• YAKHAK HOEJI
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• v.56 no.3
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• pp.198-203
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• 2012

Chrysin and chlorogenic acid are natural products derived from Scutellariae Radix and Lonicerae Flos, respectively. We examined whether chrysin and chlorogenic acid affect airway mucin production induced by TNF-${\alpha}$ in NCI-H292 cells. Cells were pretreated with each agent for 30 min and then stimulated with TNF-${\alpha}$ for 24 h. Of the two compounds, chrysin suppressed airway MUC5AC mucin production. Also, chrysin suppressed MUC5AC mucin gene expression and translocation of NF-${\kappa}B$ p65 induced by TNF-${\alpha}$. This result suggests that chrysin can regulate the production and gene expression of mucin induced by TNF-${\alpha}$ through the inactivation of NF-${\kappa}B$ in airway epithelial cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.2
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• pp.135-140
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• 2008

Loss of E-cadherin (E-cad) expression has been found in multiple cancers and is postulated to facilitate tumor cell dissociation and metastais. Promotor methylation may provides an alternative pathway for loss of gene function. This study evaluated the role of hypermethylation in the down-regulation of E-cad in oral squamous cell carcinoma (OSCC). We examined the E-cad expression by immunohistochemical staining and detected methylation status by methylation-specific polymerase chain reaction (MSP) in 20 OSCC tissues. Overally, 12 (60%) cases of hypermethylation of E-cad were detected and we found there were no correlation between methylation and age, histologic grade, lympn node metastasis, tumor size and clinical stage. However, Eleven (73.3%) of 15 samples which was negative for E-cad staining showed hypermethylation of E-cad promotor region. On the other hand, only one (20%) of 5 E-cad positive sample was observed with methylated status. The underexpression of E-cad was found to be related to promotor hypermethylation (p=0.035). In conclusion, we suggest that hypermethylation play a role in inactivation of E-cad gene and may be a appreciable biomarker for diagnosis and treatment of OSCC.

• Genomics & Informatics
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• v.3 no.4
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• pp.154-158
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• 2005

Germ-line mutations of the BRCA1 gene confer an increased risk for breast and ovarian cancers. BRCA1 in female cells is directly related with the maintenance of the inactive X chromosome (Xi). The effect by the loss of the BRCA1 function on the X chromosome gene expression remains unclear in cancer cells. We attempted to investigate the expression pattern of the X-linked genes by performing BRCA1 knockdown via RNA interference in the MCF7 breast cancer cell line. The transcriptional and translational levels of BRCA1 were decreased over 95% in the MCF7 cells after BRCA1 knockdown. The expression patterns of one hundred ninety X-linked genes were profiled by the X chromosome-specific cDNA arrays. A total of seven percent of the X-linked genes (14/190) were aberrantly expressed by over 2-fold in the MCF7-BRCA1 knockdown cells, which contained two up-regulated genes (2/190, 1 %) and 12 down-regulated genes (12/190, 6.3%). It is interesting that 72% of the aberrantly expressed X-linked genes were located on the Xq (10/14,) region. Our data suggests that BRCA1 may not be important to maintain X chromosome inactivation in cancer because the BRCA1 knockdown did increase the expression of the only one percent of X-linked genes in the human breast cancer cells.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.245-254
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• 2018

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

• Development and Reproduction
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• v.24 no.4
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• pp.249-261
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• 2020

Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.

• Journal of fish pathology
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• v.22 no.3
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• pp.375-382
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• 2009

