• Molecules and Cells
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• 제21권2호
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• pp.206-212
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• 2006

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and $p16^{INK4a}$ functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and $p16^{INK4a}$ pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

• Tuberculosis and Respiratory Diseases
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• 제72권2호
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• pp.132-139
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• 2012

Background: Periostin is preferentially expressed in periosteum, indicating a potential role in bone formation. Recently, there have been emerging controversies about its role in invasion and metastasis of human malignancies. We attempted to determine the clinicopathological significance of periostin expression in non-small cell lung carcinoma (NSCLC). Methods: Immunohistochemical staining of periostin protein from 91 cases of NSCLCs was performed using tissue microarray blocks. The results were correlated with clinicopathological parameters. Results: Positive reaction to periostin was predominantly noted in the tumor stroma. The strongest reaction presented as a band-like pattern just around the tumor nests. Non-neoplastic lung tissue and most in-situ carcinomas did not show a positive reaction in their stroma. With respect to tumor differentiation, moderate to poor differentiated tumors (47/77) revealed even higher periostin expression than the well-differentiated ones (4/14) (p=0.024). High periostin expression was positively correlated with E-cadherin and p53 expression, but was not related with patient age, sex, tumor type, PCNA index, b-catenin, cyclin D1, pTNM-T, pTNM-N, stage, and patient survival (p>0.05). Conclusion: These results suggest that periostin might play a role during the biological progression of NSCLC, but may not be related to the clinical prognostic parameters.

• Molecules and Cells
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• 제37권11호
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• pp.812-818
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• 2014

This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1.

• Tuberculosis and Respiratory Diseases
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• 제56권2호
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• pp.187-197
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• 2004

연구배경 : Nitric Oxide(NO)는 매우 다양한 생물학적 조절기능을 수행하는 분자로서 심혈관계, 신경계, 면역기능 등에 관여함은 물론 최근 세포 사에도 직 간접적으로 영향을 미치고 있음이 알려져 있다. NO의 이렇게 복잡한 생물학적 기능 수행은 reactive oxygen species(ROS), metal ions 및 단백질 등과 복잡한 상호작용에 의한 것이며 NO가 나타내는 생물학적 효과는 용량-의존적이며, 세포-특이적이라고 밝혀져 있다. NO는 간세포 및 현관내피세포에서는 아포프토시스를 억제하지만 종양세포 및 신경세포 등에서는 아포프토시스를 유도하는 것으로 보고되고 있다. NO는 여러 호흡기질환의 병태생리에도 관여하는 것으로 알려져 있는바 천식과 같은 염증성 기도 질환에서 호기 NO가 증가되어있는 반면 흡연자나 일차성 폐 고혈압 환자에서는 감소되어 있다고 보고되고 있다. 이러한 배경에서 NO가 폐 상피 세포의 세포 사에 미치는 영향과 신호전달 경로를 밝히기 위하여 본 연구를 시행하였다. 방 법 : 폐 상피 세포로는 A549 세포 주를, NO donor로서는 SNAP (S-nitroso-N-acetyl-penicillamine)과 SNP(sodium nitroprusside)를 사용하였다. 세포 독성 검사는 crystal violet assay를 이용하였고 아포프토시스 assay는 Hoechst 33342와 propium iodide(PI) 이중 염색 후 형광현미경을 이용하여 핵의 형태학적변화를 관찰함으로써 괴사(necrosis)와 감별하였다. 철에 의한 NO 유도성 세포 사 억제 효과를 관찰하기 위하여 RBC와 FeSO4를 이용하였다. NO 유도성세포사의 신호전달 경로에 bcl-2와 p53이 미치는 영향을 평가하기 위하여 bcl-2 과 발현 세포 주 (A549-bcl-2)와 p53 knock out 세포 주 (A549-E6)를 대상으로 세포독성을 비교하였고 p53 활성화는 Western blot을 이용하여 확인하였다. 결 과 : A549 세포 주에서 SNAP과 SNP 모두 농도-의존적 세포독성을 관찰할 수 있었다. 아포프토시스 assay에서 SNAP은 저 농도에서는 아포프토시스를, 고농도에서는 괴사를 유도함을 관찰하였고 SNP는 농도에 상관없이 세포사가 괴사의 형태를 나타냄을 확인하였다. 이는 SNP가 순순한 NO donor가 아니라 cyanide에 의한 세포독성의 결과라고 생각되며 고농도의 SNAP에 의한 괴사 유도는 peroxynitrite 생성에 의한 결과임을 시사한다. SNAP에 의한 세포 사는 RBC와 FeSO4등 철에 의해 억제됨을 확인하였고 bcl-2에 의해서 억제되었으며 p53을 활성화시키고 p53 knock out에 의해 차단되었다. 결 론 : 폐상피세포에서 NO는 저 농도에서는 아포프토시스를 고농도에서는 괴사에 의한 세포 사를 유도하며 철이 중요한 억제제이며 bcl-2 및 p53이 신호전달 경로에 있어서 중요한 역할을 담당하는 것으로 생각된다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권5호
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• pp.2743-2746
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• 2013

