Methylation Status Of E-cadherin Research Articles

Influence of E-cadherin methylation on prognosis in children with acute lymphoblastic leukemia

To study the significance of E-cadherin and the association between E-cadherin methylation status and prognosis in children with acute lymphoblastic leukemia (ALL) by examining the mRNA and protein expression of E-cadherin and its gene methylation status in bone marrow mononuclear cells of children with ALL. The samples of 5 mL bone marrow blood were collected from 42 children with ALL who were diagnosed for the first time at diagnosis (pre-treatment group) and on day 33 of induction chemotherapy (post-treatment group). RT-qPCR, Western blot, and methylation-specific PCR were used to measure the mRNA and protein expression of E-cadherin and the methylation level of the E-cadherin gene. The changes in each index after induction chemotherapy were compared. The mRNA and protein expression levels of E-cadherin in the post-treatment group were significantly higher than those in the pre-treatment group (P<0.05), while the positive rate of E-cadherin gene methylation in the post-treatment group was significantly lower than that in the pre-treatment group (P<0.05). At the end of the test, the children with negative methylation had significantly higher overall survival rate and event-free survival rate than those with positive methylation (P<0.05). E-cadherin expression is associated with the development of ALL in children, and its decreased expression and increased methylation level may indicate a poor prognosis.

Methylation status of p15 and E-cadherin gene in a cohort of Egyptian acute monocytic leukemia patients

BACKGROUND: Acute monocytic leukemia (AML) is a common hematological malignancy with different subtypes. AML with monocytic involvement (AML M5) is the most frequent subtype among adults. Many genetic and epigenetic abnormalities were described in adult AML. Methylation status in AML is studied for classification, as prognostic marker and even for therapeutic implications. p15 and E-cadherin promotor region methylation are frequently implicated in different kinds of hematological and nonhematological malignancies. AIM OF THE WORK: The aim is to detect methylation status of p15 and E-cadherin in a group of adult Egyptian monocytic leukemia patients and relate it to further events; relapse and central nervous system (CNS) infiltration. Patients and methods: The study was conducted on 76 acute monocytic leukemia patients and 50 healthy controls. PCR specific methylation for p15 and E cadherin was done for both groups. RESULTS: p15 and E-cadherin aberrant methylation are very frequent in adult acute monocytic leukemia. p15 methylation is related to CNS infiltration, while p15 and E-cadherin methylation status is related to relapse. CONCLUSIONS: Methylation of p15 and E-cadherin can be added to prognostic markers of acute monocytic leukemias.

Hypoxia reduces the E-cadherin expression and increases OSCC cell migration regardless of the E-cadherin methylation profile

ObjectiveThe purpose of the current study is to investigate the association between E-cadherin methylation status, hypoxia and OSCC. MethodsHaCat and SCC9 cell lines were submitted to hypoxic treatment, followed by methylation profile analysis (MS-PCR) and analysis of the expression of mRNA gene E-cadherin (RT-PCR). Study group samples comprise individuals affected by potentially malignant lesions Potential Malignant Oral Lesion (PMOL, n=18) and oral squamous cell carcinoma (OSCC, n=28). The control group oral mucosa (OM, n=15) of patients with an oral mucocele. Cell migration ability was evaluated a scratch wound assay in SCC9 and HaCat cell lines ResultsE-cadherin mRNA expression in the cell lines SCC9 and HaCat was significantly reduced under hypoxia, regardless of the methylation profile, when compared to the control group. No differences in methylation profile of the E-cadherin were observed among the groups OM, PMOL and OSCC. HaCat and SCC9 presented increases in cell migration rates under hypoxia. ConclusionThe current study demonstrates that hypoxia reduces E-cadherin expression and increase cell migration, regardless of the methylation profile. Additionally, no differences in E-cadherin methylation patterns were observed among OM, PMOL and OSCC.

