncogene. Abnormal transcription is driven by the antisense promoter of a Line-1 repetitive element present within an intron. The element is a recent addition to the genome and is absent in animal models of cancer.

Retrotransposition and Structural Variation in the Human Genome

New assays are revealing that the diploid human genome contains extensive amounts of structural variation. Genome-wide approaches described in three papers in this issue (Beck et al., 2010; Huang et al., 2010; Iskow et al., 2010) paint a dynamic portrait of our genome, revealing a prominent role for repetitive sequences in shaping its structural variation.

Abstract 4900: MAGE-A11 expression in castration-recurrent prostate cancer: association with a global epigenetic defect

Abstract Despite the initial clinical response to androgen deprivation therapy, most prostate cancer patients subsequently develop castration-recurrent disease. Elucidation of the mechanisms driving this process is expected to lead to improved strategies to prevent and treat advanced prostate cancer. Epigenetic alterations, including DNA hypomethylation, play a key role in human cancer development. DNA hypomethylation is thought to promote oncogenesis in part by driving oncogene expression, but its potential role in castration-recurrent prostate cancer is unknown. Melanoma Antigen A-11 (MAGE-A11) is a cancer germline (CG) antigen that stabilizes the androgen receptor (AR), facilitates AR association with co-activators, and promotes AR transcriptional activity; thus it appears to play an oncogenic role in castration-recurrent disease. We have recently reported that MAGE-A11 is over-expressed in prostate cancer cell lines, prostate cancer xenografts, and human prostate cancer tissues; in each setting MAGE-A11 overexpression is associated with DNA hypomethylation at the MAGE-A11 promoter CpG island (Karpf et al., Molecular Cancer Research 7; 523-535, 2009). Critically, MAGE-A11 expression and promoter hypomethylation correlate with castration-recurrent cancer growth, both in the CWR-22 xenograft model and in primary human prostate cancer tissues. In the current study we examined whether MAGE-A11 expression and hypomethylation in prostate cancer is an isolated molecular event, or alternatively whether it coincides with a global DNA hypomethylation defect. To test this, we analyzed the global DNA methylation status of prostate cancer cell lines, CWR-22 cancer xenografts, and primary prostate cancer, in which MAGE-A11 expression and methylation was also characterized. We assessed global DNA methylation by measuring total genomic 5-methyl-2′-deoxycytidine (5mdC), LINE-1 repetitive DNA element methylation, and the expression and methylation of several other CG antigen genes, including MAGE-A1, NY-ESO-1, XAGE-1, and BORIS/CTCFL. Taken together, our data provide evidence for a global DNA hypomethylation phenotype in castration-recurrent prostate cancers that express MAGE-A11. Ongoing studies are using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP assay) to define the epigenome of castration-recurrent prostate cancer displaying MAGE-A11 expression. In summary, our data provide evidence that global DNA hypomethylation contributes to castration-recurrent prostate cancer, at least in part by promoting MAGE-A11 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4900.

Abstract 159: LINE-1 hypomethylation and patient prognosis in 1118 colorectal cancers

Abstract Background: Global DNA hypomethylation plays an important role in activation of oncogenes in a genome-wide scale, chromosomal instability and colorectal carcinogenesis. DNA methylation in LINE-1 (L1) repetitive element has been correlated with global DNA methylation level. We examined LINE-1 methylation in colorectal cancer and patient survival. Methods: Utilizing 1118 colorectal cancers in two prospective cohorts, we quantified methylation in a LINE-1 by Pyrosequencing. Kaplan-Meier method and Cox proportional hazard models were used to assess differences in colon cancer-specific and overall mortalities in univariate analysis and multivariate analysis that adjusted for patient characteristics and tumoral molecular features, including BRAF mutation, the CpG island methylator phenotype and microsatellite instability. Results: LINE-1 hypomethylation in 875 colon cancers was independently and linearly associated with high colon-cancer specific mortality (Ptrend&amp;lt;0.0001) and overall mortality (Ptrend=0.0004). Compared to patients with ≥75% LINE-1 methylated tumors, the adjusted hazard ratio (HR) for colon cancer-specific mortality were 1.36 [95% confidence interval (CI), 0.76-2.41] for 60-75% LINE-1 methylated tumors, 1.98 (95% CI, 1.10-3.55) for 45-60% LINE-1 methylated tumors, and 3.83 (95% CI, 1.87-7.84) for &amp;lt;45% LINE-1 methylated tumors. Similar findings were noted for overall mortality. Kaplan-Meier analysis demonstrated shorter survival among patients with LINE-1 hypomethylated tumors (log-rank p&amp;lt;0.0001). The prognostic influence of LINE-1 hypomethylation in colon cancer was consistent across the two independent cohort studies as well as across strata of clinical or molecular characteristics. In contrast, LINE-1 hypomethylation was not significantly associated with survival of 243 rectal cancer patients (adjust HR 1.02; 95% CI, 0.48-2.19; for a 30% decrease of LINE-1 methylation; Ptrend=0.96). Conclusions: Tumoral LINE-1 hypomethylation is associated with shorter survival among colon cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 159.

