Aberrant DNA Methylation Status Research Articles

DNA demethylation of promoter region orchestrates SPI-1-induced ADAMTS-5 expression in articular cartilage of osteoarthritis mice.

Osteoarthritis (OA) is one of the most prevalent joint diseases in aged people and characterized by articular cartilage degeneration, synovial inflammation, and abnormal bone remodeling. Recent advances in OA research have clearly shown that OA development is associated with aberrant DNA methylation status of many OA-related genes. As one of most important cartilage degrading proteases in OA, a disintegrin and metalloproteinase with thrombospondin motifs subtype 5 (ADAMTS-5) is activated to mediate cartilage degradation in human OA and experimental murine OA models. The pathological factors and signaling pathways mediating ADAMTS-5 activation during OA development are not well defined and have been a focus of intense research. ADAMTS-5 promoter is featured by CpG islands. So far there have been no reports concerning the DNA methylation status in ADAMTS-5 promoter during OA development. In this study, we sought to investigate DNA methylation status in ADAMTS-5 promoter, the role of DNA methylation in ADAMTS-5 activation in OA, and the underlying mechanisms. The potential for anti-OA intervention therapy which is based on modulating DNA methylation is also explored. Our results showed that DNA methyltransferases 1 (Dnmt1) downregulation-associated ADAMTS-5 promoter demethylation played an important role in ADAMTS-5 activation in OA, which facilitated SPI-1 binding on ADAMTS-5 promoter to activate ADAMTS-5 expression. More importantly, OA pathological phenotype of mice was alleviated in response to Dnmt1-induced DNA methylation of ADAMTS-5 promoter. Our study will benefit not only for deeper insights into the functional role and regulation mechanisms of ADAMTS-5 in OA, but also for the discovery of disease-modifying OA drugson the basis of ADAMTS-5 via modulating DNA methylation status.

ALDH2 as a potential stem cell-related biomarker in lung adenocarcinoma: Comprehensive multi-omics analysis

Lung adenocarcinoma (LUAD) is the most prevalent lung cancer and one of the leading causes of death. Previous research found a link between LUAD and Aldehyde Dehydrogenase 2 (ALDH2), a member of aldehyde dehydrogenase gene (ALDH) superfamily. In this study, we identified additional useful prognostic markers for early LUAD identification and targeting LUAD therapy by analyzing the expression level, epigenetic mechanism, and signaling activities of ALDH2 in LUAD patients. The obtained results demonstrated that ALDH2 gene and protein expression significantly downregulated in LUAD patient samples. Furthermore, The American Joint Committee on Cancer (AJCC) reported that diminished ALDH2 expression was closely linked to worse overall survival (OS) in different stages of LUAD. Considerably, ALDH2 showed aberrant DNA methylation status in LUAD cancer. ALDH2 was found to be downregulated in the proteomic expression profile of several cell biology signaling pathways, particularly stem cell-related pathways. Finally, the relationship of ALDH2 activity with stem cell-related factors and immune system were reported. In conclusion, the downregulation of ALDH2, abnormal DNA methylation, and the consequent deficit of stemness signaling pathways are relevant prognostic and therapeutic markers in LUAD.

Genome-wide DNA methylation profiling of stomach cancer in the ethnic population of Mizoram, North East India

Stomach cancer is the fifth most common cancer in terms of prevalence and incidence and the fourth leading cause of mortality in men and women worldwide. It is well-established that aberrant DNA methylation in cells can lead to carcinogenesis. The primary objective of our study was to investigate the aberrant DNA methylation status of genes associated with stomach cancer with a particular reference to the ethnic population of Mizoram, North East India. The site-level analysis identified 2883 CpG sites differentially methylated, representing ∼922 genes. Out of which 476 Differentially Methylated Positions (DMPs) were promoter-associated, 452 DMPs were hypermethylated, and 24 were hypomethylated. The region-level analysis identified 462 Differentially Methylated Regions (DMRs) corresponding to ∼320 genes, of which ∼281 genes were hypermethylated and ∼40 genes were hypomethylated. TCGA analysis showed that some of the genes had been previously implicated in other cancers including stomach cancer. Five hypermethylated genes were selected as candidate genes for further investigations and they have shown to be novel and could serve as candidate hypermethylation biomarkers for stomach cancer in this particular ethnic group.

