• Asian Pacific Journal of Cancer Prevention
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• 제16권15호
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• pp.6445-6449
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• 2015

Background: NSCLC is a disease involving uncontrolled cell growth, which could result in metastases into nearby tissues beyond the lungs. Materials and Methods: The aim of the present study was to analyze the influence of epidermal growth factor receptor (EGFR) gene expression on metastasis and survival in NSCLC patients. The present case-control study included 100 cases of NSCLC patients and 100 age and sex matched controls. EGFR gene expression was analyzed by quantitative real time PCR using serum RNA. Association with NSCLC patient survival was analyzed by the Kaplan-Meier method. Results: We analyzed EGFR gene expression and observed mean increased gene expression of 13.5 fold in NSCLC patients. Values reflected overall survival of patients with a median of 15.8 months in the cases of <13 fold increased gene expression vs 6.7 months with >13 fold increased EGFR gene expression (p=0.005). Distant metastatic patients with <13 fold increased EGFR gene expression had 7.9 months of median survival time while>13 fold increased EGFR gene expression had only 5 months of median survival time (p=0.03). Non metastatic patients with <13 fold increased EGFR gene expression had 18 months of median survival time as compared to only 7.1 months with >13 fold increased expression. Conclusions: Higher cell free EGFR mRNA expression may play an important role in causing distant metastases and reducing overall survival of NSCLC patients in the Indian population.

• 대한핵의학회지
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• 제33권2호
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• pp.152-162
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• 1999

목적: 다약제내성인자가 과발현된 백혈병세포에서 $^{99m}Tc$-MIBI가 인지되는 지를 알아보기 위하여 다약제내성 유전자가 발현된 세포와 발현되지 않은 세포에서의 $^{99m}Tc$-MIBI의 섭취 정도를 측정하고, P-당단백의 길항제로 알려진 verapamil, cyclosporin 그리고 dipyridamole을 첨가하였을 경우 $^{99m}Tc$-MIBI의 세포 내 섭취에 어떤 영향을 주는지 알아보고자 하였다. 대상 및 방법: 다약제내성 인자가 발현되지 않은 대조군 세포로 murine leukemia cell인 L1210세포를 사용하였고, 다약제내성 세포는 L1210 세포에 adriamycin과 vincristine을 첨가하여 유도하였다. 다약제내성의 발현은 RT-PCR로 증명하였고 vera-pamil은 0, 1, 10, 50, 100, $200{\mu}M$의 농도로, cyclosporin은 0, 0.1, 1, 10, 50, $100{\mu}M$의 농도로, 그리고 dipyridamole은 0, 10, 50, 100, 200, 500 ${\mu}M$의 농도로 각각 사용하여 각각의 농도에서의 $^{99m}Tc$-MIBI의 섭취를 감마 카운터로 측정하였다. 결과1) 저용량의 adriamycin 혹은 vincristine을 in-vitro 에서 처리하여 mdr1 유전자를 성공적으로 유도할 수 있었다. 2) mdr1 유전자가 발현된 세포에서 보다 발현되지 않은 세포인 L1210 세포에서 $^{99m}Tc$-MIBI의 섭취가 더 높았고, $4^{\circ}C$에서보다 $37^{\circ}C$에서 섭취가 더 높았다. 3) P-당단백의 작용을 길항시키는 약제로 알려진 verapamil 과 cyclosporin 그리고 dipyridamole을 첨가한 경우 mdr1 이 발현된 세포에서 $^{99m}Tc$-MIBI 섭취의 증가정도가 더 컸다. 또한 다약제 내성이 발현되지 않은 세포인 L1210 세포에서도 적은 정도이기는 하나 $^{99m}Tc$-MIBI의 섭취가 증가하였다. 결론: 다약제내성이 발현된 백혈병 세포에서는 $^{99m}Tc$-MIBI의 세포 내 섭취가 감소되었고, 다약제내성 극복제로 알려진 verapamil, cyclosporin과 dipyridamole은 세포 내 $^{99m}Tc$-MIBI 섭취를 증가시켰다. 본 연구의 결과로 보아 P-당단백에 의해 인지되는 $^{99m}Tc$-MIBI는 암세포에서 P-당단백의 발현을 밝히고, P-당단백 길항제들의 작용을 규명하는 데 유용하게 이용될 수 있을 것으로 판단된다.