Abstract 4919: Body mass index interacts with DNA global methylation levels to predict bladder cancer in The Shanghai Bladder Cancer Study

Abstract Background: Bladder cancer is a relatively common and costly disease, and although most frequently associated with tobacco smoking, there are several environmental and occupational exposure contaminants associated with bladder cancer which are mostly found in urban settings. As nations become more developed they may become more susceptible to bladder cancer as a result of urbanization, as well as changes in dietary and lifestyle risk factors associated with developed countries. Recent research suggests that in addition to mutagenic effects, environmental exposures may be linked to epigenetic alterations, including changes in global DNA methylation, which have previously been associated with risk of bladder cancer. Therefore, we have sought to examine the association between environmental exposures and global DNA methylation, and risk of bladder cancer in in Shanghai, China. Methods: The Shanghai Bladder Cancer Study is a population-based case-control study in Shanghai with 581 bladder cancer patients, 25-74yrs of age at diagnosis between 1995-1998, and 604 healthy community control subjects comparable by gender and age. In addition to an in-person interview to gather information on body weight, height, smoking history, and other lifestyle factors, blood and urine samples were collected from all subjects. We utilized quantitative bisulfite pyrosequencing of the LINE-1 repetitive elements of DNA extracted from peripheral blood samples as a surrogate measure for genome-wide methylation. Odds ratios and 95% confidence intervals for bladder cancer associated with LINE-1 values were calculated using unconditional logistic regression methods for all study subjects as well as for subgroups stratified by body mass index (BMI) and smoking status. Results: Among individuals with BMI &amp;gt;25 kg/m2, 28% of bladder cancer cases versus 15% controls possessed the highest quintile of LINE-1 values (&amp;gt;83.38) (P = 0.098). This difference in global DNA methylation between cases and controls became more apparent among current smokers (P=0.03), and in long-term (&amp;gt;20 years) smokers (P=0.009). Conclusion: We found that BMI and smoking are significant risk factors for bladder cancer, and have the ability to predict case status in our study. Using logistic regression analysis, we also found a significant interaction between BMI and global DNA methylation that is a significant predictor of case status. This association between BMI and global DNA methylation is especially prominent among smokers. BMI is a known risk factor for various cancers, although the mechanisms underlying this association are not well understood. These data suggest that smoking and BMI might influence bladder cancer etiology through epigenetic mechanisms. Due to rapidly rising rates of global obesity, it is important that we understand the impact of BMI on DNA methylation, and how it relates to cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4919.

Abstract 190: p53 target gene expression, DNA hypomethylation, and clinical response in MDS patients undergoing decitabine therapy

Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of blood disorders characterized by peripheral cytopenias, a cellular bone marrow with dysplasia and increased intramedullary apoptosis, and are associated with risk of progression to acute myelogenous leukemia (AML). MDS are often fatal due to complications from cytopenias and/or progression to AML. DNA methyltransferase inhibitors (DNMTi), including 5-azacytidine and 5-aza-2’-deoxycytidine (decitabine) have recently assumed a major role in the clinical management of MDS, and are two of only three FDA-approved treatments for these disorders. The mechanistic basis for the activity of DNMTi in MDS is unclear. We recently reported that decitabine treatment induces the expression of p53-inducible ribonucleotide reductase p53R2/RRM2B in tumor cell lines and MDS patient bone marrow (Link et al., Cancer Research 68; 9358-9366, 2008). Importantly, p53R2 induction was independent of DNA hypomethylation at its promoter region, and the level of p53R2 induction correlated with clinical response in a cohort of 13 patients. These data suggest that important pharmacodynamic responses to DNMTi may be associated with DNA damage and/or p53 activation. To better understand the overall nature of the molecular responses occurring in the bone marrow of MDS patients treated with decitabine, in the current study we examined a variety of pharmacodynamic endpoints including: p53 target gene expression, global DNA methylation, tumor antigen gene expression and methylation, and tumor suppressor gene expression. Specifically, we measured the expression of three classical p53 target genes (p21, GADD45-α, NOXA), LINE-1 repetitive element methylation, the expression and methylation of cancer-germline (CG) antigens including MAGE-A1, NY-ESO-1, XAGE-1, and CTCFL/BORIS, and the expression of the tumor suppressor genes (TSGs) p15INK4b and PDLIM4. Our data reveal that decitabine treatment causes robust induction of p53 target genes in MDS patient bone marrow, which coincides with LINE-1 hypomethylation and clinical responses. In contrast, while CG antigen genes showed some evidence for DNA hypomethylation post-therapy, this did not coincide with CG antigen gene induction. Surprisingly, TSGs were also not induced by decitabine therapy in MDS patient bone marrow. Ongoing studies seek to define p53 mutational status in MDS patients and to determine how this relates to p53 target gene induction and/or clinical response. In summary, our data reveal a connection between p53 target gene activation, global DNA hypomethylation, and clinical response in MDS patients undergoing decitabine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 190.