Association between toxic and essential metals in blood and global DNA methylation among electronic waste workers in Agbogbloshie, Ghana.

Aberrant global DNA methylation status is a known biomarker for increased disease risk, especially cancer. There is little published data on the association between toxic and essential metal mixtures and global DNA methylation in electronic waste (e-waste) workers. We aimed to establish the association between toxic and essential metals in blood and the effect of their interactions on global DNA methylation among e-waste recyclers and a reference group in Ghana. We used ICP-MS to measure the level of five metals (Se, Zn, Mn, Cd, and Pb) in the blood of 100 e-waste workers and 51 controls. We quantified blood DNA methylation levels of LINE-1 as an indicator of global DNA methylation. Cd, Mn, and Se levels were significantly higher in the reference group than in e-waste workers. Only Pb was significantly higher in the e-waste workers compared to the controls. Our linear regression analysis results showed a significant inverse association between Zn and LINE-1 DNA methylation (βZn = - 0.912; 95% CI, - 1.512, - 0.306; p = 0.003) which corresponds to a 0.009 decrease in %LINE-1 methylation (95% CI, - 0.015, - 0.003; p = 0.003) for a 1% increase in Zn concentration. Potential interactions between Cd and Zn on global DNA methylation were observed. In summary, co-exposure to toxic and essential metals is associated with global (LINE-1) DNA methylation.

Downregulation of LINC00886 facilitates epithelial-mesenchymal transition through SIRT7/ELF3/miR-144 pathway in esophageal squamous cell carcinoma.

LINC00886 has been reported to be down-regulated in laryngeal squamous cell carcinoma, and aberrant DNA methylation status of it has been screened in several tumor types. However, the roles of LINC00886 in esophageal squamous cell carcinoma (ESCC) remained unclarified. The present study was to investigate the expression level, epigenetic inactivation mechanisms, and functions of LINC00886 in ESCC tumorigenesis. Frequent down-regulation of LINC00886 was verified in esophageal cancer cells and ESCC tissues. There are CpG islands spanning the promoter and exon 1 regions of LINC00886 gene, and DNA hypermethylation of proximal promoter led to transcriptional inhibition of LINC00886, moreover, histone modification also played certain roles in LINC00886 transcription. LINC00886 functioned as a tumor suppressor by inhibiting proliferation, migration, and invasion of esophageal cancer cells. LINC00886 was down-regulated following TGF-β1 treatment in esophageal cancer cells and participated in epithelial-mesenchymal transition (EMT) process by regulating EMT-related genes, especially ZEB1 and ZEB2. ELF3 was proved to be one of the downstream target genes of LINC00886. LINC00886 may interact with and recruit SIRT7 to decrease acetylation level of H3K18 on the promoter region of ELF3 to inhibit its expression. Furthermore, ELF3 may promote EMT process via promoting ZEB1 and ZEB2 expression through binding to the promoter region of miR-144 to suppress miR-144-3p transcriptional activity in ESCC. These data suggest that LINC00886 may act as a tumor suppressor gene in ESCC and its down-regulation through epigenetic mechanisms promotes EMT process via SIRT7/ELF3/miR-144 pathway in ESCC. Thus, LINC00886 may be a potential therapeutic target for ESCC treatment.

Elevation of ADAM12 facilitates tumor progression by enhancing metastasis and immune infiltration in gastric cancer.