• 동의신경정신과학회지
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• 제20권1호
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• pp.147-164
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• 2009

Objectives : We investigated the effects of Sopungsungi-won(Shu!engshunqiyuan) (SSEx1, SSEx2) to treat the metabolic syndrome by the molecular mechanism of regulation of PPAR and modulation of mitochondrial MCAD, VLCAD mRNA expression. Methods : Mouse NMu2Li liver cells and C2C12 skeletal muscle myogenic progenital cells were transiently transfected with expression plasmids for PPAR(PPAR${\alpha}$, PPAR${\delta}$), a luciferase reporter gene construct containing 3 copies of the PPRE from the rat acyl-CoA oxidase gene and ${\beta}$-galactosidase gene. Cells were treated with several concentrated kinds of SSEx1, SSEx2 at the initial time of culture and analyzed PPAR${\alpha}$, PPAR${\delta}$ reporter gene activity using spectrophotometer (405 nm). Total RNA was extracted from SSEx1, SSEx2 and measured mRNA levels of mitochondrial MCAD, VLCAD. Representative RT-PCR bands are shown. Results : 1. SSEx1 increased the expression of PPAR${\alpha}$ reporter gene activities at 0.1 ${\mu}$g/ml (p${\mu}$g/ml (p<0.05), SSEx2 at 0.1 ${\mu}$g/ml (p${\mu}$g/ml (p<0.05) significantly in NMu2Li liver cell lines. 2. SSEx1 increased the expression of PPAR${\alpha}$ reporter gene activities at 1 ${\mu}$g/ml (p${\mu}$g/ml (p${\alpha}$ reporter gene activities in C2C12 skeletal muscle cells. 4. SSEx1 increased the modulation of mitochondrial MCAD mRNA expression (p<0.05) significantly in NMu2Li liver cell lines. 5. SSEx1, SSEx2 both increased the modulation of mitochondrial MCAD mRNA expression (p<0.05) significantly in C2C12 skeletal muscle cells. Conclusions : These results show the SSEx1, SSEx2 can be used as therapeutic agent for metabolic syndrome and it's molecular mechanisms of PPAR more contribute to the activation of PPAR${\alpha}$ then PPAR${\delta}$ reporter gene activities and it's total RNA more contribute to the modulation of mitochondrial MCAD then VLCAD mRNA expression.

• 대한한의학회지
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• 제25권3호
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• pp.123-136
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• 2004

Objectives : The purpose of this investigation is to evaluate the effects of Woohwangcheongsim-won (WC) on the in vitro neuronal development and alteration in gene expression in a hypoxia model using cultured rat cortical cells. Methods : E/sub 18/ rat cortical cells were grown in a neurobasal medium containing B27 supplement and various concentration of WC. Initial development of growth cone was investigated by phase-contrast microscopy, while dendritic spine formation and synaptogenesis were investigated by immunocytochemistry with SynGAPα(a postsynaptic marker) and synaptophysin (presynaptic marker) antibodies. Alteration in gene expression was analyses by microarray using rat 5K-TwinChips. Results : WC suppressed the development of growth cones and WC increased the number of dendritic spines at 20 and 50㎍/mL concentration but there was no statistical significance. Instead, it significantly decreased the number at 100㎍/mL. The expression of anti-apoptosis gene Bcl2-like 1 (Bcl211) increased (Global M=0.46), while Akt1 decreased. Proapoptosis genes Bad and PDCD2 increased. The expression of hemoglobin alpha 1 (probably neuroglobin) increased (Global M=0.93). The expression of antioxidants such as catalase, heme oxygenase (HO), and PRKAG2 gene increased. The expression PKC gene increased. The expression of retinoic acid receptor alpha (RARα) increased significantly (Global M=1.0). Conclusions : These data suggest that WC trends to suppress cellular activity slightly in normoxia and increases the expression of apoptosis-, antioxidation-, oxygen capture-related genes in hypoxia, but increases Bcl111 that anti-apoptosis gene, on the other hand increases Bad, PDCD2 that pro-apoptosis genes, too..