Repetitive element DNA methylation and circulating endothelial and inflammation markers in the VA normative aging study

BACKGROUND: Lower blood DNA methylation has been associated with atherosclerosis and high cardiovascular risk. Mechanisms linking DNA hypomethylation to increased cardiovascular risk are still largely unknown. In a population of community-dwelling elderly individuals, we evaluated whether DNA methylation in LINE-1 repetitive element, heavily methylated sequences dispersed throughout the human genome, was associated with circulating Vascular Cell Adhesion Molecule-1 (VCAM-1), Inter- Cellular Adhesion Molecule-1 (ICAM-1), and C-reactive protein (CRP). METHODS AND RESULTS: We measured LINE-1 methylation by bisulfite PCR-Pyrosequencing on 742 blood DNA samples from male participants in the Boston area Normative Aging Study (mean age=74.8 years). Mean serum VCAM-1 increased progressively in association with LINE-1 hypomethylation (from 975.2 to 1063.4 ng/ml in the highest vs. lowest methylation quintiles; ptrend= 0.004). The association between VCAM-1 and LINE-1 hypomethylation was significant in individuals without ischemic heart disease or stroke (n=480; p=0.001), but not in those with prevalent disease (n=262; p=0.57). Serum ICAM-1 and CRP were not associated with LINE-1 methylation (p-trend=>0.25). All results were confirmed by multivariable analyses adjusting for age, BMI, smoking, pack-years, and ischemic heart disease/stroke. CONCLUSIONS: LINE-1 element hypomethylation is associated with higher serum VCAM-1. Our data provide new insights into epigenetic events that may accompany the development of cardiovascular disease.

A Phase I Biological Study of Azacitidine (Vidaza) to Determine the Optimal Biological Dose and Route of Administration.