A disintegrin and metalloprotease 12 (ADAM12), an essential transmembrane protein with metalloprotease, cell binding and intracellular signal-regulating capabilities, has been reported to play a crucial role in various types of cancers. However, the biological function of ADAM12 in gastric cancer (GC) remains unclear. Bioinformatic and experimental analyses were used to determine the expression level and prognostic value of ADAM12 in GC. The level of DNA methylation and the competing endogenous RNA (ceRNA) network was identified using MethSurv, Starbase3.0, miRNet2.0 and experimental analyses. Then, the co-expression profiles of ADAM12 were determined and subjected to enrichment analysis using the LinkedOmics database. The protein-protein interaction network and the docking model of ADAM12 were constructed using the GeneMANIA, STRING, and HDOCK webservers. The role of ADAM12 in tumor metastasis and immune infiltration was investigated using in vitro assays and TIMER database exploration. It was found that ADAM12 was overexpressed and was correlated with a poor prognosis of GC patients. In addition, the aberrant DNA methylation status and ceRNA regulation may contribute to the upregulation of ADAM12 in GC. Moreover, the enrichment analysis revealed that ADAM12 is involved in multiple vital biological functions and pathways, such as 'macrophage activation', 'extracellular matrix binding' and 'ECM-receptor interaction'. Subsequently, the protein-protein interaction network and molecular docking model demonstrated that follistatin like 3 (FSTL3) is a potential binding partner of ADAM12. Finally, it was demonstrated that ADAM12 promotes tumor metastasis, immune infiltration and M2 macrophage polarization in GC. In summary, these results highlight the potential of ADAM12 to be used as a therapeutic target for GC.

Elevated placental histone H3K4 methylation via upregulated histone methyltransferases SETD1A and SMYD3 in preeclampsia and its possible involvement in hypoxia-induced pathophysiological process.

Disturbance in placental epigenetic regulation contributes to the pathogenesis of preeclampsia (PE). Although aberrant placental DNA methylation status in PE has been thoroughly studied, the role of histone modifications, including histone methylation, in PE remains unclear. Moreover, no study has ever reported the association between PE and placental histone methylation status by focusing on histone methyltransferases. The present study aimed to investigate the possible involvement of placental epigenetic regulation by histone methylation via histone methyltransferases in the pathophysiology of PE. Placental mRNA expression of histone methyltransferases was examined using quantitative RT-PCR. Protein expression of histone methyltransferases and histone methylation status in placentas and trophoblast cell lines were assessed by immunoblotting and immunohistochemistry. Expression profile of histone methyltransferases in the placentas using quantitative RT-PCR revealed that the mRNA expression levels of histone 3 lysine 4 (H3K4) methyltransferases, SETD1A and SMYD3, were significantly increased in placentas from PE patients. Immunoblotting and immunohistochemistry revealed that not only protein expression levels of SETD1A and SMYD3, but also H3K4 methylation status was increased in the trophoblasts from PE placentas. In vitro studies using HTR-8/SV-neo and BeWo cells showed that hypoxia induced the expression levels of SETD1A and SMYD3, and subsequently enhanced H3K4 methylation. Furthermore, the overexpression of SETD1A and SMYD3 in HTR-8/SV-neo cells enhanced H3K4 methylation in response to hypoxia. Our study results suggest that placental epigenetic alteration by enhanced histone H3K4 methylation through upregulated SETD1A and SMYD3 might play a role in the pathophysiological process of PE associated with hypoxia.

Investigation of the Effect of Decitabine on Hep-2 Cell Line at Chromosomal Level

