• 대한의생명과학회지
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• 제11권2호
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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.

• 생명과학회지
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• 제27권4호
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• pp.398-407
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• 2017

BTG1 (B-cell translocation gene 1)은 APRO family (anti-proliferative protein family)에 속하며, 이들은 공통의 생물학적 기능은 세포증식을 억제하는 것으로 알려져 있다. 본 연구에서, 굴의 gill cDNA library를 random sequencing을 통한 EST 분석과정에서 BTG1 clone을 확보하였으며, 분자생물학적 특성을 조사하였다. 굴의 BTG1은 182 개의 아미노산으로 구성되며, zebrafish와 57%, human과 56%의 상동성을 나타냈으며, 사람이나 설치류와 달리 ORF (open reading frame) 내에 intron이 존재하지 않았다. Genomic DNA walking을 통해 굴의 BTG1의 predicted promoter를 확인하였으며, 분석결과 AP-1 element와 SRE (serum response element) 부위가 존재하였으며, 5'flanking region에 cAMP response element (CRE) 부위가 확인되었다. 굴의 BTG1의 조직별 유전자발현 수준을 확인하기 위해 real-time PCR을 수행하였으며, 6 개 조직 모두에서 BTG1의 유전자발현이 나타났으며, 그 중에서 heart와 mantle에서 높은 수준의 mRNA 발현을 확인할 수 있었다.

• Journal of Gastric Cancer
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• 제17권4호
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• pp.295-305
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• 2017

Purpose: We previously found that the histone methyltransferase suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3 (SMYD3) is a potential independent predictive factor or prognostic factor for overall survival in gastric cancer patients, but its roles seem to differ from those in other cancers. Therefore, in this study, the detailed functions of SMYD3 in cell proliferation and migration in gastric cancer were examined. Materials and Methods: SMYD3 was overexpressed or suppressed by transfection with an expression plasmid or siRNA, and a wound healing migration assay and Transwell assay were performed to detect the migration and invasion ability of gastric cancer cells. Additionally, an MTT assay and clonogenic assay were performed to evaluate cell proliferation, and a cell cycle analysis was performed by propidium iodide staining. Furthermore, the expression of genes implicated in the ataxia telangiectasia mutated (ATM) pathway and proteins involved in cell cycle regulation were detected by polymerase chain reaction and western blot analyses. Results: Compared with control cells, gastric cancer cells transfected with si-SMYD3 showed lower migration and invasion abilities (P<0.05), and the absence of SMYD3 halted cells in G2/M phase and activated the ATM pathway. Furthermore, the opposite patterns were observed when SMYD3 was elevated in normal gastric cells. Conclusions: To the best of our knowledge, this study provides the first evidence that the absence of SMYD3 could inhibit the migration, invasion, and proliferation of gastric cancer cells and halt cells in G2/M phase via the ATM-CHK2/p53-Cdc25C pathway. These findings indicated that SMYD3 plays crucial roles in the proliferation, migration, and invasion of gastric cancer cells and may be a useful therapeutic target in human gastric carcinomas.

• 미생물학회지
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• 제52권2호
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• pp.157-165
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• 2016