Integration and expression of a target gene into chromosomal genomes of host cell by retrovirus mediated gene transfer system usually require complicate and laborious procedures. In the present study, we investigate a simple method to integrate a target gene into genome of BF-2 cells using ultraviolet (UV)-inactivated snakehead retrovirus (SnRV), a fish retrovirus. First of all, an optimization of transfection condition was determined with BF-2 cells using Lipofectamine 2000 and Transome. Using 0.5 $\mu\ell$ Lipofectamine 2000 resulted in 33.8, 40.6 and 40.2% of transfection efficacy with high survival rate (minimum 80%) in 0.5, 1 and 2 $\mu{g}$ DNA, respectively, and those of Transome were all less than 5%. It was confirmed that UV-treatment for 5 min was enough to inactivate infectivity of SnRV. Next, a cassette composed of GFP (green fluorescent protein) gene flanked by LTR (long terminal repeats) sequences derived from SnRV was constructed and transfected into BF-2 cells followed by treatment with UV-inactivated SnRV for optimization of integration and expression of the cassette gene. As the results, the fluorescence was expressed in BF-2 cells treated with UV-inactivated SnRV 3 and 5 times, while there was no expression in BF-2 cells with once and non treatment. Accordingly, it was confirmed that GFP gene was integrated into chromosomal genome of BF-2 cells with UV-inactivated SnRV.

• IMMUNE NETWORK
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• v.1 no.2
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• pp.151-161
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• 2001

Background: Inactivation in p53 tumor suppressor gene through a point mutation and deletion is one of the most frequent genetic changes found in human cancer, with 50% of an incidence. This high rate of mutation mostly suggests that the gene plays a central role in the development of cancer and the mutations detected so far were found in exons 5 to 8. Mutation of p53 locus produced accumulation of abnormal p53 protein, and negative regulation of cell proliferation and transcriptional activation as a suppressor of transformation were lost. In addition, inhibition of its normal cellular function of wild-type by mutant is an important step in tumorigenesis. Method: 4 colon cancer cell lines (SNU C1, C2A, C4, C5) were examined for mutation in exons 5 to 8 of the p53 tumor suppressor gene by PCR-SSCP analysis and expression pattern by western blotting and immunoprecipitation. p53-mediated transactivation ability were examined by CAT assay and base substitution of p53 in SNU C2A cell were detected by DNA sequencing. Results: 1) SNU C2A cell and SNU C5 cell were detected mobility shifts each in exon 5 and exon 7 of p53 gene by the PCR-SSCP method, implicating being of p53 mutation. 2) 3 colon cancer cell lines (SNU C1, SNU C2A, SNU C5) expressed wild type and mutant type p53 protein. 3) In northern blot experiment, SNU C2A and SNU C5 cell expressed high level of p53 mRNA. 4) Results of p53-mediated transactivation in colon cancer cell lines by CAT assay represented only SNU C2A cell has transcriptional activity. 5) DNA sequencing in SNU C2A cell showed missense mutation in codon 179 of one allele, histidine to arginine and wild type p53 in the other allele. Conclusion: Colon cancer cell lines showed correlation with mutation in p53 gene and accumulation of abnormal p53 protein. Colon cancer cell SNU C2A retained p53-mediated transactivation as heterozygous p53 with one mutant allele in 179 codon and the other wild-type allele.

• Journal of Korean Neurosurgical Society
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• v.29 no.4
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• pp.471-476
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• 2000

Objective : p16/INK4a, a kind of tumor suppressor genes, encodes a specific inhibitor of the cyclin D-dependent kinases CDK4 and CDK6. This prevents the association of CDK4 with cyclin D1, and subsequently inhibits phosphorylation of retinoblastoma tumor suppressor protein(pRb), thus preventing exit from the G1 phase. According to previous reports, over 50% of glioma tissue and 80% of glioma cell lines have been demonstrated inactivation of p16/INK4a gene. The purpose of this study was to determine whether recombinant adenovirus-p16 virus is a suitable candidate for gene replacement therapy in cases of glioma. Methods : Three human glioma cell lines(U251MG, U87MG and U373MG) that express mutant p16 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p16 cDNA into the cells ; control cells were infected with the Ad-${\beta}$-gal expressing ${\beta}$-galactosidase. To monitor gene transfer and the expression of exogenous genes, we used Western Blotting analysis. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. Results : We showed here that restoration of p16/INK4a expression in p16 negative U87MG, U251MG and partially deleted U373MG by Ad-CMV-p16 induced growth suppression in vitro. Flow cytometric study revealed that Ad-CMV-p16 infected U87MG cells were arrested during the G0-G1 phase of the cell cycle. Expression of p16 transferred by Ad-CMV-p16 in glioma cells was highly efficient and maintained for more than seven days. Conclusions : Our results suggest that Ad-CMV-p16 gene therapy strategy is potentially useful and warrants further clinical investigation for the treatment of gliomas.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.6
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• pp.453-459
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• 2010