Background: Dilantin sodium (phenytoin) is an antiepileptic drug, which is routinely used to control generalized tonic clonic seizure and partial seizure episodes. A few case reports of oral squamous cell carcinomas arising from regions of phenytoin induced gingival overgrowth (GO), and overexpression of mitogenic factors and p53 have presented this condition as a pathology with potential to transform into malignancy. We recently investigated the genetic status of p53 and H-ras, which are known to be frequently mutated in Indian oral carcinomas in GO tissues and found them to only contain wild type sequences, which suggested a non-neoplastic nature of phenytoin induced GO. However, besides p53 and H-ras, other oncogenes and tumor suppressors such as PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$, are frequently altered in oral squamous cell carcinoma, and hence are required to be analyzed in phenytoin induced GO tissues to be affirmative of its non-neoplastic nature. Methods: 100ng of chromosomal DNA isolated from twenty gingival overgrowth tissues were amplified with primers for exons 9 and 20 of PIK3CA, exons $1{\alpha}$, $1{\beta}$ and 2 of p16INK4a and p14ARF, and exon 2 of $p21^{Waf1/Cip1}$, in independent reactions. PCR amplicons were subsequently gel purified and eluted products were sequenced. Results: Sequencing analysis of the twenty samples of phenytoin induced gingival growth showed no mutations in the analyzed exons of PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$. Conclusion: The present data indicate that the mutational alterations of genes, PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$ that are frequently mutated in oral squamous cell carcinomas are rare in phenytoin induced gingival growth. Thus the findings provide further evidence that phenytoin induced gingival overgrowth as a non-neoplastic lesion, which may be considered as clinically significant given the fact that the epileptic patients are routinely administered with phenytoin for the rest of their lives to control seizure episodes.

• 한국미생물·생명공학회지
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• 제38권1호
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• pp.40-45
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• 2010

Agarose의 $\beta$-1,4결함을 분해하는 Zobellia galactanivorans 유래의 $\beta$-agarase 유전자(agaB)는 클로닝 되었고, AOX1(alcohol oxidase 1, methanol inducible) promoter 하류에 Saccharomyces cerevisiae mating factor alpha-1 secretion signal($MF{\alpha}1$)를 연결하여 $MF{\alpha}1$-AgaB를 구축하였다. 구축된 plasmid pPIC-AgaB(9 kb)를 Pichia pastoris genome에 HIS4 gene 위치에 integration하였고, colony PCR을 통해 확인하였다. Methanol 첨가 배지에서 자란 형질전환체는 iodine solution의 첨가에 의해 red halos를 보였으며, P.pastoris에서 agaB의 효율적 분비 발현을 확인하였다. SDS-PAGE와 zymographic analysis에서 $\beta$-agarase의 분자량은 약 53 kDa으로 추정되었으며, 15% 정도의 N-linked glycosylation이 일어났음을 알 수 있었다. P.pastoris GS115/pPIC-AgaB의 48시간 baffled flask culture에서 세포외 $\beta$-agarase의 활성은 각각 0.1, 0.5, 1% methanol의 유도에 의해 1.34, 1.42 그리고 1.53 units/mL의 활성을 보였다. 대부분의 $\beta$-agarase의 활성은 세포 외에서 관찰되었고, 분비효율은 98%였으며 분비시의 glycosylation에 의해 열안정성도 증가되었다.

