사람 백혈구 및 위 조직중의 8-Hydroxy-2'-Deoxyguanosine 측정에 관한 연구

강호일(Hoil Kang),엄미옥(Mi Ok Eom),박미선(Misun Park),염태경(Tai Kyung Ryeom),지승완(Seung Wan Jee),전해명(Hea Myung Jeon),김옥희(Ok Hee Kim) 한국환경성돌연변이발암원학회 2005 한국환경성돌연변이·발암원학회지 Vol.25 No.1

In the present study, we have measured 8-hydroxy-2'-deoxyguanosine in DNA of stomach cancers, adjacent stomach cancer tissues, normal stomach tissues and peripheral blood leukocytes of the same stomach cancer patients (n=48) to investigate their etiological association with gastric cancer and possibility whether peripheral blood leukocytes can use surrogate marker for early stomach cancer diagnosis by HPLC/ECD system. In normal stomach tissues, we found that 8-hydroxy-2'-deoxyguanosine levels in tissues infected with Helicobacter pylori were 1.4 fold higher than those in tissues without infected with Helicobacter pylori. However, in adjacent stomach cancer tissues, we found that 8-hydroxy-2'-deoxyguanosine levels in tissues infected with Helicobacter pylori were 1.5 fold lower than those in tissues without infected with Helicobacter pylori. In stomach cancer tissues, 8-hydroxy-2'-deoxyguanosine levels in tissues infected with Helicobacter pylori were not significantly different from those in tissues without infected with Helicobacter pylori. In Helicobacter pylori-negative specimens, 8-hydroxy-2'-deoxyguanosine levels of adjacent stomach cancer tissues were found to be significantly higher than those of normal stomach and cancer tissues. The 8-hydroxy-2'-deoxygnanosine levels of female were 1.7 fold higher than those of male in peripheral blood leukocytes of the same stomach cancer patients. The 8-hydroxy-2'-deoxyguanosine levels in Helicobacter pylori-negative specimens among adjacent stomach cancer tissues were found to be reversely correlated with those in peripheral blood leukocytes, suggesting that 8-hydroxy-2'-deoxyguanosine in peripheral blood leukocytes may not use as surrogate marker for the early diagnosis of human stomach cancer.

Nicotine 및 Tobacco-Specific Nitrosamine이 발암과정에 미치는 영향

강호일(Hoil Kang),박미선(Mi Sun Park),김옥희(Ok Hee Kim) 한국환경성돌연변이발암원학회 2005 한국환경성돌연변이·발암원학회지 Vol.25 No.3

Nicotine has been implicated as a potential factor in the pathogenesis of human lung cancer, however its mechanism of action in the development of lung cancer remains largely unknown. To explore the role of nicotine in the development of lung cancer, we first investigated the effects of nicotine on the expression of tumor associated genes by treating Sprague-Dawley rats with nicotine (10 ㎎/㎏) by gavage once daily for 10 days. We determined the expression of proteins and mRNAs of the ras, raf, myc, jun, fos oncogenes and p53, Rb tumor suppressor genes by Western and Northern blotting, respectively. We did not detect any changes on the levels of proteins and mRNAs of these tumor associated genes in the lung of Sprague-Dawley rats from 3 days to 12 weeks after the last treatment of nicotine, indicating that nicotine appears to have no effect on expression of these oncogenes and tumor suppressor genes at an early stage in multistage chemical carcinogenesis. In a second experiment, we investigated the possibility that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) could be formed endogenously by treating with nicotine and sodium nitrite. We treated groups of Fischer 344 rats with nicotine (60 μmol/㎏) and sodium nitrite (180 μmol/㎏), nicotine, sodium nitrite and NNK (120 nmol/㎏) alone by gavage once daily for 7 days, respectively and determined the 8-hydroxydeoxyguanosine (8-OHdG), as an indicator of NNK formation, in the lungs of rats 24 hours and 48 hours after the last treatment by HPLC/ECD method. We detect increased level of 8-OHdG in the lungs of rats treated with NNK, but in the case of nicotine plus sodium nitrite, nicotine and sodium nitrite alone we could not detected any changes of 8-OHdG, respectively.