Effect of MTHFR C677T Polymorphism on Methylation Status of E-Cadherin in Gastric Cancer

Background: MTHFR gene is one of the main and effective factors in genes methylation. This gene has a common polymorphism in cordon 677, which can affect its activity. Changing activity rate of this gene can affect methylation rate of tumor suppressor genes and cell membrane proteins and other genes in addition to their expression rate. Therefore, it can be considered as one of the effective factors on producing cancer. Methods: In this descriptive-cross sectional study, 34 cancer and non-cancer tissue samples were studied. Initially, DNA was extracted from samples and then E-cadherin and MTHFR c677t polymorphism were amplified by the polymerase chain reaction (PCR). Methylation status of E-cadherin was evaluated by adding methylation restriction enzyme HpaII and polymorphism of MTHFR c677t was assessment by the restriction fragment length polymorphism (RFLP) method. Results: Methylation status of E-cadherin gene numbers of methylated cases in the cancer group were equal to one and unmethylated cases equal to 33 and numbers of unmethylated cases in the non-cancer group was equal to 34 while there were no methylated cases. In assessment of methylation status of E-cadherin and different genotypes of MTHFR in codon number 677 in cancer samples, number of CC, CT and TT genotypes were equal to 0, 1 and 0 in the methylated group and equal to 6, 27 and 1 in the unmethylated group, respectively. Conclusions: According to this observation, another factor such as oncogenes activity may cause cancer in samples but CT genotype of MTHFR can be considered as an effective factor in creating cancer.

Differences in Expression Patterns of DNMTs and TSG Proteins in Lymphoid Tissue Section Play an Important Role in Their Association

In situ, patterns of expression of DNMTs (DNA methytransferases) in normal reactive tonsillar tissue have been examined. Difference in pattering of expression of DNMTs and TSG (Tumor suppressor genes) proteins in lymphoid tissue section is an important question in relation to their association with each other as well as relationship to mRNA gene expression level. In order to examine this issue, we examined DNMTs and TSG proteins expression by immunohistochemistry in sections of paraffin-embedded specimens obtained from 33 subjects of lymphoma and 16 subjects of Non-malignant tissues after receiving written informed consent. The specimens were stained with anti-DNMTs (DNMT1, DNMT3A and DNMT3B) and anti-TSG (E-cadherin, H-cadherin and ADAMTS18) antibodies. In addition, using fresh-frozen optimal cutting temperature (OCT) compound-embedded tissue specimens before any treatment, we examined mRNA expression levels and promoter methylation status of E-cadherin (CDH1), H-cadherin (CDH13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS18) using quantitative real-time PCR (qRT-PCR) and methylation-specific PCR (MSP), respectively. The expression of nuclear DNMTs proteins (DNMT1 and 3A) in lymphoma section was observed in [17/33 (51%); 27/33 (81%)], whereas in non-malignant tissues was [14/16 (87.5%); 13/16 (81%)], respectively. The DNMT3B protein expression was not detected in our tissue samples, which might be explained by the fact that DNMT3B characterized by alternative splicing as shown previously. Membrane proteins (E-cadherin, H-cadherin and ADAMTS18) showed low expression [12/33 (36%); 10/33 (30%); 6/33 (18%), respectively], when compared to non-malignant tissue sections [12/16 (75%); 7/16 (43%); 8/16 (50%), respectively]. The expression levels of CDH1, CDH13 and ADAMTS18 mRNAs were non-significantly reduced in their corresponding protein negative expression compared to the levels in cases with positive protein expression (p =0.112, p =0.378, p =0.077, respectively). We could not find any correlation between mRNA/protein expression levels of DNMTs and the methylation status of CDH1, CDH13 and ADAMTS18. Importantly, by immunostaining especially in non-malignant lymphoid tissues, we found that DNMT1 was highly detected in germinal center B cells (GC B cells) with gradual decrease or no expression in the mantle, marginal, interfollicular and T cells zones. Whereas DNMT3A was preferentially and scattered like expressed in the cells of the surrounding zones out of the germinal centers. Furthermore, E-cadherin, H-cadherin and ADAMTS18 proteins expression were detected on the cell surface membrane of the cells outside the GC but at rates somehow more than those cells inside the GC (Fig. 1). This is supported by the significant association observed between the frequency of DNMT3A with both E-cadherin and ADAMTS18, protein expressions (Chi square: p <0.05), while no association with H-cadherin protein expression. In addition, DNMT1 protein expression did not show significant association with the protein expressions of E-cadherin, H-cadherin and ADAMTS18. Moreover, the mRNA expression levels of DNMT3A and 3B showed high significant levels (p <0.05) in cases with negative protein expressions of both E-cadherin and ADAMTS18 when compared to cases with positive protein expressions (Fig. 2A and C). The DNMT1 mRNA expression level did not show any significant difference between the negative and positive protein expressions of E-cadherin, H-cadherin and ADAMTS18 (Fig. 2B). Furthermore, there was no significant association between the mRNA levels of DNMTs and H-cadherin protein expression. Expression of H-cadherin protein was frequently observed in the endothelial venules and trabeculae of the lymphoid tissues (Fig. 1) which might cause of its lack of association with both DNMT1 and DNMT3A. In conclusion, these results indicate that as a result of differences in pattering of DNMTs and TSG protein expressions detected in lymphoid tissues by immunohistochemistry staining, it might be one of the reasons of the association with each other and their mRNA expression levels across the spectrum of lymphomas and non-malignant lymphoid tissues. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Methylation of Integrin α4 and E-Cadherin Genes in Human Prostate Cancer.

Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

Abstract 389: Epigenetic and proteomic analysis of gastric tumor and its histologically free proximal and distal margins

Abstract Introduction: Gastric cancer (GC) corresponds to the fourth most common malignancy among men and sixth among women in Brazil. GC is a multifactorial disease that results from individual genetic predisposition and exposure to ambient factors such as diet, alcohol consumption, smoking, chronic Helicobacter pylori infection or Epstein-Barr virus (EBV). H. pylori and EBV are known to up-regulate DNA methyltransferases. Aim: Compare the promoter methylation profiles of E-cadherin, p16, DAPK, and Rb genes of histologically tumor free proximal and distal margins from fresh tissues and perform a quantitative proteomic comparison of the tumor, distal and proximal resection margins profiles from the same patient. Methods: 18 samples consisting of six gastric carcinomas, their corresponding proximal margins (PM), and distal margins (DM) were obtained from six patients subjected to gastric resection at the Federal University of Rio de Janeiro, Brazil. DNA was extracted from the fresh tissues by using proteinase K digestion and Phenol-chloroform isoamilic alcohol followed by ethanol precipitation. Methylation-specific PCR analysis was used to determine the methylation status of E-cadherin, p16, DAPK, and Rb genes promoter by bisulfite modification. The presence of EBV was investigated by PCR and a shotgun proteomic analysis of all tissues from the H pilory and EBV negative patient was performed using an Orbitrap Velos. Results: 3 of 6 patients were positive for EBV viz: 3 tumors plus 6 margins. The total methylation for the 4 genes in all 9 samples (p16, E-cadherin, DAPK, and Rb genes) were: 5/36 in PM (esophagus); 5/36 in tumor, and 9/36 for DM (intestinal); in the negative EBV samples: 6/36 in PM; 4/36 in tumor and 8/36 in the DM. The proteomic analysis disclosed 786 proteins identified in the tumor fragment (58 proteins uniquely identified in this tissue), 777 to histologically normal proximal margin (87 unique proteins) and 750 to histologically normal distal margin (132 unique proteins). In all three fragments analyzed, unique proteins related to tumor progression or metastasis were identified; examples are: hepatoma- derived growth (HDGF) in the tumor, Annexin 1A in the PM and Mucin 1 in the DM. Conclusion: Our results show that histologically free tumor margins are molecularly compromised by methylation and by up regulation of proteins correlated to tumor progression and metastasis even in samples not infected with EBV or H.pylori Financial support CNPq Citation Format: Paulo Costa Carvalho, Carlos Eduardo Carvalho, Guilherme Pinto Bravo Neto, Juliana Fischer Carvalho, Thais Mac Cormick, Priscila F. Aquino, Marcelo Mota Silva, Maria da Gloria da Costa Carvalho. Epigenetic and proteomic analysis of gastric tumor and its histologically free proximal and distal margins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 389. doi:10.1158/1538-7445.AM2014-389

MiRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs

IntroductionBreast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations.MethodsUsing a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer.ResultsUnsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with p16INK4 status while only a few miRNAs were associated with BRCA1, PIK3CA/PTEN and TP53 mutation status. Twelve miRNAs were associated with DNA copy number variation of the respective locus.ConclusionLuminal-basal and epithelial-mesenchymal associated miRNAs determine the subdivision of miRNA transcriptome of breast cancer cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16INK4a or E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of these genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.

Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy

E-cadherin has been proven to be widely down-regulated and tightly associated with tumour invasion and metastasis in multiple human cancer types. Recent research demonstrated that aberrant methylation around gene promoter region attributes to E-cadherin silencing. However, the detailed information about this epigenetic inactivation in nasopharyngeal carcinoma (NPC) is rare. The aim of this study was to probe more into the basic mechanism of E-cadherin methylation in NPC and elucidate the application of demethylating agents to restore E-cadherin expression. To address this question, we initially studied E-cadherin methylation status in NPC primary tumours and cell lines by methylation-specific PCR, and compared it with E-cadherin expression. Methylated E-cadherin was detected in 13 of 20 (65%) NPC clinical specimens and 2 of 2 (100%) NPC cell lines (HNE-1 and CNE-2), which was inversely correlated with E-cadherin expression. The detailed methylation profile at individual CpGs within CpG island of E-cadherin promoter region was confirmed by bisulphite sequencing. E-cadherin gene could be demethylated and reactivated in HNE-1 and CNE-2 cells upon treatment with 5-aza-dC, a DNA demethylating agent. Our findings indicate that frequent aberrant methylation of E-cadherin may play an important role in downregulation of E-cadherin, and demethylation therapy could serve as a promising strategy for NPC patients. Furthermore, a high frequency of E-cadherin methylation (9/20, 45%) in peripheral blood of NPC patients suggests its potential clinical application as an early diagnostic or predictive marker.

Promoter methylation of DAPK1, E-cadherin and thrombospondin-1 in de novo and therapy-related myeloid neoplasms

DNA methylation is one of the major epigenetic changes in human cancers, leading to silencing of tumor suppressor genes, with a pathogenetic role in tumor development and progression in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methylation of key promoter regions, induced by cytotoxic therapy together with complex genetic changes, is important in the biology of therapy-related myeloid neoplasms (t-MN). We were interested in the characterization of the methylation pattern of AML and MDS de novo and therapy-related. We studied 385 patients (179 females, 206 males), of a median age of 66 years (range 16-98 years). There were 105 MDS, 208 de novo AML and 72 t-MN (45 MDS and 27 AML). Using a methylation-specific PCR, we studied the promoter methylation status of E-cadherin (CDH1), TSP1 and DAP-Kinase 1. These genes have been shown to be involved in the malignant transformation, interfering with angiogenesis, interaction with micro-environment, apoptosis and xenobiotic detoxification. We found no associations between promoter hypermethylation and gender or age at the time of initial diagnosis. In patients with MDS, there were no associations between hypermethylation and clinical characteristics, including IPSS score, WHO classification and cytogenetics. DAPK1 was more frequently methylated in t-MDS/AML when compared to de novo MDS and AML (39% vs 15.3% and 24.4%, p = 0.0001), while methylation of CDH1 was similar in t-MDS/AML and AML (51% and 53.4%), but less frequent in de novo MDS (29%) (p = 0.003). In the t-MDS/AML group, we found that the methylation pattern appeared to be related to the primary tumor, with DAPK1 more frequently methylated in patients with a previous lymphoproliferative disease (75% vs 32%, p = 0.006). On the other hand, methylation of CDH1 was associated to radiotherapy for the primary malignancy (84.5% vs 38%, p = 0.003). TSP1 hypermethylation was rare and not characteristic of t-MDS/AML. In 177 patients studied for concurrent methylation of several promoters, t-MN and AML de novo were significantly more frequently hypermethylated in 2 or more promoter regions than de novo MDS (20% vs 12.4%, p < 0.001). Chemotherapy and individual genetic predisposition have a role in t-MDS/AML development, the identification of specific epigenetic modifications may explain complexity and genomic instability of these diseases and give the basis for targeted-therapy. The significant association with previous malignancy subtypes may underlie a likely susceptibility to methylation of specific targets and a role for constitutional epimutations as predisposing factors for the development of therapy-related myeloid neoplasm.

