• 미생물학회지
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• 제38권3호
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• pp.221-229
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• 2002

일본뇌염바이러스(Japanese encephalitis virus, JEV) Nakayama strain을 초기 multiplicity of infection 5.0으로 Vero세포에 감염하여 1년 이상 안정적으로 바이러스를 방출하는 지속감염(persistently-infected)세포주를 확립하였다. 지속감염 세포에서 지속적으로 방출되는 총 11 개의 Plaque 형태 변이바이러스(morphology mutants)클론을 확보하였다. 분리된 변이바이러스의 복제효율을 분석한 결과 생물학적 표현형과 복제효율은 유의하게 상관하였다. 변이바이러스 RNA 게놈 양 말단의 non-coding region 및 envelop 단백질의 ORF에서는 유의한 염기서열 변화가 관찰되지 않아 JEV 약독화에 새로운 인자가 추가로 관여할 가능성을 제시하였다. 변이바이러스에 감염된 신선한 Vero세포는 wild-type JEV의 일반적 감염성상과 다르게 대다수의 세포가 유의할 만한 세포병변현상을 나타내지 않았다. 감염된 Vero세포에서 wild-type JEV 및 large plaque을 형성하는 변이바이러스의 경우 mRNA와 함께 Bcl-2의 발현은 모두 유의하게 감소하였으며 p53은 뚜렷하게 증가하였다. 반면 small plaque을 형성하는 변이바이러스의 감염세포에서는 Bcl-2와 p53 모두 유의한 변화를 볼 수 없었다. 이상의 결과들과 함께, 감염된 Vero세포의 internucleosomal DNA fragmentation과DNA profile의 유형분석에 따르면 궁극적 인 세포병변효과의 변화는 변이바이러스의 복제효율과 더불어 p53에 비의존적인 apoptosis 수위의 전반적인 감소에 기인하는 것으로 판단된다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.657-662
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• 2014

Background: The epidermal growth factor receptor (EGFR) mutation status of lung cancer is important because it means that EGFR-tyrosine kinase inhibitor treatment is indicated. The purpose of this prospective study is to determine whether EGFR mutation status could be identified with reference to preoperative factors. Materials and Methods: One hundred-forty eight patients with lung cancer (111 adenocarcinomas, 25 squamous cell carcinomas and 12 other cell types) were enrolled in this study. The EGFR mutation status of each lung cancer was analyzed postoperatively. Results: There were 58 patients with mutant EGFR lung cancers (mutant LC) and 90 patients with wild-type EGFR lung cancers (wild-type LC). There were significant differences in gender, smoking status, maximum tumor diameter in chest CT, type of tumor shadow, clinical stage between mutant LC and wild-type LC. EGFR mutations were detected only in adenocarcinomas. Maximum standardized uptake value (SUVmax:$3.66{\pm}4.53$) in positron emission tomography-computed tomography of mutant LC was significantly lower than that ($8.26{\pm}6.11$) of wild-type LC (p<0.0001). Concerning type of tumor shadow, the percentage of mutant LC was 85.7% (6/7) in lung cancers with pure ground glass opacity (GGO), 65.3%(32/49) in lung cancers with mixed GGO and 21.7%(20/92) in lung cancers with solid shadow (p<0.0001). For the results of discriminant analysis, type of tumor shadow (p=0.00036) was most significantly associated with mutant EGFR. Tumor histology (p=0.0028), smoking status (p=0.0051) and maximum diameter of tumor shadow in chest CT (p=0.047) were also significantly associated with mutant EGFR. The accuracy for evaluating EGFR mutation status by discriminant analysis was 77.0% (114/148). Conclusions: Mutant EGFR is significantly associated with lung cancer with pure or mixed GGO, adenocarcinoma, never-smoker, smaller tumor diameter in chest CT. Preoperatively, EGFR mutation status can be identified correctly in about 77 % of lung cancers.

• 대한의생명과학회지
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• 제13권2호
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• pp.75-81
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• 2007

Sodium salicylate (NaSal), a chemopreventive drug, has been shown to induce apoptosis and cell circle arrest depending on its concentrations in a variety of cancer cells. In A549 cells, low concentration of NaSal (5$\sim$10 mM) induces cell cycle arrest, whereas it induces apoptosis at higher concentration of 20 mM. In the present study, we examined the molecular mechanism for NaSal-induced cell cycle arrest. NaSal induced expression of p53, p21 (Wafl/Cipl), and p27 (Kipl) that play important roles in cell cycle arrest. p53 induction was mediated by its phosphorylation at Ser-15 that could be prevented by the PI3K-related kinase (ATM, ATR and DNA-PK) inhibitors including wortmannin, caffeine and LY294002. In addition, NaSal-induction of p2l (Wafl/Cipl) was detected in P53 (+/+) wild type A549 cells but not in p53 (-/-) mutant H1299 cells, indicating p53-dependent p21 (Wafl/Cipl) induction. In contrast, p27 (Kipl) that is a negative regulate. of cell cycle with p21 (Wafl/Cipl) was observed both in A549 cells and H1299 cells. Thus, 5 mM NaSal appeared to cause cell cycle arrest through inducing the cyclin-dependent kinase inhibitor p21 (Wafl/Cipl) via PI3K-related protein kinase-dependent p53 activation as well as by up-regulating p27 (Kipl) independently of p53 in A549 cells.

• Journal of Chest Surgery
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• 제34권1호
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• pp.64-71
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• 2001

배경: 최근 치료법의 진보에도 불구하고 진행성 식도암의 예후는 5년 생존율이 10% 이하로매우 불량하기 때문에 식도암에 대한 새로운 치료방법의 하나로 암면역 치료가 대두되고 있다. 암면역 치료를 위해서 MAGE 등 종양 특이항원이 연구의 대상이 되고 있으나 국내에서는 아직 이에 대한 연구가 없다. 대상 및 방법: 1995년 1월부터 1998년 12월까지 고신대학교 복음병원 흉부외과에서 수술 치험한 125례의 식도암중 병리조직 보관상태가 양호한 편평세포암 79례를 병기에 따라(1병기 19례, IIa병기 19례, IIb병기 10례, III병기 21례, IV병기 10례) 무작위로 추출하고 대조군으로 평활근종 20례와 정상 식도점막 20례를 대조군으로 하여 DO7 단클론 항체와 항 MAGE-3 단클론 항체 57B를 이용하여 면역조직화학검사를 시행하여 변형 p53 단백과 MAGE-3 유전자 산물의 발현율을 조사하고 식도암 조직에서 질병의 진행도를 반영하는 병기에 따른 발현율 및 변형 p53 단백과 MAGE-3 유전자 산물의 발현율간의 상관관계를 조사하였다. 결과: 식도암조직에서 변형 p53 단백과 MAGE-3 유전자 산물의 발현율은 각각 51.9%, 60.8%의 발현율을 보였으나 식도평활근종과 정상 식도점막에서는 한례도 발현되지 않아 변형 p53 단백과 MAGE-3 유전자 산물은 대조군에 비해 식도암 조직에서 의미있게 발현되었다(p<0.001). 변형 p53 단백과 MAGE-3 유전자 산물의 발현은 I병기에서 68.4%, 52.6%, IIa병기에서 57.9%, 47.6%, IIb병기에서 60%, 70%, III병기에서 33.3%, 71.4%, IV병기에서 40%, 70% 각각 발현되어 병기에 따른 발현율의 차이는 없었다(p=0.193, p=0.452). 식도암 조직내에서 변형 p53 단백과 MAGE-3 유전자 산물의 발현간에는 상관관계가 없는 것으로 나타났다(p=0.679). 결론: 이상의 결과로 변형 p53 단백과 MAGE-3 유전자 산물의 발현은 식도암에서 예후인자로서의 역할은 할수 없으나 식도 편평세포 암조직에서만 특이하게 높은 빈도로 발현됨으로써 식도암도 면역치료의 대상이 될 수 있음을 확인하였다.

• BMB Reports
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• 제47권4호
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• pp.221-226
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• 2014

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-$Ser^{392}$-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-$Ser^{392}$-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-$Ser^{392}$-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on $Ser^{392}$ presents an alternative for HCC chemotherapy.

• 대한세포병리학회지
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• 제9권1호
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• pp.15-20
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• 1998

Mutant form of the p53 gene product is abnormally accumulated in the nuclei of the tumor cells due to prolonged half life, and readily detected by immunohistochemical methods. To determine the positivity rate of p53 in body cavity fluid according the primary site and histological types of tumors and the utility of p53 immunostaining as an adjunct in the diagnosis of malignancy, we reviewed 69 effusions, including pleural effusion, ascitic fluid, and pericardial fluid, that were diagnosed as overt malignancy and 21 effusions of suspicious malignancy, immunohistochemistry was performed on paraffin-embedded cell blocks using a monoclonal antibody to p53 supressor gene product(Clone DO7) and a standard avidin-biotin complex technique with a citrate buffer antigen retrieval solution. The results were as follows; of the 46 pleural effusions with overt malignancy, 22 were immunopositive for p53 protein; of the 21 ascitic fluids with overt malignancy, 5 were positive for p53. Positivity rates according to the primary sites of tumors were 18 of 34(52.9%), 8 of 21(38.1%), 1 of 9(11.1%) cases of the tumors of the lung, GI tract, and ovary, respectively. According to the histologic types of lung cancer, 11 cases(61.6%) were positive out of 18 adenocarcinomas, 2 of 5 large cell undifferentiated carcinomas, and 1 of 2 small cell undifferentiated carcinomas. Of 21 cases of suspicious malignancy, 6 were positive for p53 and all of them(6/6) were confirmed as adenocarcinoma of the lung or GI tract. These findings indicate that p53 immunostaining using paraffin embedded cell block is useful diagnostic and prognostic marker in body fluid cytology although negative immunostaining does not exclude malignancy.

• Tuberculosis and Respiratory Diseases
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• 제64권4호
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• pp.278-284
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• 2008

연구배경: TRAIL 은 death 수용체(DR4, DR5)와 decoy 수용체(DcR1, DcR2)라는 독특한 수용체 시스템 덕분에 정상세포는 보호하고 종양선택적 살상능을 가진 유망한 항암제로 주목받고 있다. DR5 TRAIL 수용체는 wild-type p53 세포에서 DNA 손상항암제인 doxorubicine에 의해 발현 유도되는 p53-조절 death 수용체 유전자로 처음에 발견되었다. 폐암 조직에서 이러한 DR5와 p53 발현에 관연 연구 보고는 많지 않다. 본 연구에서 비소세포폐암 조직에서 면역조직화학적 방법을 이용하여 DR5 발현과 p53과의 상관관계를 중심으로 분석하였다. 방법: Avidin-biotinylated horseradish peroxidase complex를 이용하여 89예의 수술적 절제된 비소세포폐암 포르말린 고정조직에서 면역조직화학적 염색을 시행하였다. 1차 항체로는 anti-DR5 polyclonal antibody (Pro Sci Inc., Poway, CA)와 anti-p53 monoclonal antibody (DO-7, Novocastra, Inc, Manhasset, NY)를 이용하였다. DR5 발현에 대한 면역조직화학적 판정은 면역염색의 범위와 강도를 합산하여 다음과 같이 판정하였다; 0, non-staining; 1+, weak and diffuse(>50%) or focal (<50%) or moderate and focal; 2+, strong and focal or moderate and diffuse ; 3+, strong and diffuse. 2+ 이상을 고발현(high expression)으로 분류하였다. p53 발현 정도에 따른 p53 wild type과 mutant type의 구분은 3+의 강양성인 경우를 mutant type으로 판정하였고, 나머지 0, 1+, 2+ 발현은 wild type p53으로 판정하였다. 결과: 주변 정상폐조직에서의 DR5발현은 기관지점막에서 89%의 고발현율을 보였으나, type I, II 폐상피세포, 림프구, 평활근세포 등의 정상 세포에서는 거의 발현되지 않았다. 폐암 조직에서 DR5 고발현율은 편평상피암에서 28% (15/53), 선암에서 47% (15/32), 대세포암에서 50% (2/4)의 빈도를 보여 전반적으로 36%의 고발현율을 보였다. DR5 고발현율은 임상병기, 생존기간 등 임상 지표와 유의한 상관관계를 보여주지 못하였지만 p53 발현과는 유의한 역상관관계를 보여주었다. 결론: 비소세포폐암 조직에서 면역조직화학적 분석을 통하여 DR5 발현이 기관지점막을 제외하고는 종양 선택적임을 확인하였고 p53 발현과 유의한 역상관관계가 있음을 확인하였다. 이는 DR5가 p53 조절 유전자임을 in vivo에서 확인한 유력한 증거일 수 있다고 생각된다.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.287-294
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• 2008

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 ${\beta}$-glycosidase (Tca ${\beta}$-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both ${\beta}$-galactosidase and ${\beta}$-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5mM p-nitrophenyl ${\beta}$-D-galactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5mM p-nitrophenyl ${\beta}$-D-glucopyranoside at $75^{\circ}C$. Kinetic analysis with p-nitrophenyl ${\beta}$-D-galactopyranoside revealed that the $k_{cat}$ value of the H119G mutant was 76.3-fold lower than that of the wild type, but the $K_m$ of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency $(k_{cat}/K_m)$ of the mutant decreased to 0.08% to that of the wild type. The $k_{cat}$ value of the H119G mutant for p-nitrophenyl ${\beta}$-D-glucopyranoside was 5.l-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from $90^{\circ}C\;to\;80-85^{\circ}C$). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in ${\beta}$-galactosidase and ${\beta}$-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.

• Toxicological Research
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• 제20권1호
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• pp.71-82
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• 2004

Changes in cell cycle control in the lungs and liver of the B6C3F1 mice (20 males per each group) exposed to ozone (0.5 ppm), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1.0 mg/kg), and dibutyl phthalate (DBP, 5,000 ppm) after 52 weeks were examined through Western, Northern blot, and immunohistochemistry based on alterations in protein expression levels of G1/S checkpoints (cyclin D1, cyclin E, and PCNA), G2/M checkpoints (cyclin B1, cyclin G, and cyclin A), negative regulators (p53, p21, GADD45, and p27), and positive regulator (mdm2). Expression levels of cyclins D1, E, G, PCNA, mutant p53, and mdm2 proteins were higher in the lungs and livers treated with combination of toxicants than in those treated with ozone only. Expression levels of the wild-type and mutant p53, p21, GADD45, p27, and mdm2 proteins and mRNAs were higher in toxicant-treated groups than those of the control. Immunohistochemical analysis revealed staining intensities of the PCNA, cyclin D1, c-myc and mdm2 protein- treated lungs and livers were stronger than those of the control group. Our results showed that combined treatment of ozone with NNK/DBP altered the cell cycle control through instability of the wild-type p53 gene. Such pivotal p53-mediated cell cycle alterations may be responsible for the toxicity observed under our experimental condition. These results may be applied to risk assessment of mixture-induced toxicity.

• 대한두경부종양학회지
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• 제17권2호
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• pp.169-173
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• 2001

Objectives: The p53 tumor suppressor gene encodes a nuclear transcription factor that is critical regulator of cell growth and proliferation through its action in cell-cycle checkpoint control. The wide variety of stressful stmuli which include DNA damage, hypoxia, heat shock, metabolic changes activate the p53 protein, which in turn drives a series of events that culminate either in cell cycle arrest or apoptosis. Mutations of the p53 gene is the most common genetic alteration in human cancer. This gene is altered in approximately 40-60% of head and neck cancers. Whereas the wild-type form of the p53 protein plays a central role in cell-cycle control in response to DNA damage, most of the mutant forms are unable to do so. The high levels of p53 protein expression in tissues are related to the increased cellular proliferative activity and may be associated with the poor clinical outcome. To determine whether the expression of the p53 protein has prognostic significance and is associated with patterns of treatment failure in head and neck squamous cell carcinoma (HNSCC), We analyzed p53 overexpression in 40 cases of HNSCC. Materials and Methods: Immunohistochemical analysis with a monoclonal antibody (DO7) specific for p53 protein was used to detect expression of the protein in formalin-fixed, paraffin-embedded tumor samples from 40 HNSCC. We evaluated p53 protein expression and analyzed the relationship between the p53 overexpression and age, sex, primary tumor site, stage, survival rate, recurrence. All reported P values resulted from two-sided statistical tests. Results: Overexpression of p53 was detected in 20 cases(50%) among 40 cases of HNSCC. The p53 overexpression was not associated with age, sex, primary tumor site, stage, recurrence and survival rate. Conclusions: In our results, p53 was not significant prognostic factor in HNSCC. Based on many previous studies, It is evident that p53 has a certain role in tumorigenesis of HNSCC. So, the further study is needed to evaluate the prognostic significance of p53 in HNSCC.