• 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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 1998.07a
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• pp.50-51
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• 1998

guanidinoacetate methyltransferase (GAMT) catalyzes the last step of creatine biosynthesis in mammals. Creatine plays an important role in cellular energy metabolism in variety of tissues including brain and male reproductive tract. Congenital deficiency of the enzyme leads to a neurologic disorder in humans. We used an interspecific backcross DNA panel to map Gamt to the central region of mouse Chromosome (Chr) 10 near the locus of hesitant mutation affecting male fertility. We assigned the human GAMT gene to Chr 19 by PCR analysis of a human/rodent somatic hybrid cell line DNA panel, and further localized the human gene to Chr 19 at band p13.3 by PCR analysis of a human radiation hybrid DNA panel. Human chr 19p13.3 is homologous to the central part of mouse Chr 10 where mouse Gamt is located. Furthermore, this part of mouse Chr 10 contains mutant loci the phenotype of which is similar to the GAMT deficiency in human.

• Tuberculosis and Respiratory Diseases
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• v.70 no.3
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• pp.206-217
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• 2011

Background: Transcription factor FOXP3 characterizes the thymically derived regulatory T cells. FOXP3 is expressed by cancer cell itself and FOXP3 expression was induced by TGF-${\beta}$ treatment in pancreatic cancer cell line. However, the expression of FOXP3 expression is not well known in patients with lung cancer. This study was conducted to investigate the expression of FOXP3 in patients with lung cancer and to investigate the regulation of FOXP3 expression by the treatment of TGF-${\beta}$ and DNA methyltransferase inhibitor in lung cancer cell lines. Methods: FOXP3 expression in the tissue of patients with resected non-small cell lung cancer (NSCLC) was evaluated by immunohistochemistry. The regulation of FOXP3 expression was investigated by Western blot and RT-PCR after lung cancer cell lines were stimulated with TGF-${\beta}1$ and TGF-${\beta}2$. The regulation of FOXP3 expression was also investigated by RT-PCR and flow cytometry after lung cancer cell lines were treated with DNA methyltransferase inhibitor (5-AZA-dC). Results: FOXP3 expression was confirmed in 27% of patients with NSCLC. In NCI-H460 cell line, TGF-${\beta}2$ decreased FOXP3 mRNA and protein expressions. In A549 cell line, both TGF-${\beta}1$ and TGF-${\beta}2$ decreased FOXP3 mRNA and protein expressions. 5-AZA-dC increased FOXP3 mRNA expression in NCI-H460 and A549 cell lines. Moreover, 5-AZA-dC increased intracellular FOXP3 protein expression in A549 cell lines. Conclusion: It was shown that FOXP3 is expressed by cancer cell itself in patients with NSCLC. Treatment of TGF-${\beta}2$ and DNA methyltransferase inhibitor seems to be associated with the regulation of FOXP3 expression in lung cancer cell lines.

• BMB Reports
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• v.54 no.8
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• pp.413-418
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• 2021

ErbB3-binding protein 1 (EBP1) is a multifunctional protein associated with neural development. Loss of Ebp1 leads to upregulation of the gene silencing unit suppressor of variegation 3-9 homolog 1 (Suv39H1)/DNA (cytosine 5)-methyltransferase (DNMT1). EBP1 directly binds to the promoter region of DNMT1, repressing DNA methylation, and hence, promoting neural development. In the current study, we showed that EBP1 suppresses histone methyltransferase activity of Suv39H1 by promoting ubiquitin-proteasome system (UPS)-dependent degradation of Suv39H1. In addition, we showed that EBP1 directly interacts with Suv39H1, and this interaction is required for recruiting the E3 ligase MDM2 for Suv39H1 degradation. Thus, our findings suggest that EBP1 regulates UPS-dependent degradation of Suv39H1 to govern proper heterochromatin assembly during neural development.

• Molecules and Cells
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• v.19 no.1
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• pp.16-22
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• 2005

A cDNA encoding ${\gamma}-tocopherol$ methyltransferase (${\gamma}-TMT$) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce ($T_0$) containing the ${\gamma}-TMT$ transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and ${\gamma}-TMT$ activities. The ratio of ${\alpha}-/{\gamma}-tocopherol$ content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the $T_0$ plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 $T_1$ progenies of the $T_0$ transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and ${\gamma}-TMT$ activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in $T_2$ progenies of $T_1$ plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis ${\gamma}-TMT$ transgene in lettuce results in a higher enzyme activity and the conversion of the ${\gamma}-tocopherol$ pool to ${\alpha}-tocopherol$ in transgenic lettuce.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3213-3222
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• 2016

Background: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyl-donor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. Materials and Methods: The human cervical cancer cell line, C4-II, was grown in complete medium and medium depleted of folate (F-M+) and folate and methionine (F-M-). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT-PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. Results: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant down-regulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. Conclusions: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

• Journal of Applied Biological Chemistry
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• v.44 no.3
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• pp.119-124
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• 2001