Abstract 1753Poster Board I-779 BackgroundAzacitidine (5-azacytidine, Vidaza) is a DNA methylation inhibitor with used to treat myelodysplastic syndrome (MDS). The studies which led to FDA approval based dosing and administration guidelines on clinical response. In vitro studies have demonstrated that azacitidine exerts its effect by inhibiting DNA methyltransferase in hypermethylated tumor suppressor genes in malignant cells. Research to date has not linked azacitidine dosing with biochemical and clinical response in vivo. The degree of DNA repetitive element sequence methylation (such as LINE-1) has been demonstrated to correlate with global DNA methylation and may be used to determine DNA methylation changes after treatment with azacitidine. We have conducted a phase I study to link clinical and biologic response to azacitidine. This study aims to determine the optimal dose and route of administration for azacitidine to inhibit global DNA methylation levels in the peripheral blood of patients with hematologic malignancies. MethodsPatients with hematologic malignancy who provided informed consent were eligible for study inclusion, with enrollment criteria based on the specific malignancy. Patients were enrolled into one of five dose level treatment groups (25mg, 50mg, 75mg, 100mg or 150mg IV per m2 per day for 5 days) for the first course of therapy. On day 28, all patients received a course of 75mg/m2/day IV for 5 days. Subcutaneous dosing of 75mg/m2/day for 5 days was used for course three. Patients received 75mg/m2/day either SQ or IV x 5 days every 4 weeks for course four and beyond. Peripheral blood was collected on days 1, 3, and 5 during each course, and global DNA methylation was measured using bisulfite-PCR Pyrosequencing of the 6 DNA repetitive elements (LINE1, AluYb8, AluSq, Sat-alpha, D4Z4, NBL-2). Additionally, gene promoter specific DNA methylation was assessed in a subset of patients using the Illumina GoldenGate Bead Array DNA Methylation Assay which measures DNA methylation of 1505 CpG sites (807 genes). ResultsSeventeen patients were treated (3 at 25mg, 4 at 50mg, 4 at 75mg, 3 at 100mg, and 3 at 150mg/m2). Diagnosis included 5 patients with MDS, 10 patients with AML (2 untreated older patients, 7 relapsed or refractory patients), 1 patient with CML (Imatinib refractory), and 1 patient with non-Hodgkin's lymphoma (relapsed disease). At the time of submission, 14 patients were evaluable for response with 4 CR (1 mCr, 1 CRp), 1 PR, 6 SD and 3 PD reported. The median number of cycles given was 3 (range 1-14+). LINE1 DNA methylation decreased by 1.4, 2.3, 4.8, 1.9 and 4.0% on day 5 for the 25mg, 50mg, 75mg, 100mg, and 150mg/m2 course one dose levels respectively. Mean decrease in LINE1 DNA methylation with 75mg/m2 IV was 3.7% and only 2.6% by 75mg/m2 of azacitidine SQ. There was a large amount of inter-patient variability but less intra-patient variability in DNA methylation response to azacitidine. ConclusionAzacitidine is effective at inhibiting DNA methylation at multiple dose levels for both IV and SQ routes of administration. There is a high degree of patient-to-patient variability in DNA methylation changes, although 75mg/m2 lead to the greatest mean decrease in DNA methylation by a 5 day IV regimen. Measurement of DNA methylation of LINE1 and AluYb8 repetitive elements were the best surrogate markers for measuring overall changes in gene specific promoter DNA methylation when compared with 807 genes assessed by the Illumina GoldenGate platform. High-throughput gene promoter DNA methylation analysis revealed subtle changes in DNA methylation, though gene specific changes could not be linked to therapeutic activity. DisclosuresOff Label Use: Azacitidine in hematologic malignancies other than MDS. Mohrbacher:Celgene: Honoraria, Speakers Bureau. Gorospe:Novartis: Honoraria, Speakers Bureau. Yang:Celgene: Honoraria, Research Funding, Speakers Bureau.

A Study of Aberrant DNA Methylation in Refractory and Relapsed Childhood Acute Lymphoblastic Leukaemia.

Abstract 3456Poster Board III-344Refractory and relapsed Acute Lymphoblastic Leukaemia (ALL) raise difficult clinical challenges and represent a significant cause of death in childhood and adolescence. This study examines epigenetic changes in relapsed paediatric ALL to increase our understanding of the mechanisms of treatment failure in these patients. Aberrant DNA methylation, a frequent phenomenon in paediatric ALL, has been identified as an independent factor of poor prognosis. In this study, using a CGI microarray consisting of 12K clones representing 5411 unique chromosomal sequences spread throughout the genome, we analysed a cohort of 22 childhood B-precursor ALL patient samples, 11 diagnosis samples and 11 relapse samples (unmatched) from patients who experienced a hematologic relapse, and developed genome-wide methylation profiles of leukaemic cells, at diagnosis and relapse. Comparison of these profiles identified 227 genes with statistically significant methylation changes – increase and decrease of methylation levels - between the two patient groups. The methylation status and the methylation change of 5 genes (ECRG4, CDH5, IRX3, FOXD3 and ERBB4) were independently verified, using pyrosequencing, in 6 ALL patients, 3 diagnosis and 3 relapse samples. The relationship between methylation and expression of one of these genes (ECRG4) was examined in ALL cell lines (CEMC1 and Jurkat) before and after 5-Aza-2'-deoxycytidine treatment. More genes are currently under investigation. Methylation studies on the repetitive element LINE-1 showed a significant increase in methylation of repetitive sequences in relapse samples (mean 79% vs. 89%, respectively; p=0.003, Mann Whitney test). Chromosomal mapping of the 227 genes revealed significant clustering of differentially methylated targets in chromosomes 11, 13 and 15, chromosomal locations that were previously found to be associated with ALL prognosis. Functional gene relation analysis revealed a connection between 11 genes (FOXD3, HPGD, NR4A2, CAST, TBRG4, XRCC6, NAB2, SEZL6, CDH13, NR2F1, SP1) that show increased methylation in relapse ALL and NR3C1, the gene coding for glucocorticoid receptor. Currently we are investigating the role of methylation in glucocorticoid resistance in ALL cell lines. Our data indicate an underlying epigenetic mechanism responsible for the chemoresistance observed in relapsed paediatric ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Prolonged Exposure to Particulate Air Pollution and Decreased DNA Methylation

