• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1171-1176
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• 2014

Objective: The tumor suppressor gene, Ras-association domain family (RASSF)2A, is inactivated by promoter hypermethylation in many cancers. The current study was performed to evaluate the methylation status of RASSF2A in epithelial ovarian cancer (EOC) tissues and plasma, and correlations with gene expression and clinicopathologic characteristics. Method: We detected methylation of the RASSF2A gene in tissues and corresponding plasma samples from 47 EOC patients and 14 patients with benign ovarian tumors and 10 with normal ovarian tissues. The methylation status was determined by methylation-specific PCR while gene expression of mRNA was examined by RT-PCR. The EOC cell line, SKOV3, was treated with 5-aza-2'-deoxycytidine (5-azadC). Results: RASSF2A mRNA expression was significantly low in EOC tissues. The frequency of aberrant methylation of RASSF2A was 51.1% in EOC tissues and 36.2% in corresponding plasma samples, whereas such hypermethylation was not detected in the benign ovarial tumors and normal ovarian samples. The expression of RASSF2A mRNA was significantly down-regulated or lost in the methylated group compared to the unmethylated group (p<0.05). After treatment with 5-aza-dC, RASSF2A mRNA expression was significantly restored in the Skov3 cell line. Conclusion: Epigenetic inactivation of RASSF2A through aberrant promoter methylation may play an important role in the pathogenesis of EOC. Methylation of the RASSF2A gene in plasma may be a valuable molecular marker for the early detection of EOC.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2207-2212
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• 2012

Overexpression of DNA methyltransferase 1 (DNMT1) has been detected in many cancers. Tobacco exposure is known to induce genetic and epigenetic changes in the pathogenesis of malignancy. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen present in tobacco smoke; however the detailed molecular mechanism of how NNK induces esophageal carcinogenesis is still unclear. We found that DNMT1 was overexpressed in ESCC tissues compared with paired non-cancerous tissues, the overexpression being correlated with smoking status and low expression of $RAR{\beta}$. The latter could be upregulated by NNK treatment in Het-1A cells, and the increased DNMT1 expression level reflected promoter hypermethylation and downregulation of retinoic acid receptor ${\beta}$($RAR{\beta}$). RNA interference mediated knockdown of DNMT1 resulted in promoter demethylation and upregulation of $RAR{\beta}$ in KYSE30 and TE-1 cells. 3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometric analysis demonstrated that NNK treatment in Het-1A cells could enhance cell proliferation and inhibit cell apoptosis in a dose-dependent manner. In conclusion, DNMT1 overexpression is correlated with smoking status and low expression of $RAR{\beta}$ in esophageal SCC patients. NNK could induce $RAR{\beta}$ promoter hypermethylation through upregulation of DNMT1 in esophageal squamous epithelial cells, finally leading to enhancement of cell proliferation and inhibition of apoptosis.

• International journal of thyroidology
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• v.11 no.2
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• pp.123-129
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• 2018

Background and Objectives: Hypermethylation of the tumor suppressor gene RASSF1A and activating mutation of BRAF gene have been recently reported in thyroid cancers. To investigate the role of these two epigenetic and genetic alterations in thyroid tumor progression, methylation of RASSF1A and BRAF mutation were examined in thyroid tumors. Materials and Methods: During 2007 to 2017, 69 papillary carcinomas, 18 nodular hyperplasia, 3 follicular carcinomas, and 13 follicular adenomas were selected. The methylation-specific polymerase chain reaction (MSP) technique was used in detecting RASSF1A methylation and polymerase chain reaction (PCR)-single-stranded conformation polymorphism and sequencing were used for BRAF gene mutation study. Results: The hypermethylation of the RASSF1A gene was found in 84.6%, 100% and 57.9% of follicular adenomas, follicular carcinomas, and papillary carcinomas, respectively. Nodular hyperplasia showed a hypermethylation in 33.3%. The BRAF mutation at V600E was found in 60.7% of papillary carcinoma and 27.0% of nodular hyperplasia, but none of follicular neoplasms. The BRAF mutation was correlated with the lymph node metastasis and MACIS clinical stage. There is an inverse correlation between RASSF1A methylation and BRAF mutation in thyroid lesions. Conclusion: Epigenetic inactivation of RASSF1A through aberrant methylation is considered to be an early step in thyroid tumorigenesis, and the BRAF mutation plays an important role in the carcinogenesis of papillary carcinoma, providing a genetic marker.

• Journal of Korean Neurosurgical Society
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• v.65 no.1
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• pp.4-12
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• 2022

Objective : We reported the differentially methylated genes in patients with subarachnoid hemorrhage (SAH) using bioinformatics analyses to explore the biological characteristics of the development of delayed cerebral ischemia (DCI). Methods : DNA methylation profiles obtained from 40 SAH patients from an epigenome-wide association study were analyzed. Functional enrichment analysis, protein-protein interaction (PPI) network, and module analyses were carried out. Results : A total of 13 patients (32.5%) experienced DCI during the follow-up. In total, we categorized the genes into the two groups of hypermethylation (n=910) and hypomethylation (n=870). The hypermethylated genes referred to biological processes of organic cyclic compound biosynthesis, nucleobase-containing compound biosynthesis, heterocycle biosynthesis, aromatic compound biosynthesis and cellular nitrogen compound biosynthesis. The hypomethylated genes referred to biological processes of carbohydrate metabolism, the regulation of cell size, and the detection of a stimulus, and molecular functions of amylase activity, and hydrolase activity. Based on PPI network and module analysis, three hypermethylation modules were mainly associated with antigen-processing, Golgi-to-ER retrograde transport, and G alpha (i) signaling events, and two hypomethylation modules were associated with post-translational protein phosphorylation and the regulation of natural killer cell chemotaxis. VHL, KIF3A, KIFAP3, RACGAP1, and OPRM1 were identified as hub genes for hypermethylation, and ALB and IL5 as hub genes for hypomethylation. Conclusion : This study provided novel insights into DCI pathogenesis following SAH. Differently methylated hub genes can be useful biomarkers for the accurate DCI diagnosis.

• Journal of Life Science
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• v.28 no.9
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• pp.1007-1015
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• 2018

The E6-associated protein (E6AP) is known to induce the ubiquitination and proteasomal degradation of HCV core protein and thereby directly impair capsid assembly, resulting in a decline in HCV replication. To counteract this anti-viral host defense system, HCV core protein has evolved a strategy to inhibit E6AP expression via DNA methylation. In the present study, we further explored the mechanism by which HCV core protein inhibits E6AP expression. HCV core protein upregulated both the protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b to inhibit E6AP expression via promoter hypermethylation in HepG2 cells but not in Hep3B cells, which do not express p53. Interestingly, p53 overexpression alone in Hep3B cells was sufficient to activate DNMTs in the absence of HCV core protein and thereby inhibit E6AP expression via promoter hypermethylation. In addition, upregulation of p53 was absolutely required for the HCV core protein to inhibit E6AP expression via promoter hypermethylation, as evidenced by both p53 knockdown and ectopic expression experiments. Accordingly, levels of the ubiquitinated forms of HCV core protein were lower in HepG2 cells than in Hep3B cells. Based on these observations, we conclude that HCV core protein evades ubiquitin-dependent proteasomal degradation in a p53-dependent manner.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4281-4287
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• 2014

Background: Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). Materials and Methods: The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. Results: The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). Conclusions: Results of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

• Journal of Genetic Medicine
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• v.6 no.1
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• pp.1-7
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• 2009

Epigenetic changes represented by promoter CpG island hypermethylation and histone modification are an important carcinogenetic mechanism, which is found in virtually all histologic types of human cancer. About 60-70% of human genes harbor CpG islands in their promoters and 5' exonal sequences, and some of them undergo aberrant promoter CpG island hypermethylation and subsequent downregulation of gene expression. The loss of expression in tumor suppressor or tumor-related genes results in acceleration of tumorigenic processes. In addition to regional CpG island hypermethylation, diffuse genomic hypomethylation represents an important aspect of DNA methylation changes occurring in human cancer cells and contributes to chromosomal instability. These apparently contrasting methylation changes occur not only in human cancer cells, but also in premalignant cells. CpG island hypermethylation has gained attention for not only the tumorigenic mechanistic process, but also its potential utilization as a tumor biomarker. DNA methylation markers are actively investigated for their potential uses as tumor biomarkers for diagnosis of tumors in body fluids, prognostication of cancer patients, or prediction of chemotherapeutic drug response. In this review, these aspects will be discussed in detail.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6261-6265
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• 2013

Background: Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1) is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development of gastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signaling pathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 gene remains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer. Materials and Methods: Using the methylation-specific polymerase chain reaction (MSP), the methylation status of CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40 patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 in gastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was also determined after treatment with a DNA methyltransferase inhibitor, 5-aza-2'-doexycytidine (5-Aza-CdR). RAS activity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstream molecule of RAS signaling pathways, were determined by Western blotting. Results: The frequencies of RASAL1 promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30% (12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation with tumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer (all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 gene was detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell line GES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detected in three gastric cancer cell lines after treatment with 5-Aza-CdR. Conclusions: Aberrant hypermethylation of the RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylating agent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1 significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have a certain relationship with the reduced RASAL1 expression in gastric cancer.

• Biomedical Science Letters
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• v.11 no.2
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• pp.165-173
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• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.

• Proceedings of the Zoological Society Korea Conference
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• 2001.10a
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• pp.263.2-263
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• 2001

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