침수된 논토양에서는 메탄생성균이 벼 줄기를 타고 올라오는 메탄을 생성하는 것으로 알려져 있고, 그래서 논토양은 대기 메탄의 인위적인 발생원 중 하나로 알려져 있다. 또한 (분뇨)거름을 사용하면 벼로부터 메탄 배출이 증가하는 것으로 연구 결과 알려져 있다. 어떠한 기작으로 (분뇨)거름이 메탄 배출을 증가시키는지 알아보기 위하여, 무기비료를 사용한 논토양(NPK)과 돈분뇨를 처리한 논토양(PIG)에서의 미생물의 메타게놈에 대해 비교분석을 수행하였다. 미생물군집 분류 분석 결과, 메탄생성균과 메탄영양균, 메틸영양균, 초산생성균(acetogen)이 NPK에서 보다 PIG에서 더 풍부하였다. 더욱이 BLAST 비교 분석 결과 탄수화물 대사 기능유전자가 PIG에 더 풍부하였다. 메탄 대사와 관련된 유전자 중에서 메틸-조효소-M-환원효소(mcrB/mcrD/mcrG)와 트리메틸아민-코리노이드 단백질 Co-메틸전달효소(mttB)가 PIG 시료에 더 풍부하였다. 그와는 상대적으로, 트리메틸아민 모노산소첨가효소(tmm)와 포스포세린/호모세린 인산전달효소(thrH) 같은 메탄 배출을 하향 조절하는 유전자는 NPK 시료에서 더 관찰되었다. 메탄영양과 관련된 유전자(pmoB/amoB/mxaJ)들 또한 PIG에서 더 풍부하게 발견되었다. 메탄 배출과 메탄 산화와 관련된 핵심 유전자들을 환경에서 확인함으로써, (분뇨)거름 사용에 의해 벼로부터 메탄 배출이 증가하는 기작에 대해 기초적인 정보를 얻을 수 있을 것이다. 본 연구에 제시된 내용을 통해 돈분료거름을 처리한 논토양 내 미생물의 분자적 변이를 알 수 있었다.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

• 동의생리병리학회지
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• 제17권4호
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• pp.905-913
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• 2003

Oriental medicine explains aging as the weakening of Kidney-ai, and Kidney-strengthening herbal medicines such as Yukmijihwang-tang have been studied for anti-aging effects. In Western Medicine, the hypothesis that reactive oxidant species(ROS) contribute to the aging process is generally accepted. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective constituent of Yukmijihwang-tang in decreasing ROS production in oxidative-stressed cells. The purpose of this study is to confirm the anti-oxidant effect of MCR on PC12 cells, the expression of Heme oxygenase (HO), Macrophage migradon inhibitory factor (MIF), Catechol-O-methyltransferase (COMT) using real time RT PCR. PC12 cells were treated without or with hydrogen peroxide in the presence or absence of MCR using MTS assay. Hydrogen peroxide decreased the viability of PC12 cells by 53% and MCR did not influence that of stressed PC 12 cells irrespective of dose or incubation period. However, MCR showed an inhibitory effect on production of ROS in stressed cells, both dose and incubation time dependently. In particular, 1 ㎎/㎖ of MCR for 24 h culture almost returned to normal level. In the quantiation of anti-aging related gene expression, MCR at 1 ㎎/㎖ increased the expression of HO by 370%, MIF by 180% and COMT by 280% through real time RT PCR. In conclusion, MCR treatment protected PC12 cells from hydrogen peroxide and decreased ROS production and enhanced anti-oxidative gene expression such as HO, COMT and MIF, which suggests that MCR is involved in controlling anti-aging of nerve cells through elimination of cytotoxic stimuli.

• 생명과학회지
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• 제17권3호통권83호
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• pp.420-426
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• 2007

본 연구에서는 유전자 발현에 중요한 조절 역할을 하는 DNA 메틸화를 식이성 폴리페놀의 하나인 녹차의 대표적인 추출물 EGCG로 억제하였을 때 C2C12 myoblast 세포에 일어나는 현상을 살펴보았다. 그 결과 smooth muscle의 지표인 transgelin, smooth muscle ${\alpha}-actin$ mRNA와 단백질이 현저히 증가함을 보였고, 형태학적으로도 smooth muscle의 전형적인 모습을 보였다. 식이에 포함된 DNA 메틸화 억제제인 EGCG가 C2C12 myoblast를 smooth muscle로 분화를 유도하였으며, 암 예방 차원에서의 EGCG의 역할 외에 혈관질환과 같은 smooth muscle에 관련된 예방과 치료차원에서 EGCG의 역할이 있을 것으로 사료된다. 본 연구는 C2C12 myoblast를 smooth muscle로 유도하는 결정적인 신호전달 역할을 하는 유전자에 대한 연구는 수행하지 못하였다. 따라서 EGCG에 의해 변화되는 유전자에 대한 기전연구가 필요하다고 하겠다.