DNA methylation is an epigenetic event that is defined as the binding of a methyl group via DNA methyl transferases (DNMTs) to the fifth carbon of cytosine in the CpG dinucleotide. Regulation of the expressions of tissue-specific genes, regulation of gene expressions during development, X inactivation and imprinting events are caused by DNA methylation. The aberrant DNA methylation associated with various diseases, especially cancers, is one of the most studied epigenetic mechanisms. Decitabine, a nucleoside analogue, inhibits DNMT enzymes and causes hypomethylation in the genome. There are many studies investigating the aberrant DNA methylation status in the genome of cells treated with Decitabine. Karyotype analysis is a cytogenetic method, which has been used successfully for the detection of both numerical and structural chromosomal abnormalities for many years. Hep-2 is a laryngeal cancer cell line, which is, used to investigate various genetic and epigenetic changes at the molecular level that revealed important information about the initiation and development of laryngeal cancer. However, the number of studies showing the chromosomal structure of Hep-2 cells is insufficient. The effects of Decitabine on cell morphology in different cell lines, its relationship with proliferation of the cells and its effects on chromosomes have been studied. However, the number of studies showing the effect of Decitabine at the chromosomal level in Hep-2 cell lines is also very limited. In current study, we investigated the chromosomes of Hep-2 cells cultured 72 hours with 1 μM and 10 μM Decitabine and without Decitabine. Keywords : Cell lines, Hep-2 cell line, Karyotyping, Decitabine, DNA methylation DOI : 10.7176/JSTR/5-4-11

Genome-wide analysis of DNA methylation in endometriosis using Illumina Human Methylation 450 K BeadChips.

Endometriosis is a common chronic gynecologic disorder characterized by the presence and growth of endometrial-like tissue outside of the uterine cavity. Although the exact etiology remains unclear, epigenetic modifications, such as DNA methylation, are thought to contribute to the pathogenesis of endometriosis. Here, we used the Illumina Human Methylation 450 K BeadChip Array to analyze the genome-wide DNA methylation profiles of six endometriotic lesions and six eutopic endometria from patients with ovarian endometriosis and six endometria of women without endometriosis. Compared with the eutopic endometria of women with endometriosis, 12,159 differentially methylated CpG sites and 375 differentially methylated promoter regions were identified in endometriotic lesions. GO analyses showed that these putative differentially methylated genes were primarily associated with immune response, inflammatory response, response to steroid hormone stimulus, cell adhesion, negative regulation of apoptosis, and activation of the MAPK activity. In addition, the expression levels of DNMT1, DNMT3A, DNMT3B, and MBD2 in endometriotic lesions and eutopic endometria were significantly decreased compared with control endometria. Our findings suggest that aberrant DNA methylation status in endometriotic lesions may play a significant role in the pathogenesis and progression of endometriosis.

Uterine ERα epigenetic modifications are induced by the endocrine disruptor endosulfan in female rats with impaired fertility

High ERα activity may disrupt the window of uterine receptivity, causing defective implantation. We investigated whether implantation failures prompted by endosulfan are associated with aberrant ERα uterine expression and DNA methylation status during the pre-implantation period. ERα-dependent target genes that play a crucial role in the uterine receptivity for embryo attachment and implantation were also investigated. Newborn female rats received corn oil (vehicle, Control), 6 μg/kg/d of endosulfan (Endo6) or 600 μg/kg/d of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5, and 7. On PND90, females were made pregnant and on gestational day 5 (GD5, pre-implantation period) uterine samples were collected. ERα expression was assessed at protein and mRNA levels by immunohistochemistry and real time RT-PCR, respectively. ERα transcript variants mRNA containing alternative 5’-untranslated regions (5′UTRs) were also evaluated. We searched for predicted transcription factors binding sites in ERα regulatory regions and assessed their methylation status by Methylation-Sensitive Restriction Enzymes-PCR technique (MSRE-PCR). The expression of the ERα-dependent uterine target genes, i.e. mucin-1 (MUC-1), insulin-like growth factor-1 (IGF-1), and leukemia inhibitory factor (LIF), was assessed by real time RT-PCR. Both doses of endosulfan increased the expression of ERα and its transcript variants ERα-OS, ERα-O, ERα-OT and ERα-E1. Moreover, a decreased DNA methylation levels were detected in some ERα regulatory regions, suggesting an epigenetic up-regulation of it transcription. ERα overexpression was associated with an induction of its downstream genes, MUC-1 and IGF-1, suggesting that endosulfan might alter the uterine estrogenic pathway compromising uterine receptivity. These alterations could account, at least in part, for the endosulfan-induced implantation failures.

