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Molecular Genetics Laboratory

Molecular Genetics Laboratory. Professor Guhung Jung E-mail : drjung@snu.ac.kr Tel : 02-880-7773 Room # : 18-201 Homepage: mgl.snu.ac.kr. HBV HCC. Our interests. 분자유전학 실험실. 1. HBV replication & encapsidation. Journal of Biological Chemistry 2003. Journal of Virology , 2001. 이승근. 강희용.

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Molecular Genetics Laboratory

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  1. Molecular Genetics Laboratory Professor Guhung Jung E-mail : drjung@snu.ac.kr Tel : 02-880-7773 Room # : 18-201 Homepage: mgl.snu.ac.kr HBV HCC

  2. Our interests 분자유전학 실험실 1. HBV replication & encapsidation Journal of Biological Chemistry 2003 Journal of Virology, 2001 이승근 강희용 2. HBV and Host interaction Gastroenterology 2005 FEBS Letters 2006 박성규 박사 정찬 강항 3. Mechanism of Hepatocarcinogenesis Clinical Cancer Research 2003 김민숙 김현수 임승외 구진모

  3. 우리 나라의 B형 간염 실태 • 우리나라 인구의 약 5-8%가 감염되어 있음. • 만성 B형 간염 환자 중 20년 후 48%가 간경변증 발병. • 가장 사회적으로 활동력과 영향력이 높은 4, 50대 남성의 경우는 간질환이 사망 원인 2위. • 전체 간질환의 66.6%가 B형 간염바이러스가 원인임. • 한국인 호발성 바이러스 질환에 대한 선택과 집중이 절실함.

  4. 국내 만성 간질환의 질환별 점유율 (대한 소화기학회 총서2, 간염, 18개 대학병원:20,964명 2000년)

  5. 만성 B형 간염과 간암의 관련성 • 만성 간염 분포 지역과 간암 발생 지역의 일치 • 간암 환자의 상당수가 HBsAg 양성(70%) • HBV 감염에 의한 간암 발달 증가 • HBV 보균자의 높은 간암 발생율(비보균자에 비해 223배의 위험성을 가짐) • HBV 백신에 의한 소아간암 발생율 감소 • 감염자 중 약 10%가 간암으로 발병(우리나라 암 사망률 중 3위) Lancet. 1981; Gastroenterology. 2004.

  6. Hepatocellular carcinoma (HCC) • 1년에 간암으로 백만명 이상(전세계) 만 명(국내) 이상이 사망. • 암 종류별 사망 원인 중 간암이 3위. • 초기에는 임상 증상이 거의 없어 조기 발견이 힘들고 효과적인 치료제가 없어 아직까지는 수술에 의한 제거가 가장 효율적임. • 유방암이나 대장암에 비해 연구가 부족하여 간암 발달 기작은 자세히 밝혀져있지 않음. 간암

  7. Research HBV 2. HBV and host interaction Hepatitis Cirrhosis HCC 1. HBV replication 3. Hepatocarcinogenesis Hepatocyte

  8. Approach <Molecular Biology> Protein-protein interaction Part. 1 HBV replication & encapsidation <Cell Biology> Immunofluorescent Apoptosis Part. 2 <Biochemical approach> Protein purification Enzyme assay HBV & Host interaction <2DE> Part. 3 <DNA chip> Mechanism of Hepatocarcinogenesis <ROS analysis> <Clinical case study> <Bioinformatics> System biology

  9. Hepatitis B virus(HBV) Life Cycle HBV gene products Polymerase: P Core : C Surface protein: S X protein : X N Engl J Med 2004;350:1118-29.

  10. HBV Core Assembly Part. 1 Core Dimer-Dimer interaction

  11. Comparison of assembled core levels between WT and mutant core proteins in vitro Part. 1 The mutant core particles were assembled like WT particles. WT WT+PKA Mut (S87G)

  12. Identify protein specific binding site Part. 1 Interesting protein C-terminal Deletion protein

  13. Liver cell transformation by HBV via signal transduction Part. 2 Immunocyte Inducer? Inflammation HBV NIK NIK NIK nuclear localization NF-kB activity? NF-kB activation Nucleus Phenomenon NIK nuclear localization specific to HBV Phenomenon NIK nuclear localization specific to HBV Problems Inducer? Relationship with NF-kB activity? Problems Inducer? Relationship with NF-kB activity? • Hypothesis • Immune response • Inhibition of NF-kB activity • Hypothesis • Immune response • Inhibition of NF-kB activity ? IFN-g Cytokine treatment • Experiments • Screen of inducer • NLS deletion of NIK & estimation of NF-kB activity • Experiments • Screen of inducer • NLS deletion of NIK & estimation of NF-kB activity • Results • IFN-g is inducer • NIK nuclear localization inhibits NF-kB activity • Results • IFN-g is inducer • NIK nuclear localization inhibits NF-kB activity Inhibition of NF-kB activation ?

  14. Immunohistochemistry of Normal & Tumorous Human Liver Tissues Part. 2 Nuclear Translocation Candidate Protein

  15. 간암 발생 과정 Part. 3 ROS stress Chemical Selective Clonal expansion Genetic change Genetic change Genetic change Genetic change Normal cell Initiated cell Invasion metastasis Malignant Tumor Clinical liver cancer Pre-neoplastic lesion Virus HBV HCV Inflammatory / Reactive oxygen/ NO species (Toxicology 2002; 181–182: 43–47) 여러 단계(multi-step)를 거쳐 일어나며, 각 step에서 ROS stress가 관여함.

  16. 암 진행 과정 Part. 3 Normal epithelium Dysplasia Carcinoma EMT vascularinvasion EMT Metastasis (EMT: epithelial-mesenchymal transition) (Nature Review cancer 2002; 2: 442) 암세포는 EMT를 통해 invasion 및 metastasis가 가능해짐.

  17. 임상 조직을 대상으로 한 실험 Part. 3 간암 분화도별 조직 Proteome analysis (2-DE & MS) Transcripteome analysis (Microarray) 간암 관련 유전자 발굴 Antioxidant enzyme, Iron metabolic protein, cytoskeleton protein, heat shock protein … Case study (Immunoblot, IHC) ROS 관련 유전자 및EMT 관련 유전자의 발현 변화가 크게 나타남

  18. Proteome analysis of HCC Part. 3 (Cirrhosis) (HCC G2) We have observed down-regulation of antioxidant enzymes (AOEs) in HCC by proteome analysis.

  19. 간암 분화도별 AOE와 vascular invasion 관계 Part. 3 Invasion EMT Frequency AOE HCC GIII Cirrhosis LGDN HGDN HCC GI HCC GII Histological grade Antioxidant enzymes ↓ → ROS stress ↑→ EMT ↑→ Invasion ↑

  20. ROS에 의한 EMT 유발 Part. 3 B. Hep3B A. Huh7 No treatment H2O2 4days No treatment H2O2 4days E-cad DAPI H2O2 down-regulate E-cadherin expression at day 4.

  21. The effect of ROS on tumor cell invasion Part. 3 HCC cells Matrigel PET memb. (8um pore) Matrigel invasion assay No treatment H2O2 treatment Invaded cells (Hep3B) ROS level Invasion assay: Hep3B Invasion assay: Huh7 (N=3) (N=3) (N=3) H: H2O2 H2O2 enhances tumor cell invasion.

  22. ROS up-regulate Snail expression Part. 3 (RT-PCR) O: untreated, N: NAC, H: H2O2

  23. ROS and Snail induced DNA methlyation of E-cadherin promoter Part. 3 IS1 IS2 IS3 IS1 IS2 IS3 C S C S C S O H O H O H U M U M U M U M U M U M U M U M U M U M U M U M IS: CpG island O: untreated H: H2O2 U: unmethyl M: methyl C: control S: Snail

  24. ROS induce tumor cell invasion in HCC via snail mediated epigenetic change Part. 3 • Snail ↑ • DNA methylation • E-cadherin ↓ Invasion ROS • Antioxidant (NAC) • Antioxidant enzyme ↑ • (catalase, SOD1) ROS HDAC1 DNMT1 Snail Promoter Promoter E-cad E-cad

  25. Goal of Research Inhibition of HBV Infection HBV infection Inhibition of HBV Replication Liver disease Discovery of drug target HCC therapy Hepatocellular Carcinoma

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