마우스피부암 발생과정에 있어서 텔로머레이저 활성에 관한 연구

강호일(Hoil Kang),지승완(Sung Wan Jee),김옥희(Ok Hee Kim) 한국환경성돌연변이발암원학회 2005 한국환경성돌연변이·발암원학회지 Vol.25 No.2

Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, has activity in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. Although telomerase is known to be a regulated enzyme, the factors and mechanisms involved in telomerase regulation are not well understood. In the present study, we compared the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and non-phorbol ester tumor promoters such as okadaic acid, anthralin and benzoyl peroxide on the expression of telomerase in the mouse skin carcinogenesis system, a well characterized model for studying pre-malignant and malignant progression. We found that most early papillomas harvested after 10 weeks of TPA promotion showed telomerase activity. Other papillomas harvested after 10 weeks of okadaic acid, anthralin and benzoyl peroxide promotion and after single treatment of DMBA only also showed telomerase activity, respectively. On the other hand, normal and all skins surrounded by papillomas harvested after 10 weeks of these promoters has no telomerase activity. Taken together these results, there appears to be no clear association between the level of telomerase activity and protein phosphorylation in mouse skin papillomas and telomerase may be useful as bio-markers in early detection of tumors.

사람의 정상 피부세포 및 폐세포의 발암에 미치는 2,3,7,8-tetrachlorodibenzo-ρ -dioxin의 영향

강미경(Mi Kyung Kang),염태경(Tai Kyung Ryeom),김강련(Kang Ryune Kim),김옥희(Ok Hee Kim),강호일(Hoil Kang) 한국환경성돌연변이발암원학회 2006 한국환경성돌연변이·발암원학회지 Vol.26 No.3

2,3,7,8-Tetrachlorodibenzo-ρ-dioxin (TCDD) displays high toxicity in animals and has been implicated in human carcinogenesis. Although TCDD is recognized as potent carcinogens, relatively little is known about their role in the tumor promotion and carcinogenesis. It is known that TCDD can increase of cancer risk from various types of tissue by a mechanism possibly involving the aryl hydrocarbon receptor (AhR) activation. In this study, effects of TCDD on cellular proliferation of normal human skin and lung fibroblasts, Detroit551 and WI38 cells were investigated. In addition, to enhance our understanding of TCDD-mediated carcinogenesis, we have investigated process in which expression of Erk1/2, cyclinD1, oncogene such as Ha-ras and c-myc, and their cognate signaling pathway. TCDD that are potent activators of AhR-mediated activity was found to induce significant increase of cytochrome P4501A1 mRNA expression, suggesting a presence of functional AhR. These results support that CYP1A1 enzyme may be involved in the generation of TCDD-induced toxicity. Moreover mitogen-activated protein kinases (MAPKs) phosphorylation and cyclin D1 overexpression are induced by TCDD, which corresponded with the progression of cellular proliferation. However, TCDD did not affected Haras and c-myc mRNA expression. Taken together, it seems that TCDD are could be a part of cellular proliferation in non-tumorigenic normal human cells such as Detroit551 and WI38 cells through the upregulation of MAPKs signaling pathway regulating growth of cell population. Therefore, AhR-activating TCDD could potentially contribute to tumor promotion and Detroit551 and WI38 cells have been used as a detection system of tumorigenic effects of TCDD.

에스트로겐 수용체를 통한 카드뮴 독성 및 항산화제에 의한 독성경감에 관한 연구

김태성(Tae Sung Kim),강태석(Tae Seok Kang),강호일(Hoil Kang),문현주(Hyun Ju Moon),강일현(Il Hyun Kang),이영주(Young Joo Lee),최은희(Eun Hee Choi),홍진태(Jin Tae Hong),한순영(Soon Young Han),홍진환(Jin Hwan Hong) 한국환경성돌연변이발암원학회 2006 한국환경성돌연변이·발암원학회지 Vol.26 No.1