Hypermethylation of CpG sites at the promoter region is associated with deregulation of mitochondrial ATPsyn-β and chemoresistance in acute myeloid leukemia.

Aberrant DNA methylation status of some genes has been shown to be involved in chemoresistance of acute myeloid leukemia (AML). We have recently found that down-regulation of the β subunit of mitochondrial ATP synthase (ATPsyn-β) leads to adriamycin resistance in acute and chronic myeloid leukemia cells, and hypermethylation of the ATPsyn-β gene promoter is associated with chemoresistance in chronic myeloid leukemia. To further investigate the relationship between methylation of ATPsyn-β gene, mRNA expression as well as chemoresistance in AML. Quantitative RT-PCR and methylation specific PCR were performed to assess mRNA expression and methylation status of ATPsyn-β gene on primary bone marrow nuclear cells (BMMCs), and cell proliferation assay was used to determine the sensitivity of BMMCs to adriamycin. Hypermethylation status of ATPsyn-β gene promoter existed in those relapsed/refractory AML patients, and this hypermethylation of the gene was associated with a suppressed mRNA expression levels. Four patients at diagnosis and relapse underwent gene methylation status shift from hypermethylation to hypomethylation, which was accompanied by reduced mRNA expression of the gene. 5-azacitidine(5-Aza)- a demethylating agent, could restore ATPsyn-β mRNA expression and increase the adriamycin sensitivity of primary leukemic cells from seven relapsed/ refractory AML patients. Hypermethylation of ATPsyn-β gene promoter is associated with a down-regulated mRNA expression and chemoresistance in AML patients.

Improved Noninvasive Bladder Cancer Diagnosis using Urine Sediments and Novel DNA Methylation Biomarker Panels.

Aberrant DNA methylation status early in carcinogenesis represents a potential indicator of tumor detection. We would like to establish a DNA methylation biomarker panel for bladder cancer detection in the research. Seven candidate genes with known cancer associations were selected for this study. The DNA methylation status of the candidate genes was analyzed by methylation-specific polymerase chain reaction assays to evaluate the relationship between bladder cancer and target gene methylation status in the urine sediments of participants. 112 bladder cancer patients, 10 healthy volunteers, and 17 glandular cystitis patients were enrolled. There were significant differences in the methylation status of p14ARF, RUNX3, RARβ, DAPK, and HPP1 between the healthy control, glandular cystitis, and bladder cancer groups (p = 0.027, p < 0.001, p < 0.001, p = 0.030 and p = 0.003, respectively). A panel composed of all five genes with significant methylation differences yielded an area under the receiver-operating characteristic curve (AUC) of 0.936 and had 98.21% sensitivity and 88.89% specificity, while a panel using just two of these genes (RUNX3 and RARP) yielded an AUC of 0.918 with 96.64% sensitivity and 88.89% specificity. Another panel of two genes (p14ARF and HPP1) had 100% specificity, but an AUC of 0.688 and 37.50% sensitivity. Using the urine of bladder cancer patients and healthy controls, we assessed several novel DNA methylation biomarker panels that demonstrated good bladder cancer detection capability. Based on the results of this study we recommend the RUNX3 and RARβ panel as the first choice for bladder cancer detection. In suspicious or difficult to diagnose cases, the p14ARF and HPP1 panel could be used as an additional diagnostic tool due to its high specificity.

Analysis of DNA methylation in plasma for monitoring hepatocarcinogenesis.