Dynamic changes in E-cadherin gene promoter methylation during metastatic progression in papillary thyroid cancer

Thyroid cancer cells undergo epithelial to mesenchymal transition (EMT) in the invasive front of tumor. To determine whether the mesenchymal feature of invasive cancer cells is maintained in metastatic sites, we examined E-cadherin methylation and E-cadherin expression in 66 papillary thyroid cancer (PTC) samples and in 34 corresponding lymph node metastases (LNM). Relationships between E-cadherin and cell motility were evaluated using thyroid cancer cell lines. Hypermethylation of the E-cadherin gene promoter was detected in 39.3% of the PTCs, and loss of E-cadherin expression correlated with lymphocytic infiltration, extrathyroidal invasion and the presence of metastases. Comparing primary PTCs to the corresponding LNM, E-cadherin methylation status was identical in 60% of the cases. The switch in E-cadherin promoter status from unmethylated in PTCs to hypermethylated in LNM was detected in 5%; and from hypermethylatated in PTCs to unmethylated in LNM in 35%. Loss of epigenetic silencing in LNM was associated with a gain of E-cadherin expression. Hypermethylation of the E-cadherin gene promoter was detected in thyroid cancer cell lines with mesenchymal-like morphology. Loss of E-cadherin expression in these cells correlated with high migratory ability. Inhibition of RAS/ERK or PI3K/AKT signaling decreased the migratory ability of these cells but did not induce E-cadherin expression. In the cells with epithelial-like morphology, treatments with phorbol-ester or tumor necrosis factor (TNF)-α resulted in translocation of membranous E-cadherin to the cytoplasm and induction of migration. E-cadherin promoter methylation status and E-cadherin expression were not affected by TNF. Demethylating agents induced apoptosis in the mesenchymal-like cells but had no effect on E-cadherin expression or on migratory ability. Together, dynamic changes in E-cadherin methylation occur during metastatic progression in thyroid cancer. Epigenetic mechanisms and TNF-inducible signaling independently contribute to the regulation of E-cadherin expression and localization. These mechanisms may play a role in the induction of EMT in primary tumors and in the conversion from the mesenchymal to the epithelial phenotype in metastases.

Epigenetic Changes Induced by Reactive Oxygen Species in Hepatocellular Carcinoma: Methylation of the E-cadherin Promoter

In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells. HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses. We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation. These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.

