• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Tuberculosis and Respiratory Diseases
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• v.55 no.4
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• pp.378-387
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• 2003

Background : Promoter methylation of tumor suppressor genes is one of the key epigenetic changes in many human cancers. The aim of this study was to evaluate the promoter methylation status of the Death-associated protein(DAP) kinase gene, which played an important role in lung cancer, in the serum DNA of primary lung cancer patients. Methods : This study investigated the aberrant methylation of DAP kinase in the serum of 65 primary lung cancer patients by methylation-specific PCR (MSP). Results : Methylation in the serum was detected in 29 of 65(44.6%) for DAP kinase. There was no statistical association between methylation of DAP kinase and age, smoking history, histologic type, or stage. Methylation of DAP kinase was found more frequently in men (p=0.044). Conclusions : This study suggests that the aberrant methylation of the DAP kinase promoter is readily detectable in the serum DNA of lung cancer patients using MSP analysis.

• Journal of Chest Surgery
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• v.40 no.2 s.271
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• pp.114-121
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• 2007

Background: Apoptosis plays a crucial role in carcinogenesis, as well as in development and tissue homeostasis. Terminal deoxyribonucleotidyl transferase mediated neck end labelling (TUNEL) and in situ nick end labelling (ISEL) have been used to investigate the apoptosis in tissues. Since the introduction of the M30 monoclonal antibody to overcome drawbacks of TUNEL and ISEL, the apoptosis in various tumors, with the exception of pulmonary carcinomas, has been studied. In this study, attempts were made to examine the correlation of apoptosis in non-small cell carcinomas, using both M30 and the expression of p53 protein, with the clinicopathological factors. Material and Method: Forty five patients with surgically resected non-small cell carcinomas were included. Immunohistochemical staining with M30 and p53 monoclonal antibody were peformed, and their expressions compared with the clinicopathological features. The overall survival time and recurrence-free survival time were calculated, and the factors influencing the survival time analyzed using a univariate analysis. The effects of the expression stati of M30 and p53 on the risks of cancer related to both death and recurrence were evaluated using a multivariate analysis. Result: The p53 positive group had many more M30 positive cells than the p53 negative group (p53 positive group; $61.7{\pm}26.8$ cells vs. p53 negative group; $45.6{\pm}29.6$ cells, p=0.005) and significantly more p53 positive patients showing at least 10 positive cells (apoptotic index, $Al{\ge}1$) on M30 staining (p53 positive group; 52.4% (11/21) vs. p53 negative group 16,7% (4/24), p=0.025). In the univariate analysis, the survival times in relation to smoking (pack-year), performance status (PS) and Al showed significant differences. The multivariate analysis demonstrated the relative risk (R.R) of cancer death increased almost 7.5-fold (R.R 7.482; 95% Cl $1.886{\sim}29.678$; p=0.004) and the risk of recurrence almost 3,8-fold (R.R 3.795; 95% Cl: $1.184{\sim}12.158$; p=0.025) in the high Al (${\ge}1$) compared to the low Al (<1) group. There was no prognostic effect of p53 expression on the survival time or risk of cancer death and recurrence. Conclusion: In non-small cell lung carcinomas, M30 immunohistochemistry was an excellent method for analyzing apoptosis; the high apoptotic index could be an adverse prognostic predictive factor.

• Tuberculosis and Respiratory Diseases
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• v.48 no.2
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• pp.180-190
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• 2000

Background: Genomic instability, which is manifested by the replication error(RER) phenotype, has been proposed for the promotion of genetic alterations necessary for carcinogenesis. Merlo et al. reported frequent microsatellite instability in primary small cell lung cancers. However, Kim et al. found that instability occurred in only 1% of the loci tested and did not resemble the replication error-positive phenotype. The significance of microsatellite instability in the tumorigenesis of small cell lung cancer as well as the relationship between microsatellite instability and its clinical prognosis was investigated in our study. Methods: Fifteen primary small cell lung cancers were chosen for this study. The DNAs extracted from paraffin-embedded tissue blocks with primary tumor and corresponding control tissue were investigated. Forty microsatellite markers on chromosome 1p, 2p, 3p, 5q, 6p, 6q, 9p, 9q, 13q, and 17p were used in the microsatellite analysis. Results: Thirteen(86.7%) of 15 tumors exhibited LOH in at least one of the tested microsatellite markers. Three of 13 tumors exhibiting LOH lost a larger area in chromosome 9p. LOH was shown in 72.7% on chromosome 2p, 40% on 3p, 50% on 5q, 46.7% on 9p, 69.2% on 13q, and 66.7% on 17p(Table 1). Nine(60%) of 15 tumors exhibited shifted bands in at least one of the tested microsatellite markers. Nine cases exhibiting shifted bands showed altered loci ranging 2.5~52.5%(mean $9.4%\pm16.19$)(Table 2). Shifted bands occurred in 5.7% (34 of 600) of the loci tested(Table 2). Nine cases with shifted bands exhibited LOH ranging between 0~83.3%, and the median survival duration of those cases was 35 weeks. Six cases without shifted bands exhibited LOH ranging between 0~83.3%, and the median survival duration of those cases was 73 weeks. There was no significant difference between median survival durations of the two groups(p=0.4712). Conclusion: Microsatellite instability as well as the inactivation of several tumor suppressor genes may play important roles in the development and progression process of tumors. However, the relationship between microsatellite instability and its clinical prognosis in primary small cell lung cancer could not be established.

