• Radiation Oncology Journal
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• 제19권2호
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• pp.171-180
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• 2001

목적 : Tissue inhibitor of matrix metalloproteinase (TIMP)는 matrix metalloproteinase (MMP)에 작용하여 암세포의 침윤과 전이를 억제하고 염증, angiogenesis, fibrosis에 중요한 역할을 한다. TIMP 유전자는 여러 cytokine 및 signal molecule에 의하여 조절되는 유전자이므로 방사선에 의한 TIMP의 발현을 측정하고 전사 조절 기전을 연구하고자 하였다. 대상 및 방법 : 두경부암 환자의 병변에서 유도하여 확립한 두경부암 세포주를 이용하여 방사선에 의한 TIMP 유전자 발현을 측정하였다. 각 세포주의 방사선 민감도를 측정하고 transwell을 이용한 invasion assay로 전이성을 측정하였다. TIMP1, TIMP2 발현은 conditioned medium을 취해 ELISA assay로 측정하였다. 방사선조사는 2 Gy, 10 Gy 군으로 나누어 관찰했고 조사 후 시간 간격은 24, 48시간이었다. MTT assay로 생존세포 수를 측정하여 방사선 세포치사로 인한 발현 변화를 보정하였다. hTIMP1 promoter region을 PCR하여 pGL2-basic luciferase reporter vector에 cloning하여 인간 두경부암 세포주에 이입하여 functional TIMP1 발현이 증가하는지 확인하였고 protein kinase C (PKC) activator인 PMA (phorbol 12-myristate 13-acetate)와 Ras에 의한 TIMP1 발현이 유도되는지 확인하였다. 결과 : HN-1, HN-2, HN-3, HN-5, HN골 세포주의 $D_0$는 각각 1.55 Gy, 1.8 Gy, 1.5 Gy, 1.55 Gy, 2.45 Gy 이었다. 각 세포주의 방사선조사 후 MTT assay에 의한 cell viability는 24, 48시간에서 2 Gy인 경우 모두 $94\%$ 이상 그리고 10 Gy에서는 $73\%$ 이상의 생존 세포를 확인하였다. TIMP1, TIMP2 단백의 basal 농도는 24시간 48시간에서 점점 증가하여 세포에서 계속 합성되어 분비되고 있음을 확인하였다. 2 Gy 조사 후 24시간에서 TIMP2는 HN-1, HN-9 세포주에서 감소하였으나, 10 Gy 조사 후에는 두 세포주에서 모두 증가하여 방사선량에 따라 반응이 달랐고, 방사선조사 후 48시간에는 HN-9 세포주에서는 증가하나 HN-9 세포주에서는 감소하여 세포주에 따라 반응이 달랐다. 그러나 방사선에 의한 TIMP1 발현 변화는 미미하였다. TIMP1 reporter gene을 인간 두경부암 세포주에 transfection하고 PMA (100 ng/ml)을 가한 경우 HN-1세포주에서는 유의하게 증가하고 HN-9 세포주에서는 감소하였다. Ras 발현 벡터와 co-transfection한 경우 TIMP1 promoter가 활성화 되었다. 결론 : 모두 두경부 암에서 유래된 세포주 이지만 방사선에 의한 TIMP의 발현 및 전사조절 기전은 세포주 마다 차이가 있었고 이온화 방사선의 용량에 따라서, 방사선조사 후의 시간 경과에 따라서도 TIMP 발현에 차이가 있었다. 이 결과는 TIMP의 전사 및 발현이 여러 종류의 signal molecule에 의하여 영향을 받고, 이 signal molecule들이 각 세포주 마다 다르기 때문으로 사료된다.

• Journal of Microbiology
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• 제38권4호
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• pp.255-259
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• 2000

For rapid and sensitive measurement of antiviral activities, application of a recombinant virus containing firefly luciferase gene was attempted. Recombinant human cytomegalovirus (HCMV) containing luciferase gene driven by HCMV late gene pp28 promoter (HCMV/pp28-luc) was used to test the antiviral activities of three known compounds and the result was compared with results from the conventional plaque assay for measuring the production of infectious viruses. When human fibroblast cells were infected with HCMV/pp28-luc, luciferase activity was observed at 2 days after infection and reached maximum at 6 days after infection, whereas the production of infectious virus was maximal at 4 days after infection. The antiviral activities of ganciclovir, acyclovir, and papaverine were measured in HFF cells infected with HCMV/PP28-luc and the luciferase activity was compared with the infectious virus titers. Luciferase activity decreased as the concentration of ganciclovir or papaverine increased, while there was a slight decrease in luciferase activity with acyclovir. The level of the decrease in Luciferase activity was comparable to the level of decrease in the production of infectious virus. Therefore, the antiviral assay using recombinant virus HCMV/pp28-luc resulted in sensitivity similar to the conventional plaque assay with a significant reduction in assay time.