To explore whether the aberrant DNA methylation status in plasma could be used as a biomarker for hepatocellular carcinoma (HCC) screening among high-risk individuals. The promoter methylation status of ELF, RASSF1A, p16, and GSTP1 was investigated by methylation-specific polymerase chain reaction (PCR) in 34 paired HCC and nontumor liver tissue from HCC patients and 10 tissues from patients with liver cirrhosis (LC). Plasma samples from 31 HCC patients, 10 LC patients as well as 7 patients with benign hepatic conditions were also collected and characterized using the same method. Among liver specimens, HCC tissues displayed a significantly higher methylation frequency of each gene compared with nontumor tissue (p<0.05). Moreover, the frequency was much higher in tumor tissues than in nontumor tissue, when the data from two or three genes were combined (p=0.001 and p<0.001, respectively). Among plasma samples, either the frequency of at least one methylated gene (p<0.001) or the average number of methylated genes (p<0.05) demonstrated a stepwise increase in patients with benign lesions, LC, and HCC. Furthermore, when positive results, that is, plasma methylation status of at least one gene were combined with the elevated AFP400 level (serum alpha-fetoprotein [AFP] level at a cutoff of 400 ng/mL), the diagnostic sensitivity of HCC could increase to 93.55%. These results suggested that the methylation of tumor suppressor genes may participate in the development and progression of HCC. Additionally, it may be useful to combine the plasma DNA methylation status of a panel of gene markers and the serum AFP for HCC screening.

Genome-wide analysis of histone modifications by ChIP-chip to identify silenced genes in gastric cancer.

The present study aimed to identify novel histone modification markers in gastric cancer (GC) by chromatin immunoprecipitation microarray (ChIP-chip) analysis and to determine whether these markers were able to discriminate between normal and GC cells. We also tested for correlations with DNA methylation. We probed a human CpG island microarray with DNA from a GC cell line (MKN45) by chromatin immunoprecipitation (ChIP). ChIP-reverse-transcriptase quantitative polymerase chain reaction PCR (RT-qPCR) was used to validate the microarray results. Additionally, mRNA expression levels and the DNA methylation of potential target genes were evaluated by RT-qPCR and methylation-specific PCR (MSP). The moults showed that 134 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over acetylation and 46 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over H3-K4 trimethylation in MKN45 cells. The ChIP-qPCR results agreed with those obtained from the ChIP-chip analysis. Aberrant DNA methylation status and mRNA expression levels were also identified for selected genes (PSD, SMARCC1 and Vps37A) in the GC cell lines. The results suggest that CpG island microarray coupled with ChIP (ChIP-chip) can identify novel targets of gene silencing in GC. Additionally, ChIP-chip is the best approach for assessing the genome-wide status of epigenetic regulation, which may allow for a broader genomic understanding compared to the knowledge that has been accumulated from single-gene studies.

Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells.

The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an “epimutagen combination” that is effective at low concentrations as detected in maternal peripheral and cord blood.

Novel Assay for Ex Vivo Evaluation of Antiproliferative Effect of Hypomethylating Agents 5-Azacytidine and Decitabine in AML Patient Samples