• Tuberculosis and Respiratory Diseases
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• v.45 no.5
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• pp.962-974
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• 1998

Background: Alteration of p53 tumor suppressor genes is most frequently identified in human neoplasms, including lung carcinoma. It is well known that bcl-2 oncoprotein protects cells from apoptosis. Recent studies have demonstrated that bcl-2 expression is associated with favorable prognosis for patients with non-small cell lung carcinoma. However, the precise biologic role of bcl-2 in the development of these tumors is still obscure. p53 and bcl-2 have important regulatory influence in the apoptotic pathway and thus their relationship is of interest in tumorigenesis, especially lung cancer. Purpose: The author investigated to know the prognostic significance of the expression of p53 and bcl-2 in radically resected non-small cell lung cancer. Method: 84 cases of formalin-fixed paraffin-embedded blocks from resected primary non-small cell lung cancer from 1980 to 1994 at Hanyang University Hospital were available for both clinical follow-up and immunohistochemical staining using monoclonal antibodies for p53 and bcl-2. Results : The histologic classification of the tumor was based on WHO criteria., and the specimens included 45 squamous cell carcinomas(53.6%), 28 adeonocarcinomas(33.3%) and 11 large cell carcinomas(13.1 %). p53 immunoreactivity was noted in 47 cases of 84 cases(56.0%). bcl-2 immunoreactivity was noted in 15 cases of 84 cases(17.9%). The mean survival duration was $64.23{\pm}10.73$ months in bcl-2 positive group and $35.28{\pm}4$. 39 months in bcl-2 negative group. The bcl-2 expression was significantly correlated with survival in radically resected non-small cell lung cancer patients(p=0.03). The mean survival duration was $34.71{\pm}6.12$ months in p53 positive group and $45.35{\pm}6.30$ months in p53 negative group(p=0.21). The p53 expression was not predictive for survival. There was no correlation between combination of the different status of p53 and bcl-2 expression in our study. Conclusions : The interaction and the regulation of new biologic markers, such as those involved in the apoptotic pathway, are complex. bcl-2 overexpression is a good prognostic factor in non-small cell lung cancer and p53 expression is not significantly associated with the prognostic factor in non-small cell lung cancer.

• Tuberculosis and Respiratory Diseases
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• v.44 no.3
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• pp.534-546
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• 1997

Background : The p53 and retinoblastoma(Rb) tumor suppressor genes are associated with the pathogenesis of several types of human cancer. Substantial proportion of the primary lung cancers or cell lines have been reported to have the p53 and/or the Rb gene mutations. But, so far there is no report on the analysis of the Rb gene polymorphism as one of the genetic susceptibility marker. This study was undertaken to establish the gene frequencies of the polymorphic genotypes of the p53 and Rb genes in Koreans to evaluate the possible involvement of these genotypes as a risk factor of lung cancer. Methods : In this study 145 controls without previous and present tumor history and 128 lung cancer patients were subjected to analysis. The two intragenic polymorphisms of the p53 gene(exon 4/ AccII, intron 6/MspI) and one intron 17/XbaI polymorphism of the Rb gene were analysed by the method of polymersae chain reaction- restriction fragment length polymorphisms(PCR-RFLPs). The genotype of the intron 3/16 bp repeat polymorphism of p53 was determined by PCR and direct gel electrophoresis. Results : There were no significant differences in the genotype distributions of the p53 gene between lung cancer patients and controls. But heterozygotes(Arg/Pro) of the exon 4/AccII polymorphisms were slightly over-represented than controls, especially in the Kreyberg type I cancer, which was known to be associated with smoking. The intron 3/16 bp duplication and the intron 6/MspI polymorphisms were in complete linkage disequilibrium. About 95% of the individuals were homozygotes of the common alleles both in the 16 duplication and MspI polymorphisms, and no differences were deteced in the genotype distributions between lung cancer patients and controls. Overall genotype distributions of the Rb gene polymorphisms between lung cancer patients and controls were not significantly different However, the genotype distributions in the Kreyberg type I cancer were significantly different from those of controls(p = 0.0297) or adenocarcinomas(p = 0.0008). It was noticeable that 73.4% of the patients with adenocarcinomas were heterozygotes(r1/r2) whereas 39.2% of the Kreyberg type I cancer were heterozygous at this polymorphisms. In the lung cancer patients, significant differences were also noted between the high dose smokers and low dose smokers including non-smokers(p = 0.0258). The relative risk to Kreyberg type I cancer was significantly reduced in the individuals with the genotype of r1/r2(odds ratio = 0.46, 95% C.I. = 0.25-0.86, p = 0.0124). The combined genotype distribution of the exon 4 AccII of the p53 and the intron 17 Rb gene polymorphisms in Kreyberg type I cancers were significantly different from dose of controls or adenocarcinomas. The highest odds ratio were observed in the individuals with the genotypes of Arg/Pro and r2/r2(odds ratio = 1.97,95% C.I. = 0.84-4.59) and lowest one was in the patients with Arg/Arg, r1/r2 genotype(odds ratio = 0.54, 95% C.I. = 0.25-1.14). Conclusion : The p53 and the Rb gene polymorphisms modulate the risk of smoking induced lung cancer development in Koeans. However, the exact mechanism of risk modulation by these polymorphism remains to be determined. For more discrete clarification of associations between specific genotypes and lung cancer risk, the evaluations of these polymorphisms in other ethnics and more number of patients will be needed.