NTR1 gene of Brassica campestris L. ssp. perkinensis encodes a floral nectary-specific methyltransferase. In this study, the NTR1 cDNA was expressed in E. coli to examine the enzymatic characteristics of the protein product. The GST-NTR1 fusion protein was purified to near homogeneity, showing that the size of NTR1 was 44 kDa. The protein reacted specifically with jasmonic acid (JA), consuming methyl group from S-adenosyl-L-methionine (SAM). GC-MS analysis revealed that the compound produced was authentic methyl jasmonate (MeJA), suggesting that NTR1 is an S-adenosyl-L-methionine: jasmonic acid carboxyl methyltransferase. Km values of NTR1 for JA and SAM were 38.0 and $6.4{\mu}M$, respectively. Optimal activity of the NTR1 was observed at $20^{\circ}C$, pH 7.5, in the presence of 100-150 mM KCl. Thus, kinetic properties, thermal characteristics, optimal pH, and ion-dependency of the NTR1 activity were almost identical to those of Arabidopsis JA methyltransferase JMT, indicating that these two proteins are orthologues of each other.

• The Journal of the Korean bone and joint tumor society
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• v.15 no.1
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• pp.13-25
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• 2009

Purpose: The DNA repair protein, $O^6$-methylguanine-DNA methyltransferase (MGMT), removes alkyl adducts from the $O^6$ position of guanine. Epigenetic inactivation of MGMT has been found in human neoplasia and considered one of the implicated factors in chemoresistance. Materials and Methods: Sixty-two patiensts with soft tissue sarcomas (STS) were analyzed for the status of MGMT protein expression by immunohistochemistry and the promoter hypermethylation of the MGMT gene using methylation-specific PCR. Result: The loss of MGMT expression was found in 20 cases (32.3%) of total 62 STS. MGMT promoter hypermethylation rate was 25.0% (11/44 cases). The loss of MGMT expression showed significant association with high AJCC stage, high FNCLCC grade, and aggressive behavior. However,when the group who received chemotherapy was analyzed (n=27), loss of MGMT expression was correlated with worse survival in multivariate analysis (p=0.024). MGMT promoter hypermethylation is associated with high FNCLCC grade. MGMT promoter hypermethylation status had a strong correlation with loss of MGMT expression (p=0.000). Conclusion: Our results suggest that MGMT promoter hypermethylation and loss of MGMT expression had a tendency to be associated with poor prognosis and that loss of MGMT protein expression is frequently occurs via MGMT promoter hypermethylation.

• Molecules and Cells
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• v.42 no.1
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• pp.67-78
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• 2019

Methylation of HBV cccDNA has been detected in vivo and in vitro; however, the mechanism and its effects on HBV replication remain unclear. HBx derived from a 1.2-mer HBV replicon upregulated protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), 3a, and 3b, resulting in methylation of the negative regulatory region (NRE) in cccDNA, while none of these effects were observed with an HBx-null mutant. The HBx-positive HBV cccDNA expressed higher levels of HBc and produced about 4-fold higher levels of HBV particles than those from the HBx-null counterpart. For these effects, HBx interrupted the action of NRE binding protein via methylation of the C-1619 within NRE, resulting in activation of the core promoter. Treatment with 5-Aza-2′dC or DNMT1 knock-down drastically impaired the ability of HBx to activate the core promoter and stimulate HBV replication in 1.2-mer HBV replicon and in vitro infection systems, indicating the positive role of HBx-mediated cccDNA methylation in HBV replication.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5713-5718
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• 2013

Background: DNA methyltransferase-3a (DNMT3a) plays significant roles in embryogenesis and the generation of aberrant methylation in carcinogenesis. This study aimed to investigate associations between single nucleotide polymorphisms (SNPs) of the DNMT3a gene and risk of Helicobacter pylori infection, gastric atrophy and gastric cancer. Methods: The subjects comprised 447 patients with gastric cancer; 111 individuals with gastric atrophy and 961 healthy controls. Two SNPs (rs1550117 and rs13420827) of the DNMT3a gene were genotyped by Taqman assay. DNMT3a expression was analyzed in cancer tissues from 89 patients by tissue microarray technique. Odds ratio (ORs) and 95% confidence intervals were calculated by multivariate logistic regression. Results: Among healthy controls, risk of H.pylori infection was significantly higher in subjects with the rs1550117 AA genotype, compared to those with GG/AG genotypes of DNMT3a [OR=2.08, (95%CI: 1.02-4.32)]. However, no significant correlation was found between the two SNPs and risk of developing gastric atrophy or gastric cancer. In addition, no increase in DNMT3a expression was observed in the gastric cancer with H.pylori infection. Conclusions: This study revealed that DNMT3a rs1550117 polymorphism is significantly associated with an increased risk of H. pylori infection, but did not support any evidence for contributions of DNMT3a rs1550117 and rs13420827 to either gastric atrophy or gastric cancer. The biological roles of DNMT3a polymorphisms require further investigation.