Introduction: Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and aberrant DNA methylation status. One alternative treatment for AML is hypomethylating agents such as 5-Azacytidine (AZA) and Decitabine (DAC), but just around 30% of the patients obtain a clinical response. These agents are slow acting requiring 3-4 cycles to demonstrate some efficacy, for both reasons looking for biomarkers to identify sensitive patients before treatment could be very beneficial to the patients.Objective: Design an ex-vivo model using the automated flow cytometry ExviTech® platform to identify the antiproliferative effect of hypomethylating agents such as 5-Azacytidine and Decitabine in leukemic cells from AML whole bone marrow patient samples. This assay may capture changes of hypomethylating agents effect that may potentially predict ex vivo the patient clinical outcome.Methods: Bone-marrow samples from 10 patients diagnosed of AML were sent to Vivia from 24 hospitals across Spain within 24h. Whole sample were incubated for 72h in in well plates containing 8 concentrations of AZA and DAC. To induce blast proliferation, AML cells were resuspended in RPMI medium and human cytokines including GM-DSF, IL3, SCF, G-CSF, EPO and transferrin. The number of proliferative and no proliferative live leukemic cells was determined using the CFDA dye. Blast population and viability was determined labeling with monoclonal antibodies and Annexin V. Dose-response curves of AZA and DEC were measured in these 10 patient samples.Results: To corroborate the hypothesis that AZA or DAC induce cell cycle arrest and this effect is different from cytotoxicity, we evaluated the effect of DAC in an AML cell line (KG-1) that grow spontaneously with medium and serum. As shown in figure 1, a decrease in the number of cells is observed when the AML cell line (KG-1) is incubated with DAC (blue line). Interestingly, an exactly proportional increase in the Mean Fluorescent Intensity (MFI) of CFDA is observed (red line), the symmetry between these curves indicate they measure the same effect; the drug-concentration dependent decrease in proliferating cells. Green line represents the percentage of apoptosis, as a measurement of the cytotoxic activity, clearly different that the other 2 measurements and only relevant at much higher doses, indicating that DAC has an essentially antiproliferative effect in the cell line. Once tested the hypothesis, we translate the idea to fresh AML samples inducing the blast proliferation with a very complete cytokine medium. In all patients included in this study, an average population of 47.2%±16.9% proliferating cells was identified. A clear difference in the sensitivity of no proliferative vs proliferative cell subset was observed, while DAC had no activity on the no proliferating cells (flat dose response in Figure 2), it was potently (1 uM) inhibiting almost completely proliferation in the proliferating cell subset.Conclusion: Although the mechanism of action of hypomethylating agents is not still entirely clear, we hypothesize that is associated with antiproliferative effects. In this study, using our Exvitech platform we have developed an ex-vivo model to measure the antiproliferative effect of hypomethylating agents, using whole stimulated bone marrow samples from AML patients. If this new assay would correlate with patient treatment response, it could become a valuable tool to personalize hypomethylating treatments for AML patients. [Display omitted] [Display omitted] Figure 1Figure 2 DisclosuresHernandez:Vivia Biotech: Employment. Gomez:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Sanchez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity’s Board of Directors or advisory committees.

The Pleckstrin and Sec7 domain-containing gene as a novel epigenetic modification marker in human gastric cancer and its clinical significance.

The Pleckstrin and Sec7 domain-containing (PSD) gene has been recently found to participate in the progression of several types of cancer. In the present study, we identified PSD as a candidate tumor suppressor gene silenced through epigenetic modification in gastric cancer (GC). PSD mRNA expression and DNA methylation were evaluated by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and methylation-specific PCR in GC cell lines and tissue samples. Involvement of histone modification in GC cell lines was examined by chromatin immunoprecipitation assay. We also used an siRNA-mediated approach to knock down the PSD gene in SGC7901 cells, which was utilized to detect the role of PSD in GC progression, followed by analysis of cell apoptosis and invasion. PSD gene expression was reduced in all GC cell lines compared with GES1 (an immortalized normal gastric cell line). In addition, PSD expression was markedly downregulated in gastric carcinoma tissues when compared to adjacent non-tumor tissues. Our data also indicated that PSD mRNA and protein levels were associated with tumor differentiation and lymph node metastasis. Aberrant DNA methylation status and histone modification were also found in GC cell lines. Enhanced gene expression was detected when the HGC27, AGS and BGC823 GC cell lines were treated with the DNA-demethylating agent 5-aza-2'-deoxycytidine. However, treatment with trichostatin A, a histone deacetylase inhibitor, had no effect on PSD expression in any of the GC cell lines. Suppression of PSD by siRNA led to enhanced SGC7901 cell invasion. The depletion of PSD expression inhibited cell proliferation and decreased apoptosis in SGC7901 cell lines. Knockdown of the PSD expression decreased caspase-3 and -7 protein levels in SGC7901 cells. PSD gene may function as a tumor suppressor in GC suggesting a vital role for DNA methylation and histone modification in PSD silencing. PSD expression might be a useful biomarker for epigenetic-based GC early diagnosis and may lead to the identification of new targets for pharmacological intervention.

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

BackgroundStatus of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue. Materials and methodsIn this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated. ResultThe methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively. ConclusionsThis study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.