• Asian Pacific Journal of Cancer Prevention
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• 제17권6호
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• pp.3015-3020
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• 2016

Background: Numerous studies have investigated associations of DNA methyltransferase (DNMT) -149 C>T and -579 G>T polymorphisms with gastric cancer (GC) and colorectal cancer (CRC) susceptibility; however, the findings are inconsistent prompting the present meta-analysis. Materials and Methods: Related studies were identified from PubMed, Google scholar, and SID until 10 October 2015. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. Results: Eleven studies were included based on the search criteria for CRC and GC related to the DNMT3B 149 C>T (3,353 cases and 4,936 controls) and DNMT3B 579 G>T (1,387 cases and 2,064 controls) polymorphisms. There was no significant association overall between DNMT3B -149 and 579 polymorphisms and the risk of cancer. In the stratified analysis by cancer type, DNMT3B 579G>T polymorphism was associated with the risk of CRC and GC. While the DNMT3B -149C/T polymorphism was related with a significantly increased risk of CRC in two tested models, dominant (GG+GT vs. TT: OR 0.51, 95 % CI 0.38-0.69; P = 0.00, Pheterogeneity=0.69, $I^2=0%$) and heterozygote (GT vs. TT: OR 0.50, 95 % CI 0.37-0.69; P=0.00, Pheterogeneity=0.41, $I^2=0%$), no evidence of any association with GC risk was observed as in the pooled analyses. Conclusions: More studies are needed to assess associations of DNMT3B -149C/T and DNMT3B 579G>T polymorphisms with cancer in different ethnicities with large population sizes to generate comprehensive conclusions.

• Molecules and Cells
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• 제44권3호
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• pp.146-159
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• 2021

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

• 대한화장품학회지
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• 제47권2호
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• pp.107-121
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• 2021

백색증이나 백반증에서 관찰되는 피부 저색소침착은 유전적 요인, 후성유전적 요인 및 기타 요인에 의해 멜라닌 합성이 감소할 때 발생한다. 세포에서 멜라닌 합성을 촉진 할 수 있는 약물 후보를 확인하기 위해 141개의 세포 투과성 저분자 약물로 구성된 후성유전적 조절제 라이브러리를 스크리닝했다. B16/F10 쥐 흑색종 세포를 0.1 𝜇M에서 각 약물로 처리하고 멜라닌 합성 및 세포 생존력을 모니터링했다. 그 결과, (-)-네플라노신 A, 3-디아자네플라노신 A (DZNep) 및 DZNep 염산염이 세포 독성을 일으키지 않고 멜라닌 합성을 증가시키는 것으로 나타났다. 이 세 가지 구조적으로 관련된 약물은 세포 멜라닌 합성 및 세포 생존력에 유사한 용량 의존적 효과를 나타내었기 때문에 DZNep을 추가 실험을 위한 대표 약물로 선택하였다. DZNep는 세포내 멜라닌 함량과 티로시나제(TYR) 활성을 증가 시켰다. DZNep은 또한 mRNA와 단백질 수준에서 TYR, 티로시나제 관련 단백질 1 (TYRP1) 및 도파크롬 토토머라제 (DCT)의 발현을 유도했다. DZNep는 또한 멜라닌 합성의 주요 조절자인 소안구증 관련 전사 인자(MITF)의 mRNA와 단백질 발현을 유도했다. DZNep은 S-아데노실 호모시스테인 가수분해효소의 선택적 억제제이며 히스톤 메틸화효소를 저해하는 S-아데노실 호모시스테인의 세포내 축적을 유발하였다. 이 연구는 특정 세포 상황에서 S-아데노실 호모시스테인 가수분해효소를 표적함으로써 멜라닌 생성이 조절될 수 있음을 시사한다.