• International Journal of Oral Biology
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• 제40권1호
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• pp.19-25
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• 2015

Osteocytes may function as mechanotransducers by regulating local osteoclastogenesis. Reduced availability of oxygen, i.e. hypoxia, could occur during disuse, bone development, and fracture. Receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) is an osteoblast/stromal cell derived essential factor for osteoclastogenesis. The hypoxia induced osteoclastogenesis via increased RANKL expression in osteoblasts was demonstrated. Hypoxic regulation of gene expression generally involves activation of the hypoxia-inducible factor (HIF) transcription pathway. In the present study, we investigated whether hypoxia regulates RANKL expression in murine osteocytes and HIF-$1{\alpha}$ mediates hypoxia-induced RANKL expression by transactivating RANKL promoter, to elucidate the role of osteocyte in osteoclastogenesis in the context of hypoxic condition. The expression levels of RANKL mRNA and protein, as well as hypoxia inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) protein, were significantly increased in hypoxic condition in MLO-Y4s. Constitutively active HIF-$1{\alpha}$ alone significantly increased the levels of RANKL expression in MLO-Y4s under normoxic conditions, whereas dominant negative HIF-$1{\alpha}$ blocked hypoxia-induced RANKL expression. To further explore to find if HIF-$1{\alpha}$ directly regulates RANKL transcription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity, whereas mutations of putative HIF-$1{\alpha}$ binding elements in RANKL promoter prevented this hypoxia-induced RANKL promoter activity in MLO-Y4s. These results suggest that HIF-$1{\alpha}$ mediates hypoxia-induced up-regulation of RANKL expression, and that in osteocytes of mechanically unloaded bone, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in osteocytes.

• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5181-5185
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• 2015

Background: CCAT1 has been reported to be linked with pathogenesis of malignancies including colon cancer and gastric cancer. However, the regulatory effect of CCAT1 in hepatocellular carcinoma (HCC) remains unclear. The purpose of this research was to identify any role of CCAT1 in the progression of HCC. Materials and Methods: Real time-PCR was performed to test the relative expression of CCAT1 in HCC tissues. A computation screen of CCAT1 promoter was conducted to search for transcription-factor-binding sites. The association of c-Myc with CCAT1 promoter in vivo was tested by Pearson correlation analysis and chromatin immunoprecipitation assay. Additionally, Kaplan-Meier analysis and Cox proportional hazards analyses were performed. Results: c-Myc directly binds to the E-box element in the promoter region of CCAT, and when ectopically expressed increases promoter activity and expression of CCAT1. Moreover, Kaplan-Meier analysis demonstrated that the patients with low expression of CCAT1 demonstrated better overall and relapse-free survival compared with the high expression group. Cox proportional hazards analyses showed that CCAT1 expression was an independent prognostic factor for HCC patients. Conclusions: The findings demonstrated CCAT1, acting as a potential biomarker in predicting the prognosis of HCC, is regulated by c-Myc.

• Animal cells and systems
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• 제4권2호
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• pp.181-186
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• 2000

The expression of $p21^{WAF1/ClP1/SDl1}$, one of the cyclin-dependent kinase inhibitors, is regulated by a variety of transcription factors including p53 and STAT. Heat shock induces the expression of p21 in a temperature- and time-dependent manner. Although the p21 induction by heat shock has been reported to be controlled by p53, a p53-independent mechanism Is also involved. To understand the p53-independent regulation of heat shock-induced p21 expression, we searched the promoter region of p21 gene and found one or two heat shock element (HSE)-like sequences in human, rat, and mouse. Electromobility shift assay (EMSA) showed that heat shock factor (HSF) could bind to these HSE-like sequences In response to heat shock, even though to a lesser extent than to HSE. In addition, p21 promoter deletion analysis revealed that heat shock activated a p21 deletion promoter construct containing the HSE-like sequences but lacking p53-binding sites, but not a promoter construct containing neither HSE-like sequences nor the p53-responsive element. Furthermore, the p21 induction by heat shook was significantly inhibited in confluent cells in which heat shock-induced HSF activation was reduced. These results suggest that the transcriptional regulation of p21 by heat shock may be mediated through activation and binding to HSE-like sequences of HSF.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1380-1389
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• 2015

Prx1, an AhpF-dependent 2-Cys peroxiredoxin (Prx), was previously identified in Vibrio vulnificus, a facultative aerobic pathogen. In the present study, transcription of the V. vulnificus prx1ahpF genes, which are adjacently located on the chromosome, was evaluated by analyzing the promoter and intergenic region of the two genes. Northern blot analyses revealed that transcription of prx1ahpF results in two transcripts, the prx1 and prx1ahpF transcripts. Primer extension analysis and a point mutational analysis of the promoter region showed that the two transcripts are generated from a single promoter. In addition, the 3' end of the prx1 transcript at the prx1ahpF intergenic region was determined by a 3'RACE assay. These results suggested that the prx1ahpF genes are transcribed as an operon, and the prx1 transcript was produced by transcriptional termination in the intergenic region. RNA secondary structure prediction of the prx1ahpF intergenic region singled out a stem-loop structure without poly(U) tract, and a deletion analysis of the intergenic region showed that the atypical stem-loop structure acts as the transcriptional attenuator to result in the prx1 and prx1ahpF transcripts. The combined results demonstrate that the differential expression of prx1 and ahpF is accomplished by the cis-acting transcriptional attenuator located between the two genes and thereby leads to the production of a high level of Prx1 and a low level of AhpF.