Folate Deficiency and Aberrant Expression of DNA Methyltransferase 1 were Associated with Cervical Cancerization

DNA methyltransferase 1 (DNMT1) plays a significant role in maintaining DNA methylation. Aberrant DNA methylation is a recognized feature of human cancers and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also have suggested that folate deficiency was associated with many cancers. The aim of the present study was to evaluate the effect of folate deficiency and aberrant expression of DNA methyltransferase 1 (DNMT1) on cervical cancerization. The expression of DNMT1 protein and mRNA and levels of serum folate were detected in 238 women with a diagnosis of normal cervix (NC，n = 53), cervical intraepithelial neoplasia (CIN I, n = 52; CIN II/III, n = 53), and squamous cell carcinoma of the cervix (SCC; n = 80). In addition, the expression of DNMT1 protein and mRNA was measured in cervical cancer cells (Caski and C33A) treated by different concentration of folate. Serum folate levels decreased and expression levels of DNMT1 protein and mRNA increased gradually with progressive severity of the cervix lesions (P<0.001). It was found that folate was able to reduce the viability of Caski or C33A cell (r=0.978, P=0.002; r=0.984, P<0.001) and regulated aberrant expression of DNMT1 protein (r=-0.859, P=0.01; r=-0.914, P<0.001) and mRNA (r=-0.297, P=0.159; r=0.433, P=0.034) in vitro. Our findings indicated that the low-level of serum folate and high-expression of DNMT1 protein or mRNA was significantly associated with cervical carcinogenesis. Folate deficiency and aberrant expression of DNA methyltransferase 1 had additive effect on cervical cancerization. Folate supplement and recovery of aberrant DNA methylation status may offer a new strategy for prevention and therapy of cancers.

Identification of aberrant DNA methylation profiles in non-tumor liver tissues of patients with non-B, non-C hepatocellular carcinoma.

249 Background: Percentage of patients with non-B non-C (NBNC) hepatocellular carcinoma (HCC) is rapidly increasing in Japan. We have previously shown that miRNA expression profiles in non-tumor liver tissues of patients with NBNC-HCC are different from those of patients with HBV-HCC and HCV-HCC. Moreover, we have recently reported that specific miRNA expression profiles in non-tumor liver tissues can predict a risk of multicentric recurrence after hepatectomy for HCC (Hepatol Res, 2013). In this study, we performed the genome-wide analysis of aberrant DNA methylation in non-tumor liver tissues of patients with NBNC-HCC. Methods: We divided the 23 patients, who underwent hepatectomy, into 3 groups; NBNC (HBcAb-, n=7), NBNC (HBcAb+, n=8), and normal control (NC, n=8). We analyzed DNA methylation status in fresh-frozen non-tumor liver tissues using Infinium Human Methylation 450 Bead Chip (illumina). Gene-expression levels of several identified genes were analyzed by quantitative RT-PCR to validate the correlation between DNA methylation status and gene-expressions. Results: We identified 87 differentially methylated genes in HBcAb- liver tissues, and 603 genes in HBcAb+ liver tissues in comparison to NC liver tissues (Beta value&gt;0.2, P&lt;0.05). Thirty CpG sites were commonly hyper- or hypo-methylated in both HBcAb- and HBcAb+ livers. High percentages of aberrant DNA methylation were observed in gene body. One hypermethylated gene (Lrig1, an intestinal stem cell marker that functions as a tumor suppressor) was confirmed to be down-regulated in its gene-expression level by quantitative RT-PCR. One hypomethylated gene (LEF1, a molecule enhancing hepatocarcinogenesis though Wnt/beta-catenin signaling) was also confirmed to be up-regulated. Ingenuity Pathway Analysisrevealed the association of Wnt/beta-catenin signaling with aberrant DNA methylation status. Conclusions: Genome-wide analysis of aberrant DNA methylation in non-tumor liver tissues may provide not only molecular mechanisms of epigenetic modulation during hepatocarcinogenesis but also early molecular diagnosis in patients with NBNC-HCC.