• Journal of Chest Surgery
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• 제39권11호
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• pp.828-837
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• 2006

배경: 악성종양에서 신생혈관 생성 및 당분해의 증가는 저산소 상태의 미세환경을 나타내며, 이는 종양의 침습성, 전이 및 환자의 예후와 관련이 있는 것으로 알려져 있다. Hypoxia-inducible factor 1(HIF-1)는 당원 수송체, 당분해 효소, 혈관내피세포 성장인자 등의 유전자의 전사를 활성화한다고 알려져 있다. 그리고 HIF-1의 전사 활성도는 HIF-$1{\alpha}$ 아단위의 표현이 조절되는 정도에 의존한다. 비소세포 폐암에서 HIF-$1{\alpha}$의 발현이 혈관 생성능, 종양세포 증식능 및 이상형 p53의 축적 등 종양의 생물학적 특성에 미치는 영향과 환자의 수술 후 예후와의 관계를 규명하고자 한다. 대상 및 방법: 1997년부터 1999년까지 비소세포 폐암으로 진단받고 전폐절제술 혹은 폐엽절제술을 시행 받은 59명의 폐암 환자들에서 얻어진 파라핀 조직 블록을 대상으로 하였다. ABC(avidin-biotin complex) 방법에 기초한 면역조직화학검사를 이용하여 암조직과 정상조직에서 HIF-$1{\alpha}$, VEGF(vascular endothelial growth factor), p53 단백의 발현을 조사하고, Ki-67의 발현을 이용한 증식지수를 측정하였다. HIF-$1{\alpha}$ 발현과 환자의 생존기간을 포함한 임상적-병리학적 변수들과의 상관관계, VEGF, p53의 발현과 증식지수와의 상관관계를 분석하였다. 결과: HIF-$1{\alpha}$의 과발현은 40.7%(24예/59예)였다. HIF-$1{\alpha}$의 과발현은 병리학적 TNM병기(p=0.004), T병기(p=0.020), N병기(p=0.004), 림프관/혈관 침범(p=0.019) 등과 관련이 있었다. 또 혈관내피세포 성장인자의 발현(p<0.001) 및 이상형의 p53의 발현(p=0.040)과 관련성이 있었다. Kaplan-Meier 생존분석에서 HIF-$1{\alpha}$의 과발현이 있는 환자의 5년 생존울은 22%로 HIF-$1{\alpha}$의 저발현 환자의 5년 생존율 61%에 비해 불량한 생존율을 보였고, 단변량분석과 다변량분석에서 HIF-$1{\alpha}$의 발현은 불량한 예후를 나타내는 인자로 관찰되었다. 결론: 이상의 결과로 비소세포 폐암 환자에서 HIF-$1{\alpha}$의 과발현은 종양내 신생혈관의 생성과 림프절 전이와 관련이 있는 표지자로 여겨지며, 수술 후 불량한 예후를 나타내었다.

• Journal of Genetic Medicine
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• 제3권1호
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• pp.33-37
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• 1999

Recombinant adenovirus vector systems with strong promoters have been used to achieve high level production of recombinant protein. However, this overexpression system cause some problems such as disturbance of cell physiology and increment of cellular toxicity. Here, we showed a tetracycline-regulated adenovirus expression vector system. Our results showed that the expression level of transgene(p-53) was high and easily regulated by tetracycline. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system was higher than that of the wild type CMV promoter system. Therefore, tetracycline-regulated adenoviral vector system could be applicable for regulatory high-level expression of toxic gene. Also, this system will be useful for functional studies and gene therapy.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제26권4호
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• pp.355-362
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• 2000