• 대한방사성의약품학회지
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• 제1권2호
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• pp.118-122
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• 2015

Suicidal gene therapy is based on the transduction of tumor cells with "suicide" genes encoding for prodrug-activating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4-ipomeanol (4-IPO) or 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate.In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/2-AA or 4-IPO system were evaluated in HT-29 (human colon cancer cell). pcDNA-CYP4B1 vector was transfected into HT-29 by lipofection and stable transfectant was selected by treatment of hygromycin ($500{\mu}g/mL$) for 3 weeks. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed for confirmation of CYP4B1 expression in CYP4B1 gene transduced cell. The cytotoxic effects of CYP4B1 transduced cell were determined using dye-exclusion assay after treatment of 2-AA or 4-IPO for 96 hrs. Dye-exclusion assay showed that $IC_{50}$ of HT-29 and CYP4B1 transduced HT-29 was 0.01 mM and 0.003 mM after 4-IPO or 2-AA treatment at 96 hrs exposure, respectively. In conclusion, CYP4B1 based prodrug gene therapy probably have the potential for treatment of colorectal adenocarcinoma.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.107-107
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• 1997

Cytochrome P450 enzymes have been intensively investigated in hepatic tissues and several mammalian cell lines. Compared to most studies about cytochrome P450 isozymes in liver in vivo and hepatic, cell lines in vitro, the study of cytochrome P450IA1 in human breast cancer cells could be very important to understand the mechanism of the regulation of CYPIA1 gene expression and cell growth. MCF-7 human breast cancer cells are well characterized to study estrogen and antiestrogen action due to the fact that they contain high level of estrogen receptor and have biological markers characterized. And also MCF-7 cells express high level of arylhydrocarbon hydroxylase activity and human cytochrome P450IA1 cDNA was cloned from MCF-7 cells. Ah receptor was characterized in many breast cancer cell lines and polycyclic aromatic hydrocarbon such as 3-MC induced the expression of CYPIA1 gene and cytochrome P450- dependent monooxygenase activity. We undertook a study to examine the effect of estrogens and other chemicals on the regulation of human CYPIA1 gene expression in MCF-7 cells via RTPCR analysis, that might help us to understand the mechanism of the regulation of CYPIA1 gene expression and MCF-7 cell growth. Expression vector containing the functional 5'-regulatory region of human CYPIA1 fused to the CAT reporter gene was transfected into estrogen receptor positive MCF-T cells or estrogen receptor negative MDA-MB-231 cells. After these cells were treated with various chemicals, RTPCR was carried out to measure both CYPIA1 mRNA and CAT mRNA levels. 1nM 3-MC increased in both P450 and CAT mRNA levels over those of control by two folds in MCF-7 cells but does not in MDA-MB-231 cells. Estrogen or tamoxifen or retinoic acid or chrysin decreased in both P450 and CAT mRNA levels that were induced by 3-MC in MCF-7 when each chemical was administered with 3-MC concomitantly. These results suggested that the level of CYPIA1 gene expression is modulated with estrogen-related molecules and make it possible to speculate that ER is related to CYPIA1 gene expression and cell growth in breast cancer cells. [Supported by grants from the Korean Ministry of Education ]

• BMB Reports
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• 제42권4호
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• pp.217-222
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• 2009