• 생명과학회지
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• 제27권8호
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• pp.857-863
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• 2017

FosB (FBJ murine osteosarcoma viral oncogene homolog B) 유전자는 사람의 19번 염색체에 위치하고 있으며 약 43 KD의 단백질을 코딩하며, 발생 및 분화과정, 개체 유지, 발병 진행 등을 조절한다고 알려져 왔다. 본 연구에서는 바이오 마커 등의 가능성이 있다고 보고된 FosB 유전자의 프로모터를 클로닝하여 활성도를 분석하고자 하였다. FosB genomic DNA 서열을 확인한 결과, TSS upstream 방향의 약 1 Kb 안쪽 부위에 FosB 유전자 발현을 위한 중요한 요소들이 있을 것으로 추정하였고, 따라서 FosB genomic DNA의 upstream -1,555 부위부터 exon 1의 +73까지 부위에 대한 PCR 증폭을 수행하였다. 또한 클로닝 성공을 높이기 위하여 일차로 $TA-1^{st}FosBp$ plasmid를 얻은 후, 다시 $TA-1^{st}FosBp$ plasmid를 template로 Kpn1과 Nhe1 제한 효소 절단부위를 프라이머에 삽입한 후 제작하여 2차 PCR을 수행하였으며, $TA-2^{nd}FosBp$ 플라스미드를 제작한 후 제한 효소로 절단하여 pGL3-luc vector로 subcloning하였다. 제작된 pGL3-FosBp-luc를 이용하여 항암제에 대한 활성도를 분석하고자 A549 사람 폐암세포주에 pGL3-FosBp-luc 플라스미드를 transfection 한 후 luciferase 활성도 분석을 수행하였다. Luciferase 활성도 증가는 doxorubicin, taxol 등을 처리한 후 단백질 발현 양상과 비교 하였을 때도 일치되는 결과를 얻을 수 있었다. 그러므로 FosB프로모터 클로닝은 향후 유전자 발현 연구, 마커분석 등에 유용할 것으로 사료된다.

• 한국균학회지
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• 제26권4호통권87호
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• pp.450-457
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• 1998

Cryparin은 Cryphonectria parasitica의 세포벽에 풍부한 소수성 단백질에 속한다. cryparin은 비록 하나의 유전자에 의해 발현되지만 액체배양 후 48시간이 지나면 발현된 전체 유전자중에서 22%를 차지할 정도의 높은 발현 양상을 나타낸다. 또한 cryparin은 RNA mycovirus인 Cryphonectria hypovirus 1의 감염에 의해 발현이 현저히 억제되는 유전자로 알려졌다. 이미 지난 실험(Kim et al., 1999)에서 상동염색체간의 재조합을 이용하여 cryparin 유전자를 항생제 hygromycin B 저항성 유전자로 치환한 치환체를 제조하였다. 발현율이 매우 높으면서도 virus에 의해 밀접하게 영향받는 cryparin 유전자의 promoter 분석을 위하여서는 대상이 되는 유전자 치환을 위한 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 어느 한곳에만 삽입되도록 하는 성질을 가진 균주의 개발이 필요하다. 그러나 지난번 실험의 결과 얻어진 cryparin 치환체는 치환용 vector외에도 무작위로 삽입된 vector가 존재하고 나아가 새로운 vector들이 어느 한곳에만 삽입되도록 하는 성질을 갖지 못하였다. 따라서 본 실험에서는 cryparin 유전자 치환체와 영양요구성 돌연변이체인 균주간의 교잡을 이용하여 분석 대상이 되는 유전자의 치환에 이용된 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 genome내의 어느 한곳에만 삽입되도록 하는 성질을 가진 균주를 제조하였다.

• 한국식품저장유통학회:학술대회논문집
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• 한국식품저장유통학회 2007년도 학술발표회
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• pp.235-235
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• 2007

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2056-2060
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• 2007

Transcription factor GATA-3 is the critical transcription factor for Th2 cell differentiation. In spite of its importance in Th2 cell differentiation, the molecular mechanism for its action in Th2 differentiation is poorly understood. Previous studies have suggested that GATA-3 may be involved in the chromatin remodeling in the Th2 cytokine locus. To determine whether GATA-3 exerts its effect on its target sites in the extrachromosomal status, cell transfection assay was performed. In this assay, 800 bp IL4 promoter-luciferase constructs linked with GATA-3 target sites were transfected into the M12 B cell line, D10 mouse Th2 cell lines, and human T lymphoma Jurkat cell lines with or without the GATA-3 expression vector. The GATA-3 effects on its target sites were minimal in the extrachromosomal status, supporting the previous propositions that GATA-3 functions at the chromatin level by remodeling chromatin structure.