Introduction: Ameloblastic carcinoma is a rare malignant lesion, and may arise from either carcinoma ex-ameloblastoma or de novo carcinoma. Aberrant promoter hypermethylation of the tumor-associated genes leading to their inactivation is a common event in many cancer types. The p16/CDKN2/INK4A gene and p16 5 protein are involved directly in regulating the cell cycles. Cadherins are cell adhesion molecules that modulate the epithelial phenotype and regulate tumor invasion. The aim of this study was to evaluate the roles of p16 and E-cadherin methylation and loss of p16 and E-cadherin expression in the malignant transformation of an ameloblastoma. Materials and Methods: Eight cases of ameloblastoma, including 4 benign ameloblastomas without recurrence, 2 benign ameloblastomas with recurrence and 2 carcinoma ex-ameloblastomas, were examined. The promoter hypermethylation profile of the p16 and E-cadherin genes was studied using methylation-specific polymerase chain reaction (MSP) and immunohistochemical staining for p16 and E-cadherin expression. Results: 1) Aberrant CpG island methylation of the p16 gene was detected in 3 of the 4 benign ameloblastomas without recurrence and 1 of the 2 benign ameloblastomas with recurrence. 2) Aberrant CpG island methylation of the E-cadherin gene was found in 1 of the 4 benign ameloblastomas without recurrence. 3) A loss of p16 expression was noted in 1 of 4 benign ameloblastomas without recurrence and 1 of 2 carcinoma ex-ameloblastomas. 4) A loss of E-cadherin expression was noted in 2 of the 4 benign ameloblastomas without recurrence, 1 of the 2 benign ameloblastomas with recurrence and 2 of the 2 carcinoma ex-ameloblastomas. 5) A loss of p16 expression was observed in 1 of the 4 cases showing aberrant methylation of the p16 gene. 6) A loss of E-cadherin expression was observed in 3 benign ameloblastoma case showing aberrant methylation of the E-cadherin gene. Conclusion: These results suggest that loss of E-cadherin expression related to the other genetic pathway (not methylation) might be an adjuvant indicator predicting the malignant transformation of an ameloblastoma. However, the number of samples in this study was too small and the relationship between the treatment methods and clinical course were not defined. Therefore, further study will be needed.

• Journal of Gastric Cancer
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• v.3 no.4
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• pp.186-190
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• 2003

Purpose: The balance between cell proliferation and apoptosis is crucial for homeostatic maintenance in a cell population. Decreased apoptosis or uncontrolled proliferation can lead to cancer. The Fas receptor signal through a cytoplasmic death domain is very important in the apoptotic pathway. To identify the effect of the death domain of the Fas gene in the development and/or progression of gastric cancer, we examined the apoptotic potential of five known Fas mutants detected in gastric cancers. Materials and Methods: A wild-type Fas gene was cloned with cDNA from normal liver tissue and full length Fas was sequenced. Mutants of the gene were generated with sitedirected mutagenesis by using the wild-type gene and specific primers. Wild- and mutant-type genes were transfected to HEK293 cells. Forty-eight hours after transfection the cells were stained with DAPI and cell death was counted under fluorescent microscopy. Results: In wild-type Fas-transfected cells, the percentage of apoptotic cells was $85.9\pm3.6\%$, and significant cell death and classic morphologic signs of apoptosis were observed. However, the percentages of apoptotic cells transfected with N239D, E240G, D244V, and R263H of tumor-derived mutant Fas were $29.5\pm2.08\%,\;28.5\pm3.34\%,\;25.225\pm2.06\%,\;and\;36.625\pm4.49\%$, respectively. Conclusion: These results suggest that inactivation of Fas caused by mutations in the death domain of the Fas gene may be one of the possible escape mechanisms against Fas-mediated apoptosis and that inactivating mutation of the Fas may contribute to the development or progression of gastric cancers.