Cadmium, a human carcinogen, can induce toxicity in various cell lines and organs. Despite extensive research, the mechanisms of cadmium-induced cell toxicity and estrogenic potential in human are not clear. This study was performed to investigate cadmium-induced toxicity on human breast cancer cells :MCF-7 cells, an estrogen receptor (ER) positive breast cancer cells, and MDA-MB-231 cells, an ER negative breast cancer cells. MCF-7 cells was proved to be more sensitive than the other cell lines (IC50 = 50 μM at MCF-7 cells and 120 μM at MDA-MB-231). The expression of JNK and AP-1 transcription factors such as c-Jun and c-Fos dependent transcription were increased by cadmium treatment. Inhibition of ER activation by ER antagonist (tamoxifen or ICI 182,780) significantly recovered the viablity and inhibited apoptotic cell death. This suggested that cadmium-induced cell death in ER (+) cells was mediated by JNK/AP-1 pathway and this pathway was more stimulated by ER activated by cadmium. Co-treatment of antioxidants such as selenium (Se), butylated hydroxyanisole (BHA), glutathione (GSH), or N-acetyl-L-cysteine (NAC) recovered the cadmium-induced cell death in MCF-7 cells. Cadmium-induced lipid peroxidation was decreased by GSH, NAC, or BHA in MCF-7 cells. The expression of SOD protein was decreased by cadmium (100 μM) but recovered by GSH, NAC, BHA, or Se. Our data showed that the cadmium-induced cell toxicity in human breast cancer cells could be protected by the antioxidants (Se, BHA, NAC, GSH, or NAC) and ER antagonist (tamoxifen or ICI 182,780). Therefore, toxicity of cadmium in breast cancer were mediated by oxidative stress and ERα.

마우스 피부암 발생과정에 있어서 2,3,7,8-Tetrachlorodibenzo-p-Dioxin(TCDD)처리에 의한 유전자발현 변화 연구

염태경(Tai Kyung Ryeom),김옥희(Ok Hee Kim),강미경(Mi Kyung Kang),박미선(Misun Park),지승완(Seung Wan Jee),엄미옥(Mi Ok Eom),강호일(Hoil Kang) 한국환경성돌연변이발암원학회 2005 한국환경성돌연변이·발암원학회지 Vol.25 No.1

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) displays high toxicity in animals and has been implicated in human carcinogenesis. Although the mechanism of carcinogenesis by TCDD is unclear, it is considered to be a non-genotoxic compound and tumor promoter. In our experiment, we investigated the effects of TCDD on gene expression in mouse skin carcinogenesis. We used cDNA microarray to detect the differential gene expression in tumors induced in hairless mouse skin by MNNG plus TCDD protocol. We found that erb-2, c-ets2 and p27^(kip1) were significantly up-regulated, but TNFR2, AKT-1, integrin β1, maspin, IGF-1, c-raf-1, Rb were significantly down-regulated, in tumor region, respectively. We also found that the expression of 53 genes involved in cell cycle, signal transduction, apoptosis, adhesion molecule, angiogenesis, and invasion, were changed two fold more, in tumor surrounding region, These data suggest that TCDD alters the expression of a large array of genes involved in apoptosis, cytokine production and angiogenesis in mouse skin carcinogenesis.

2,3,7,8-TCDD의 세포형질전환 및 내성획득에 관여하는 세포내 인자에 관한 연구

염태경(Tai Kyung Ryeom),최영실(Young Sill Choi),김옥희(Ok Hee Kim),강호일(Hoil Kang) 한국환경성돌연변이발암원학회 2006 한국환경성돌연변이·발암원학회지 Vol.26 No.1

To enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression, we have investigated genes whose expressions are changed after treatment with TCDD and/or MNNG in human Chang liver cell. First, we treated with MNNG and TCDD for two weeks to transform human Chang liver cell. We obtained cell looks like to be transformed and compared the differential gene expression by using cDNA chip (Macrogen) which carrys genes related with signal transduction pathways, oncogenes and tumor suppressor genes, etc. We found that TCDD up- or down-regulated 203 and 111 genes including oncogenes and tumor suppressor genes in human Chang liver cell two fold or more, respectively. Second, we compared the differential gene expression after treatment with TCDD only by using cDNA chip (Superarray) which carrys genes related with cell cycle regulations, and found that TCDD up regulated genes related with cell proliferation as well as cell growth inhibition in human Chang liver cell two fold or more, respectively. These results suggest that toxicity induced by TCDD may reflect sustained alterations in the expression of many genes and that the changes reflect both direct and indirect effects of TCDD.