Methylation of tumor associated genes in tissue and plasma samples from liver disease patients

To investigate whether aberrant hypermethylation in plasma DNA could be used as diagnosis makers for hepatocellular carcinoma (HCC), we performed methylation-specific PCR (MSP) to check the methylation status of five tumor associated genes in 36 cases of tissue and 42 cases of plasma samples from HCC and liver cirrhosis patients, respectively. The hypermethylation frequency of GSTP1 and RASSF1A showed significant difference between HCCs and liver cirrhosis with or without HBV infection ( P < 0.05), but differences of the hypermethylation status of APC, E-cadherin, and P16 were not statistically significant. There were no significant differences in the hypermethylation status of five genes between the groups of cirrhosis with and without HBV infection. The significant differences of E-cadherin, GSTP1, P16, and RASSF1A in methylation between HCCs and liver cirrhosis were not observed in the plasma samples. Furthermore, the inconsistent results of MSP and real-time quantitative PCR for the paired samples of tissue and plasma suggested that plasma DNA could not fully stand for tissue DNA. In conclusion, hypermethylation of some specific, but not all, tumor associated genes may be involved in hepatocarcinogenesis; examination of the methylation status of E-cadherin, GSTP1, P16, and RASSF1A in the plasma samples might have limited usage for HCC diagnosis.

An Inverse Correlation between Interleukin-6 and Select Gene Promoter Methylation in Patients with Gastric Cancer

Background: Both serum IL-6 levels and CpG island methylation have been shown to have prognostic significance in gastric cancer, it was suggested that an important link existed between IL-6 and methylation of cancers. Aim: To investigate the prognostic value of IL-6 serum level and the association between serum IL-6 levels and CpG island methylation at p16, DAPK, MGMT and E-cadherin in patients with gastric cancer. Patients and Methods: Methylation status was assessed by MSP in 75 surgical specimens of gastric adenocarcinoma. IL-6 serum levels were measured by chemiluminescent enzyme immunoassay (CLEIA). Results: Methylation of p16, DAPK, MGMT, and E-cadherin were present in 53, 48, 32, and 59% of patients. Patients with tumors methylated at p16 and DAPK had lower serum levels of IL-6 compared to unmethylated tumors (1.8 vs. 4.8 pg/ml, p = 0.01 for p16; 1.5 vs. 6.2 pg/ml, p = 0.0001 for DAPK). But there was no difference with MGMT and E-cadherin methylation status. Serum IL-6 levels were also associated with TNM stage (p = 0.001), depth of tumor invasion (p = 0.002), lymphatic invasion (p = 0.01), vascular invasion (p = 0.008), metastasis (p = 0.002) and signet cell histology (p = 0.001). Conclusion: IL-6 is of prognostic value for patients of gastric cancer. Low serum IL-6 levels were associated with p16 or DAPK gene methylation in patients with gastric cancer.

Stability of E‐cadherin methylation status in gastric mucosa associated with histology changes

We have previously shown reversal of E-cadherin methylation in gastric mucosa from patients with dyspepsia at week 6 after Helicobacter pylori-eradication therapy. But the long-term methylation status of these patients was unknown. To investigate the methylation status at E-cadherin at year 3 after H. pylori-eradication therapy. 35 patients (25 with and 10 without H. pylori eradicated) enrolled in our previous study were recruited into the present study (year 3 analysis). Methylation at E-cadherin was evaluated by methylation-specific polymerase chain reaction method. There was no difference in age and sex distribution in the two groups. Methylation at E-cadherin in patients with H. pylori eradicated at weeks 0, 6 and year 3 were 52%, 20% and 20%, respectively. Concordant methylation status at week 6 and year 3 was 92%. Methylation at E-cadherin in patients without H. pylori at weeks 0, 6 and year 3 were 50%, 60% and 60%, respectively. Concordant methylation status between week 6 and year 3 was 90%. Stability of E-cadherin methylation status was associated with histological changes. No association between E-cadherin methylation status and age was observed. The methylation pattern is stable for a long period, thus suggesting the effect of environment on methylation.