Neoplastic growth is characterized by alterations of oncogenes and antioncogenes. The interaction between activated oncogenes and functional deletion of antioncogene appears to be the driving force directing normal cells to uncontrolled growth resulting in tumor. In addition to those genes mentioned, other genes controlling the entry of cells into the cell cycle have recently been implicated in cancer development. The overexpression of the cyclin D1 gene, which has been mapped to 11q13, either by gene rearrangement or amplification has been noted in various malignant tumors. The product of the cyclin D1 gene forms a complex with cyclin-dependent protein kinases(CDK4) that governs a key transition in the cell cycle. The relationships between the overexpression of cyclin D1 assessed by immunihistochemistry and the amplification of the cyclin D1 gene by differential polymerase chain reaction(DPCR) using primers for dopamin D2 receptor gene in 13 cases of squamous cell carcinomas of the oral cavity have been studied. The semiquantitative assay of cyclin D1 amplification has been made by cyclin D1/dopamin D2 receptor(CD/DR) ratio. The results were as follows; 1. In the normal tissue and the tumor, the CD/DR ratios were 0.82 and 1.36 respectively. This implicates 1.65-fold amplification of cyclin D1 gene in tumor compared to that in normal tissue. 2. The tumor tissue which showed overexpression of cyclin D1 by immunohistochemistry revealed 2-fold amplification of cyclin D1 compared to the normal tissue. 3. The tumor tissue which showed mild expression of cyclin D1 by immunihistochemistry revealed 1.7-fold amplification of cyclin D compared to the normal tissue. 4. The cyclin D1 was overexpressed in the tumor tissue at the rate of 38%. Above results suggest that cyclin D1 has close correlation with the development of carcinoma in the oral cavity. But further studies were needed to elucidate the carcinogeneic mechanisms by comparative studies among cyclin D1, pRb and p53.

• Journal of Trauma and Injury
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• 제21권1호
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• pp.59-65
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• 2008

Purpose: The aim of this study was to evaluate the effect of overexpression of heat shock protein 70 (HSP70) on the expression of inducible nitric oxide synthase and on the concentration of nitric oxide and to determine the mechanism for the relationship between HSP70 and inducible nitric oxide synthase (iNOS) in sepsis. Methods: Experiments were performed on male Sprague-Dawley rats, and sepsis was induced by using cecal ligation and puncture (CLP). Glutamine (GLN) or saline was administered 1 h after initiation of sepsis. We acquired serum and lung tissues from the rats 12 h or 24 h after initiation of sepsis. We analyzed the concentration of nitric oxide, the expression of HSP70 in the lung, and the gene expression of iNOS in the lung. Results: In CLP+GLN, glutamine given after initiation of sepsis enhanced the expression of HSP70 in the lung at 12 h (CLP+GLN vs. CLP:: $47.19{\pm}10.04$ vs. $33.22{\pm}8.28$, p = 0.025) and 24 h (CLP+GLN vs. CLP: $47.06{\pm}10.60$ vs. $31.90{\pm}4.83$, p = 0.004). In CLP+GLN, glutamine attenuated the expression of iNOS mRNA in the lung at 12 h (CLP+GLN vs. CLP: $4167.17{\pm}951.59$ vs. $5513.73{\pm}1051.60$, p = 0.025) and 24 h (CLP+GLN vs. CLP: $9,437.65{\pm}2,521.07$ vs. $18,740.27{\pm}8,241.20$, p = 0.016) and reduced the concentration of nitric oxide in serum at 12 h (CLP+GLN vs. CLP: $0.86{\pm}0.48$ vs. $3.82{\pm}2.53{\mu}mol/L$, p = 0.016) and 24 h (CLP+GLN vs. CLP: $0.39{\pm}0.25$ vs. $1.85{\pm}1.70{\mu}mol/L$, p = 0.025). Conclusion: The overexpression of HSP70 induced by the administration of glutamine in sepsis attenuated the gene expression of iNOS and reduced the concentration of nitric oxide.