Chrysin의 유전독성에 관한 연구

지승완(Seungwan Jee),김창환(Changhwan Kim),박미선(Misun Park),엄미옥(Miok Eom),염태경(Taikyung Ryeom),김옥희(Okhee Kim),강호일(Hoil Kang) 한국독성학회 2005 Toxicological Research Vol.21 No.1

Chrysin (5,7-dihydroxyflavone) is a flavonoid compound contained in many fruits, vegetables and honey. In our experiment, we investigated genotoxicity of chrysin using bacterial reverse mutation assay, chromosomal aberration test, in vivo micronucleus test. In bacterial reverse mutation assay, chrysin did not induce mutagenicity in Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA102 with and without metabolic activation. In chromosome aberration test, chrysin did not also induce structural and numerical abberations regardless of metabolic activation in Chinese hamster lung fibroblast cells. In mouse micronucleus test, no significant increase in the occurrence of micronucleated polychromatic erythrocytes (MNPCE) was observed in ICR male mice orally administered with chrysin at the dose of 0.5, 1.0, 2.0 g/kg body weight. Taken together these results, chrysin has no mutagenic potential in our experiment.

폐암세포주에서 아데노바이러스 매개 p16 유전자 전달로 인한 유전자 발현의 변화

박미선(Misun Park),김옥희(Okhee Kim),박현신(Hyunshin Park),지승완(Seungwan Jee),엄미옥(Miok Eom),엄태경(Taikyung Ryeom),강호일(Hoil Kang) 한국독성학회 2004 Toxicological Research Vol.20 No.1

For the safety evaluation of adenovirus-mediated gene transfer, we investigated differential gene expressions after transfecting adenoviral vector containing p16 tumor suppressor gene (Ad5CMV-p16) into human non-small cell lung cancer cells. In the previous study, we showed adenovirus-mediated p16INK4a gene transfer resulted in significant inhibition of cancer cell growth. We investigated gene expression changes after transfecting Ad5CMV-p16, Ad5CMV (null type, a mock vector) into A549 cells by using cDNA chip and oligonucleotide microarray chip (1200 genes) which carries genes related with signal transduction pathways, cell cycle regulations, oncogenes and tumor suppressor genes. We found that p16INK4a gene transfer down regulated 5 genes (cdc2, cyclin D3, cyclin B, cyclin E, cdk2) among 26 genes involved in cell cycle regulations. Compared with serum-free medium treated cells, Ad5CMV-p16 changed 27 gene expressions, two fold or more on oligonucleotide chip. In addition, Ad5CMV-p16 did not seem to increase the tumorigenicity-related gene expression in A549 cells. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation.

폐암세포주에서 아데노바이러스 매개 p16 유전자 전달로 인한 유전자 발현의 변화

박미선(Misun Park),김옥희(Okhee Kim),박현신(Hyunshin Park),지승완(Seungwan Jee),엄미옥(Miok Eom),염태경(Taikyung Ryeom),강호일(Hoil Kang) 한국독성학회 2004 Toxicological Research Vol.20 No.2

For the safety evaluation of adenovirus-mediated gene transfer, we investigated differential gene expressions after transfecting adenoviral vector containing p16 tumor suppressor gene (Ad5CMV-p16) into human non-small cell lung cancer cells. In the previous study, we showed adenovirus-mediated p16INK4a gene transfer resulted in significant inhibition of cancer cell growth. We investigated gene expression changes after transfecting Ad5CMV-p16, Ad5CMV (null type , a mock vector) into A549 cells by using cDNA chip and <br/> oligonucleotide microarray chip (1200 genes) which carries genes related with signal transduction pathways, cell cycle regulations, oncogenes and tumor suppressor genes. We found that p16INK4a gene transfer down regulated 5 genes (cdc2, cyclin D3, cyclin B, cyclin E, cdk2) among 26 genes involved in cell cycle regulations. Compared with serum-free medium treated cells, Ad5CMV-p16 changed 27 gene expressions, two fold or more on oligonucleotide chip. In addition, Ad5CMV-p16 did not seem to increase the tumorigenicity-related gene expression in A549 cells. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation.