보고서 정보
주관연구기관 |
가톨릭대학교 Catholic University of Korea |
연구책임자 |
박종섭
|
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2002-05 |
주관부처 |
보건복지부 |
사업 관리 기관 |
가톨릭대학교 Catholic University of Korea |
등록번호 |
TRKO200300001584 |
DB 구축일자 |
2013-04-18
|
키워드 |
인유두종 바이러스.자궁경부암.E6.E7 유전자.p53.Rb.IRF-1.종양억제 유전자.전사 조절.HPV.cervical cancer.E6/E7 gene.p53.Rb.IRF-1.hlmor suppressor gene.transcription factor.
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초록
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1. HPV E6 발암 단백질 p73 종양억제인자와의 반응 탐색 및 작용 기전 규명
논문: Park JS, Kim EJ, Lee JY, Sin HS, Namkoong Sung Eun, Um SJ. Functional inactivation of p73, a homologu of p53 tumor suppressor protein, by human papillomavirus E6 proteins Int J Cancer 91, 822-827 (2001).
자궁경부암은 국내에서 발생률이 높은 암으로 인유두종 바이
1. HPV E6 발암 단백질 p73 종양억제인자와의 반응 탐색 및 작용 기전 규명
논문: Park JS, Kim EJ, Lee JY, Sin HS, Namkoong Sung Eun, Um SJ. Functional inactivation of p73, a homologu of p53 tumor suppressor protein, by human papillomavirus E6 proteins Int J Cancer 91, 822-827 (2001).
자궁경부암은 국내에서 발생률이 높은 암으로 인유두종 바이러스 (HPV)의 감염에 의한 발암 물질이 체내 유전자 산물들과 반응하여 나타난 기전이다. p13 유전자는 alternative splicing에 의해 카복실 말단이 다른 p73α,p73β isoform을 가지며 DNA 결합, 전사활성부위, 아미노산 염기서열에서 p53과 구조적 상동성을 갖는다. P53과 유사한 p73의 특성을 바탕으로 HPV-E6 단백질이 어떠한 기전으로 p73을 불활성화시키는지 HPV-E6의 분자생물학적인 기전을 탐색하였다.p73은 HPV-H6 단백질이 표적하는 새로운 세포내 단백질이며, E6가 단백질 분해작용과 DNA 결합에 영향 없이 un의 아미노 말단 전사활성부위와 결합하여 전사활성을 방해함을 입증하였다. 이러한 결과들은 결론적으로 HPV-16 E6와 같이 암 발생 고위험군 바이러스와 작용하는 종양억제인자인 p53의 불활성화 기전과는 달리 p73은 양성 콘디로마를 형성하는 HPV-11 E6 단백질과도 반응하므로 종양억제인자보다는 성장억제인자라고 정의하는 것이 타당할 것이며, HPV-E6에 의한 P73의 기능적 억제는 적어도 E6에 의해 매개되는 세포 변형작용의 중요한 기전을 제시하였다.
2. 자궁경부암 질환 모텔 연구 체계 확립
■HPV E6, E7과 p53, IRF family 사이의 결합 분석 분자생물학적 체계 확립
■HPV E6, E7을 안정적으로 발현시키는 세포주 3 종 완성
■HPV-18 URR/E6/E7 유전자를 발현시키는 형질전환 생쥐의 제조 및 자궁경부암 유발 시스템 완성
■HPV 감염 및 이형증 조직 (50 개), 자궁경부종양 환자의 암 조직 (100 개) 확보
3.자궁경부암 관련 유전자 발굴
① HPV-16 E6 transfected stable cell line 2 종에서 microarrayer로 유전자 탐색, 분류:human colon carcinoma cell line과 lung carcinoma cell line에 각각 HPV-16 E6 유전자를 transfection시킨 stable cell line을 제작하여 시행하여 1024 유전자를 가진 DNA microarray 결과로 나타난 유전자 발현 상태로서 HPV-16 E6 관련 유전자들을 기능적으로분류하였음 (표 1-4. 참조).
논문: Park JS, Myoung J, Kim CJ, Rha SY, Jeong HC, Namkoong SE, Um SJ. Function릴 classification of gene expression by DNA microarray in HPV-16 E6 transfected stable cell lines. Gynecol Oncol, in Preparation
② Yeast two hybrid assay로 HPV-16 E6 binding 유전자 탐색 및 신규 관련 유전자 발굴:HPV-16 E6를 bait로 하고 HeLa CDNA library를 Prey로 하여 두 단백질 간의 결합반응여부를 실험한 결과, 현재까지 5 종의 E6 결합 반응 운전자들을 확보하였으며, 3 종은 처음으로 반응 사실이 밝혀진 것임.