ISEE-0332 Background and Objective: Acute and chronic exposure to particulate air pollution is associated with increased cardiovascular morbidity and mortality. Epigenetic mechanisms are a potential pathway linking environmental exposures to disease. Lower blood DNA methylation, an epigenetic mechanism, has been found in processes related to cardiovascular disease. Recently, acute air pollution exposure has been associated with changes in DNA methylation in heavily methylated sequences with high representation throughout the human genome. We hypothesized that prolonged exposure to particulate pollution might also modify DNA methylation and be a potential mechanism by which chronic air pollution exposure adversely affects health. Methods: We measured DNA methylation of LINE-1 and Alu repetitive elements by quantitative PCR-Pyrosequencing of 985 blood samples from 680 elderly participants in the Boston Normative Aging Study. We used covariate-adjusted mixed models to account for repeated measures. We estimated effects on DNA methylation of ambient pollutants (black carbon, PM2.5, and sulfate) in multiple exposure windows (ranging from 1 month to 1 year) prior to the examination. Results: We found that a 1μg/m3 increase in black carbon over a 60-day period was associated with a 0.8% (95% CI: 0.004%, 1.6%) decrease in methylation of LINE-1 repetitive elements. A 1 μg/m3 increase in black carbon over a 90-day period was associated with a 0.6% (0.001%, 1.1%) decrease in Alu repetitive elements. A 1 μg/m3 increase in sulfate over a 90-day period was associated with 0.3% (0.02%, 0.6%) decrease in LINE-1 repetitive elements. The association with longer exposure windows persisted when we fit models simultaneously adjusting for 1-week moving averages of pollutant exposure. There was no association with PM2.5. Conclusion: Exposure to particulates over 2 to 3 months is associated with decreased repetitive element methylation. This may be a mechanism by which chronic exposure to air pollution increases risk of morbidity and mortality.

Global and gene‐specific promoter methylation changes are related to anti‐B[a]PDE‐DNA adduct levels and influence micronuclei levels in polycyclic aromatic hydrocarbon‐exposed individuals

We investigated the effect of chronic exposure to polycyclic aromatic hydrocarbons (PAHs) on DNA methylation states (percentage of methylated cytosines (%mC)) in Polish male nonsmoking coke-oven workers and matched controls. Methylation states of gene-specific promoters (p53, p16, HIC1 and IL-6) and of Alu and LINE-1 repetitive elements, as surrogate measures of global methylation, were quantified by pyrosequencing in peripheral blood lymphocytes (PBLs). DNA methylation was evaluated in relation to PAH exposure, assessed by urinary 1-pyrenol and anti-benzo[a]pyrene diolepoxide (anti-B[a]PDE)-DNA adduct levels, a critical genetic damage from B[a]P. We also evaluated whether PAH-induced DNA methylation states were in turn associated with micronuclei in PBLs, an indicator of chromosomal instability.

Decline in genomic DNA methylation through aging in a cohort of elderly subjects

Loss of genomic DNA methylation has been found in a variety of common human age-related diseases. Whether DNA methylation decreases over time as individuals age is unresolved. We measured DNA methylation in 1097 blood DNA samples from 718 elderly subjects between 55 and 92 years of age (1–3 samples/subjects), who have been repeatedly evaluated over an 8-year time span in the Boston area Normative Aging Study. DNA methylation was measured using quantitative PCR-Pyrosequencing analysis in Alu and LINE-1 repetitive elements, heavily methylated sequences with high representation throughout the human genome. Age at the visit was negatively associated with Alu element methylation ( β = −0.12 %5 mC/year, p = 0.0005). A weaker association was observed with LINE-1 elements ( β = −0.06 %5 mC/year, p = 0.049). We observed a significant decrease in average Alu methylation over time, with a −0.2 %5 mC change ( p = 0.012) compared to blood samples collected up to 8 years earlier. The longitudinal decline in Alu methylation was linear and highly correlated with time since the first measurement ( β = −0.089 %5 mC/year, p < 0.0001). In contrast, average LINE-1 methylation did not vary over time [ p = 0.51]. Our results demonstrate a progressive loss of DNA methylation in repetitive elements dispersed throughout the genome.