E-cadherin and DAP kinase in pancreatic adenocarcinoma and corresponding lymph node metastases

E-cadherin and DAP kinase have been implicated as 'invasion suppressor' genes in human cancer. The aim of this study was to analyze the methylation status of E-cadherin and DAP kinase and the expression of the protein in the metastatic lesions and to compare it with the expression in the primary tumor. Methylation-specific PCR of the DAP kinase and E-cadherin promoter was performed in 28 primary adenocarcinomas of the pancreas and in 13 corresponding regional lymph node metastases. The presence of E-cadherin and DAP kinase protein was assessed by immunohistochemistry. Metastatic lymph nodes showed a significant different expression profile from the primary tumor. E-cadherin methylation was observed in 8/28 (29%) and loss of protein expression was observed in 16/28 (57%) of pancreatic carcinomas. E-cadherin methylation was observed in 7/13 (54%) and loss of protein expression was observed in 11/13 (85%) lymph node metastases (p=0.047). DAP kinase methylation occurred in 11/28 (39%) pancreatic carcinomas and loss of protein expression was observed in 13/28 (46%). DAP kinase was methylated in 6/13 (46%) lymph node meta-stases and loss of protein expression was observed in 10/13 (77%) (p=0.039). Comparing primary tumor and corresponding lymph node metastases in 13 cases, the status of E-cadherin methylation was discordant in 2 cases. The protein expression pattern of E-cadherin and DAP kinase was discordant in 4 and 3 cases respectively. Unmethylated tumor samples did not express E-cadherin in 12 and DAP kinase protein in 6 cases. Our results demonstrate that reduction of E-cadherin and DAP kinase expression is more frequent in lymph node metastases than in the primary tumor and methylation of the promoter region contributes to this reduction; however, an alternative mechanism of inactivation seems to exist.

DNA methyltransferase 1 expression and promoter methylation of E‐cadherin in mucoepidermoid carcinoma

Loss of E-cadherin expression is found frequently in many types of human malignancies, including mucoepidermoid carcinoma (MEC). CpG methylation in the promoter region has proven important in the regulation of gene expression implicated in malignant transformation. DNA methyltransferases (DNMTs) are the major enzymes involved in establishing genomic methylation patterns. The current study was designed to test the hypothesis that CpG methylation of the promoter region of the E-cadherin gene may inactivate its expression and to examine DNMT 1 (DNMT1) protein expression in MEC. Genomic DNA was obtained from paraffin embedded sections by laser microdissection in 46 MEC specimens. Methylation status of the E-cadherin promoter was examined by utilizing the methylation-specific polymerase chain reaction assay. To examine E-cadherin and DNMT1 proteins expression levels, the MEC specimens and adjacent epithelial tissues were studied immunohistochemically. Chi-square analysis was used to evaluate the correlation of protein expression and E-cadherin methylation status with clinicopathologic parameters. Comparisons of the survival rate between patients with DNMT1-positive and DNMT1-negative patients were made using Kaplan-Meier analysis. The data showed that all normal tissues expressed E-cadherin, and no promoter methylation was detected. Of the MEC samples analyzed, methylation allele was found in 33 of 46 samples (72%), and reduced E-cadherin expression was found in 21 of 46 samples (45%). DNMT1-positive expression was observed in 29 of 46 MEC samples (63%). A significant correlation was found between E-cadherin expression and the methylation status of E-cadherin promoter (P = 0.021). In addition, increased DNMT1 expression was correlated with histologic grade, clinical stage, and a poor prognosis in patients with MEC. Hypermethylation of CpG sites at the 5' promoter of E-cadherin was a common event associated with E-cadherin expression levels in MEC, suggesting an epigenetically mediated loss of E-cadherin function in these tumors. Increased DNMT1 protein expression may play a critical role in the carcinogenesis and disease progression of MEC.

