• Journal of Embryo Transfer
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• v.23 no.3
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• pp.133-139
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• 2008

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5563-5568
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• 2012

Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA down-regulation in gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms could be responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3 was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5'-AzaDC and methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysis was on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulation of ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with 5'-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did not indicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR (MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancer cell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples, 15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulation and methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA from gastric cancer patient's plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylation and methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3773-3778
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• 2013

Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with $H_2O_2$ for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in $H_2O_2$-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to $H_2O_2$. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.5
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• pp.1945-1952
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• 2015

Inflammatory bowel disease (IBD) is a disease strongly associated with colorectal cancer (CRC) as a well-known precancerous condition. Alterations in DNA methylation and mutation in K-ras are believed to play an early etiopathogenic role in CRC and may also an initiating event through deregulation of molecular signaling. Epigenetic silencing of APC and SFRP2 in the WNT signaling pathway may also be involved in IBD-CRC. The role of aberrant DNA methylation in precancerous state of colorectal cancer (CRC) is under intensive investigation worldwide. The aim of this study was to investigate the status of promoter methylation of MGMT-B, APC1A and SFRP2 genes, in inflamed and normal colon tissues of patients with IBD compared with control normal tissues. A total of 52 IBD tissues as well as corresponding normal tissues and 30 samples from healthy participants were obtained. We determined promoter methylation status of MGMT-B, SFRP2 and APC1A genes by chemical treatment with sodium bisulfite and subsequent MSP. The most frequently methylated locus was MGMT-B (71%; 34 of 48), followed by SFRP2 (66.6 %; 32 of 48), and APC1A (43.7%; 21 of 48). Our study demonstrated for the first time that hypermethylation of the MGMT-B and the SFRP2 gene promoter regions might be involved in IBD development. Methylation of MGMT-B and SFRP2 in IBD patients may provide a method for early detection of IBD-associated neoplasia.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.97-102
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• 2007

The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci. of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta. the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.

• Molecules and Cells
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• v.27 no.6
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• pp.635-640
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• 2009

Testis-derived germline stem (GS) cells can undergo reprogramming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.115-120
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• 2017

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.227-233
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• 2007

This study was conducted to examine the mRNA expression of apoptosis-related and imprinted genes and methylation pattern of the differentially methylated region (DMR) of H19 gene in day 35 of SCNT pig fetuses. The day 35 of natural mating (control) or cloned (clone) pig fetuses were recovered from uterus. Endometrium from dam and liver from fetus were obtained, respectively. mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. The Bcl-2 mRNA expression in clone was significantly lower than that of control (p<0.05). The mRNA expression of H19 gene in both endometrium and liver was significantly higher in clone than that of control, respectively (p<0.05). The level of IGF-2 mRNA in liver of clone was significantly lower than that of control (p<0.05), whereas the mRNA expression of IGF2-R gene in liver of clone was significantly higher than that of control (p<0.05). The DMR of H19 was lower methylation pattern in clone than that of control. These results suggest that the aberrant mRNA expression of apoptosis-related and imprinted genes and the lower DMR methylation pattern of imprinted gene may be closely related to the inadequate fetal development of cloned fetus.

• Applied Chemistry for Engineering
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• v.16 no.1
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• pp.86-92
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• 2005

Poly(urethaneethyl acrylate) hybrid emulsions were synthesized to improve their thermomechanical and solvent resistance properties. In the synthesis, dimethylol propionic acid was used to impart hydrophilicity to the hybrid polymers, and ethyl acrylate monomer was added to the polyurethane prepolymer after neutralization with triethylamine. After dispersion of the neutralized prepolymer, chain extension was carried out with ethylene diamine. Consequently, poly(urethaneethyl acrylate) hybrid emulsion was prepared via soap free emulsion polymerization of ethyl acrylate with reduction-oxidation initiator couple of t-butyl hydroperoxide/sodium bisulfite at $50^{\circ}C$. Tehsile strength, 100% modulus, elongation, and solvent-resistance properties of the hybrid emulsion were measured and compared with those of polyurethane homopolymer, poly(ethyl acrylate) homopolymer, and simple blended samples.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10299-10306
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• 2015

The esophageal squamous cell carcinoma (ESCC) is thought to develop through a multi-stage process. Epigenetic gene silencing constitutes an alternative or complementary mechanism to mutational events in tumorigenesis. Posttranscriptional regulation of human leukocyte antigen class I (HLA-I) and antigen processing machinery (APM) proteins expression may be associated with novel epigenetic modifications in cancer development. In the present study, we determined the expression levels of HLA-I antigen and APM components by immunohistochemistry. Then by a bisulfite-sequencing PCR (BSP) approach, we identified target CpG islands methylated at the gene promoter region of APM family genes in a ESCC cell line (ECa109), and further quantitative analysis of CpG site specific methylation of these genes in cases of Kazakh primary ESCCs with corresponding non-cancerous esophageal tissues using the Sequenom MassARRAY platform. Here we showed that the development of ESCCs was accompanied by partial or total loss of protein expression of HLA-B, TAP2, LMP7, tapasin and ERp57. The results demonstrated that although no statistical significance was found of global target CpG fragment methylation level sof HLA-B, TAP2, tapasin and ERp57 genes between ESCC and corresponding non-cancerous esophageal tissues, there was significant differences in the methylation level of several single sites between the two groups. Of thesse only the global methylation level of LMP7 gene target fragments was statistically higher ($0.0517{\pm}0.0357$) in Kazakh esophageal cancer than in neighboring normal tissues ($0.0380{\pm}0.0214$, p<0.05). Our results suggest that multiple CpG sites, but not methylation of every site leads to down regulation or deletion of gene expression. Only some of them result in genetic transcription, and silencing of HLA-B, ERp57, and LMP7 expression through hypermethylation of the promoters or other mechanisms may contribute to mechanisms of tumor escape from immune surveillance in Kazakh esophageal carcinogenesis.