■Homo sapiens protein kinase C-like 1 (PRKCLI): 2001 년 J Biol Chem 발표된 내용
■Homo sapiens interferon regulatory factor-3; 1998 년 Genes Dev 발표된 내용
■Homo sapiens PDZ/coiled-coil domain binding gene (신규 탐색된 E6 반응 유전자)
■Homo sapiens immune regulator related gene (신규 탐색된 E6 반응 유전자)
■Tumor suppressor related gene (신규 탐색된 E6 반응 유전자)
Abstract
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1. Investigation of interaction and action mechanism between HPV E6 oncoprotein and p73 tumor suppressor protein
Article; Park JS, Kim EJ, Lee JY, Sin HS, Namkoong Sung Eun, Um SJ. functional inactivation of p73, a homologu of p53 tumor suppressor protein, by human papillomavirus E6 proteins.
1. Investigation of interaction and action mechanism between HPV E6 oncoprotein and p73 tumor suppressor protein
Article; Park JS, Kim EJ, Lee JY, Sin HS, Namkoong Sung Eun, Um SJ. functional inactivation of p73, a homologu of p53 tumor suppressor protein, by human papillomavirus E6 proteins. Int J Cancer 91, 822-827 (2001).
Summary; HPV is strongly implicated as a causative agent in the etiology of cervical cancer. Of Its gene products, E6 binds to and inactivates p53 tumor suppressor Protein by ubiquitin/proteasome -dependent degradation. p73. a novel family of p53, has been identified and demonstrated to activate p21/sup WAF1/. We showed that p73 is also inactivated by HPV E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro. Transient co-expression of E6 significantly inhibited the p73-mediated activation of p21 promoter in a p53-defective C33A cell line. The inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These results may suggest that p73 is unlikely to be true tumor suppressor gene but is rather a growth regulator possibly involved in the formation of benign tumors such as genital condyloma which is tile resulted from infection by low-risk oncogenic HPV type HPV-6 or -11. These overall results. consequently, suggest that in addition to the inactivation of p53, the functional inactivation of p73 by HPV E6 may, at least in Part, contribute to E6-mediated transformation and hyperplastic proliferation of cervical cells.
2. Establishment of cervical cancer disease model
■Establishment of molecular biologic system to identify tile action mechanism between HPV E6, E7 gene and p53, IRF gene family
■Establishment of HPV E6, E7 gene expressing stable cell lines (3 types)
■Development of HPV-18 URR/E6/E7 transgenic mice and early induction of cervical neoplasia
■Collection of HPV infection and dysplasia tissues (50), cervical cancer tissues(100)
3. Detection of cervical cancer related gene
① Detection and classification of cervical cancer related genes by cDNA microarray system in HPV-16 E6 transfected stable cell lines (human colon & lung carcinoma cell lines)
Over-expressed and under-expressed genes by the result of HPY-16 E6 transfection in stable cancer cell lines were functionally classified from loaf genes of cDNA microarray (Review; Table 1-4).
Articles; Park JS, Myoung J, Kim CJ, Rha SY, Jeong HC, Namkoong SE, Um SJ. Functional classification of gene expression by DNA microarray in HPV-16 E6 transfected stable cell lines. Gynecol oncol, in preparation
② Detection of HPV-16 E6 binding new genes by yeast two hybrid assay system
Yeast two hybrid assay: cDNA sequence of HeLa cell library was introduced into the vector pBTMl16, while cDNA sequence of HPV-16 E6 was introduced into the vector pASV3 (bait; HPV-16 E6, prey; HeLa cell library). The yeast Saccharomyces cerevisiae L40 strain was transformed with a pBTMl16 plasmid and a pASV3 plasmid by the lithium-acetate procedure. Transformants were grown in 15 ml of minimal liquid medium supplemented with histidine (50 mg/liter) and adenine (20 mg/liter). Extracts were Prepared by a freezing-thawing lysis technique and analyzed for β-galactosidase activity according to a standard procedure.
We detected 5 HPV-16 E6 binding genes, and 3 of thorn were the firstly known genes.
■ Homo sapiens protein kinase C-like 1 (PRKCLI): Published in J Biol Chem (2001)
■ Homo sapiens interferon regulatory facfor-3; published in Genes Dev (1998)
■ Homo sapiens PDZ/coiled-coil domain binding gene (newly detected E6 response gene)
■ Homo sapiens immune regulator related gene (nettly detected E6 response gene)
■ Tumor suppressor related gene (newly detected E6 response gene)
목차 Contents
- I. 단독기초연구개발결과 요약문...4
- (한글)...4
- (영문)...6
- II. 단독기초연구개발과제 연구결과...8
- 1. 단독기초연구개발과제의 최종 연구개발 목표...8
- 2. 단독기초연구개발과제의 최종 연구개발 내용 및 결과...11
- 3. 단독기초연구개발과제의 연구결과 고찰 및 결론...18
- 4. 단독기초연구개발과제의 연구성과 및 목표달성도...19
- 5. 단독기초연구개발과제 활용계획...23
- 6. 첨부서류...24
- 7. 참고문헌...24
- 부록...27
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