Promoter Methylation of E-cadherin in Hepatocellular Carcinomas and Dysplastic Nodules

In order to clarify the significance of E-cadherin methylation in multistep hepatocarcinogenesis, we examined the methylation status of the E-cadherin promoter region, using methylation-specific polymerase chain reaction in 64 hepatocellular carcinomas (HCCs) and 13 dysplastic nodules (DNs), and correlated these results with E-cadherin protein expression and clinicopathologic factors of HCCs. Promoter methylation was detected in 1 of 13 (7.7%) DNs, in 5 of 13 (38.5%) Edmondson and Steiner grade I HCCs, and in 27 of 51 (52.9%) grade II or III HCCs, and a significant correlation was observed between the methylation status and the stepwise progression of hepatocarcinogenesis (p=0.004). Reduced E-cadherin immunoreactivity was found in 18 of 64 (28%) HCCs, but in none of DNs. E-cadherin methylation status in HCCs was significantly correlated with microvascular invasion (p=0.02) and tumor recurrence (p=0.04), but not with reduced E-cadherin immunoreactivity. The Kaplan-Meier method showed that methylation status did not have a significant influence on the recurrence-free survival of HCC patients (p=0.15). Our results indicate that methylation of the E-cadherin promoter region is a frequent event in HCC, which may play an important role in the stepwise progression of hepatocarcinogenesis. And the promoter methylation of E-cadherin in HCC was found to be significantly correlated with microvascular invasion and recurrence.

Tumor suppressor gene hypermethylation as a predictor of gastric stromal tumor behavior.

The growing understanding of the epigenetic changes associated with cancer, including aberrant promoter methylation of tumor suppressor genes that afford selective growth advantages to human neoplasms, suggests that the characterization of gene methylation patterns among gastrointestinal stromal tumors (GISTs) may be useful for predicting tumor behavior. Thirty-eight c-kit-positive gastric stromal tumors were subjected to methylation-specific polymerase chain reaction (MSP) to detect promoter methylation associated with 11 candidate tumor suppressor genes (p16/INK4a, APC, MGMT, hMLH1, p73, E-cadherin, RAR-beta, RASSF1A, RB, ER, and DAPK), established to have a role in tumorigenesis of several solid human organs. Aberrant methylation of any of the 11 candidate tumor suppressor genes was detected in 84% of all GISTs. In decreasing order of frequency, the six most commonly methylated genes were: MGMT (47%), p16 (45%), RASSF1A (40%), E-cadherin (37%), hMLH1 (34%), and APC (31%). For all of the GISTs, promoter methylation was less reliable than tumor mitotic rate in predicting 5-year tumor-free survival for the GISTs; however, E-cadherin methylation was a multivariate prognostic factor for early recurrence of GISTs (50% at 2 years; P=0.030). Among the mitotically active (>5 per 50 high-power field), histologically indistinguishable GISTs, E-cadherin methylation was an independent predictor of tumor-related mortality: 5-year disease-free survival was worse for the E-cadherin methylated GISTs (19%) compared to the E-cadherin unmethylated tumors (71%; P=0.010). Detection of methylation within selected genes may afford a reliable and accurate molecular marker system for predicting neoplastic behavior among GISTs. This study supports the methylation status of E-cadherin as a prognostic marker for early GIST recurrence and survival.

Multipoint methylation analysis indicates a distinctive epigenetic phenotype among testicular germ cell tumors and testicular malignant lymphomas.

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. Although a growing number of genes showing hypermethylation is being reported in human cancer, methylation profiles of tumor-related genes in testicular neoplasms have not been well elucidated. This study was designed to show the methylation profiles of multiple CpG islands in testicular germ cell tumors (TGCTs) in comparison with those in testicular malignant lymphomas. We studied the methylation status of E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 by use of TGCT tissues and testicular malignant lymphoma tissues (25 primary TGCT tissues and three primary testicular lymphoma tissues). Methylation was not observed in E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 in any of the TGCT tissues. In contrast, all three (100%) of the testicular lymphoma tissues demonstrated hypermethylation of E-cadherin, RASSF1A, and RARB, but not CDKN2B, CDKN2A, BRCA1, RB1, VHL, and GSTP1. These data demonstrate that a distinctive epigenetic phenotype underlies the TGCTs and testicular lymphomas at the CpG sites of E-cadherin, RASSF1A, and RARB; a distinctive epigenetic phenotype was not observed among seminomatous TGCTs and non-seminomatous TGCTs at the CpG sites examined.