Controlled gene expression in specific cells is a valuable tool for gene therapy. We attempted to determine whether the lentivirus-mediated Tet-On inducible system could be applied to cancer gene therapy. In order to select the genes that induce cancer cell death, we compared the ability of the known pro-apoptotreic genes, Bax and tBid, and a cell cycle inhibitor, p21cip1/waf1, and determined that Bax was the most effective. For the cancer cell-specific expression of $rtTA2^S$-M2, we tested the matrix metalloproteinase-2 (MMP-2) promoter and determined that it is highly expressed in cancer cell lines, including SNU475 cells. The co-transduction of two lentiviruses that contain sequences for TRE-Bax and $rtTA2^S$-M2, the expression of which is controlled by the MMP-2 promoter, resulted in the specific cell death of SNU475, whereas other cells with low MMP-2 expression did not evidence significant cell death. Our data indicate that the lentivirus-mediated Tet-On system using the cancer-specific promoter is applicable for cancer gene therapy.

• The Plant Pathology Journal
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• 제24권1호
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• pp.1-7
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• 2008

Fusarium verticillioides (teleomorph Gibberella moniliformis) is associated with maize worldwide causing ear rot and stalk rot, and produces fumonisins, a group of mycotoxins detrimental to humans and animals. While research tools are available, our understanding of the molecular mechanisms associated with fungal virulence and fumonisin biosynthesis in F. verticillioides is still limited. One of the restraints that hampers F. verticillioides gene characterization is the fact that homologous recombination (HR) frequency is very low (<2%). Screening for a true gene knock-out mutant is a laborious process due to a high number of ectopic integrations. In this study, we generated a F. verticillioides mutant (SF41) deleted for FvKU70, a gene directly responsible for non-homologous end-joining mechanism, with the aim of improving HR frequency. Here, we demonstrate that FvKU70 deletion does not impact key Fverticillioides phenotypes, e.g., development, secondary metabolism, and virulence, while dramatically improving HR frequency. Significantly, we also confirmed that a high percentage (>85%) of the HR mutant strains harbor a desired mutation with no additional copy of the mutant allele inserted in the genome. We conclude that SF41 is suitable for use as a type strain when performing high-throughput gene function studies in F. verticillioides.

• 한국자원식물학회지
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• 제21권3호
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• pp.210-215
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• 2008

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer $Express^{TM}$ software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.

• 동아시아식생활학회지
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• 제7권4호
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• pp.484-490
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• 1997

백삼성분이 탐식세포를 통한 면역반응조절에 어떠한 영향을 미치는가를 알아보기 위해 total saponin이나 ginsenoside Rb$_2$성분이 마우스 복강 탐식 세포의 증식과 nitric oxide(NO)분비 및 NOS 유전자 발현에 미치는 영향을 조사해 보았다. Total saponin 또는 ginsenoside Rb$_2$성분을 탐식세포 배양액에 0-256$\mu\textrm{g}$/$m\ell$ 농도로 첨가하여 배양한 후 $^3$H-thymidine incorporation법으로 분석을 실시한 결과 total saponin은 64$\mu\textrm{g}$/$m\ell$ 농도에서 세포내 DNA 합성을 증가시켰으며, ginsenoside Rb$_2$ 성분의 경우 16$\mu\textrm{g}$/$m\ell$ 또는 64$\mu\textrm{g}$/$m\ell$ 농도에서 DNA 합성이 증가되었다. 256$\mu\textrm{g}$/$m\ell$의 높은 농도에서 세포의 증식이 약간 저해되었지만 대조군에 비해 유의적인 감소는 보이지 않았다. NO 분비에 미치는 영향을 조사해 본 결과 20$\mu\textrm{g}$/$m\ell$ 농도의 백삼성분을 투여했을 때 NO분비가 대조군에 비해 유의적으로 증가하였다. Reverse transcription polymerase chain reaction (RT-PCR)을 이용하여 백삼성분에 의한NOS 유전자 발현의 정도를 조사한 결과 20$\mu\textrm{g}$/$m\ell$ 농도에서 nos gene발현이 대조군보다 증가되었다