• Korean Journal of Microbiology
• /
• v.38 no.3
• /
• pp.221-229
• /
• 2002

Japanese encephalitis virus (JEV) persistence was established and maintained in Vero cell culture for over 1 year. Eleven clones of plaque morphology mutant JEV, with large and small plaque sizes, were obtained from the cell culture supernatant. Genomic RNA replication efficiency of the mutants in naive Vero cell appeared to correspond to their different plaque sizes. No significant changes in envelop protein ORF or in non-coding regions at both ends of the RNA genome suggested that there could be an unidentified factor(s) playing role in JEV attenuation. Unlike to the replication of wild-type JEV, the mutants did not induce severe degree of cytopathic effect in Vero cells upon infection. While obvious decrease of Bcl-2 and its mRNA expression and sharp increase of p53 in naive Vero cells infected with either wild-type JEV or the large plaque-forming mutant, those changes were not observed with the small plaque-forming one. Together with these observation, internucleosomal DNA fragmentation and chromosomal DNA profile in the Vero cells infected with the mutants suggest that an overall changes in cytopathic effect in the plaque morphology mutants-infected cells should be primarily due to the reduced genomic RNA replication and the compromised degree of p53-independent apoptosis by the virus infection at least in part.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.2
• /
• pp.657-662
• /
• 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.

• Biomedical Science Letters
• /
• v.13 no.2
• /
• pp.75-81
• /
• 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
• /
• v.34 no.1
• /
• pp.64-71
• /
• 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
• /
• v.47 no.4
• /
• pp.221-226
• /
• 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.

• The Korean Journal of Cytopathology
• /
• v.9 no.1
• /
• pp.15-20
• /
• 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
• /
• v.64 no.4
• /
• pp.278-284
• /
• 2008

Background: TRAIL is a promising anticancer agent which induces selective tumor cell death due to a unique receptor system that includes death receptors and decoy receptors. DR5 TRAIL receptor is an originally identified p53-regulated death receptor gene that was induced, by doxorubicine, only in cells with a wild-type p53 status. We investigated that focused on the correlation between the DR5 and p53 expressions in non-small cell lung cancer (NSCLC). Methods: Immunohistochemical analysis, with using avidin-biotinylated horseradish peroxidase complex, was carried out in 89 surgically resected NSCLC formalin-fixed paraffin-embedded tissue sections. As primary antibodies, we used anti-DR5 polyclonal antibody and anti-p53 monoclonal antibody. A negative control was processed with each slide. The positive tumor cells were quantified twice and these values were expressed as percentage of the total number of tumor cells, and the intensity of immunostaining was expressed. The analysis of the DR5 expression was done separately in tumor area and in a nearby region of normal tissue. Results: The DR5 expression was high in the bronchial epithelium (89% of cases) but this was almost absent in type I & II pneumocytes, lymphocytes and smooth muscle cells. High DR5 expression rate in tumor was seen in 28% (15/53) of squamous cell carcinomas, in 47% (15/32) of adenocarcinomas and, in 50% (2/4) of large cell carcinomas. The DR5 expression did not show any statistical significance relationship with the T stage, N stage, or survival. However, the DR5 expression showed significant inverse correlation with the p53 expression. (p< 0.01). Conclusion: We demonstrated that the DR5 expression in NSCLC via immunohistochemical analysis is relatively tumor-specific except for that in the normal bronchial epithelium and it is significantly dependent on the p53 status. This might be in vivo evidence for the significance of the DR5 gene as a p53 downstream gene.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.2
• /
• pp.287-294
• /
• 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
• /
• v.20 no.1
• /
• pp.71-82
• /
• 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.

• Korean Journal of Head & Neck Oncology
• /
• v.17 no.2
• /
• pp.169-173
• /
• 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.