초록 ▼

○ 연구결과
① 폐암과 간암환자의 조직에서 GlcNAcT-V유전차의 클로녕 성공. 암화과정중 특성해명을 위한 재조합 단백질의 대량생산 성공: 당쇄전이효소가 간암， 폐암 등 질환발생에 관여하는 분자생물학적인 증거확보(국제논문 Arch. Biochem. Biophys. BBRC, 1997.1998의 2편발표)
② SiaJyltransferase2,3는 뇌암에서도 발현이 높음을 발견. 사람에서 폐암전이환자의 혈액과 lmelanoma B-16에 의한 mouse의 폐암전이조직에서 당쇄전이효소가 간암, 폐암등 질환발생에 관여하는 분

○ 연구결과
① 폐암과 간암환자의 조직에서 GlcNAcT-V유전차의 클로녕 성공. 암화과정중 특성해명을 위한 재조합 단백질의 대량생산 성공: 당쇄전이효소가 간암， 폐암 등 질환발생에 관여하는 분자생물학적인 증거확보(국제논문 Arch. Biochem. Biophys. BBRC, 1997.1998의 2편발표)
② SiaJyltransferase2,3는 뇌암에서도 발현이 높음을 발견. 사람에서 폐암전이환자의 혈액과 lmelanoma B-16에 의한 mouse의 폐암전이조직에서 당쇄전이효소가 간암, 폐암등 질환발생에 관여하는 분자세포생물학적인 기작해명.
③ 재조합GlcNAcT-V를 대량생산과 활성화공정개발: 국내특허출원 완료 (1998년 제4018호)
④ 암관련 시알산전이효소유전자의 분리， 발현득이성의 해명: Sialyltransferase중에서 암세포표면의 Sialic acid를 α-2.3:2-8로 전이 하는 Siaα2-3Galβ1.4GlcNAcα2,8-sialyltransferase (hST8Sia III) [Arch. Biochem. Biophys. 360. 41-46 (1998)): 암전이 및 악성화 관련 특이적인 전사발현 조절기구해석. Human ST8Sia III와 ST8Sia V의 게놈유전자의 구조해석과 전사조절영역결 . In situ hybridization으로 발생과정 중 Mouse ST3Gal III의 조직특이적 발현특성 해석: Sialy1 Lewis^x와 Sialy1 Lewis^a의 생합성에 관여하는 ST3Gal III의 조직특이적 발현특이성 해석 (Arch. Pharm. Res. 인쇄중. 1999).
⑤ GlcNAcT-V, ST9Sia V 및 ST8Sia III의 효소활성저해를 목표로 한 한방의학적 암치료 및 억제에 응용: 전통의학적으로로 사용되어온 식물약재를 탐색하여, 과루인(Trichosanthes kirilowii Maxam), 황백(Phellodendri cortex), 황연, 황금 (Scute1Jaria baicalensis Georgi), 반지 련등이 효과적임을 mRNA순준에서 확인, 유효성분의 분리 및 분석(국제지명 Glycoconjugate Journal 인쇄중 1999)(국내특허출원 제99-10881호)
⑥ 암조직과 세포내발현에 의한 암세포표면의 당단백질의 당쇄구조가 바뀌어감을 Lectin-blotting방법으로 확인 간암세포주(T-2)와 mouse의 폐암전이조직에서 GlcNAcT-V, ST9Sía V 및 ST8Sia III효소활성의 과잉발현과 질환발생에 관여하는 분자세포생물학적인 기작을 해명.

Abstract ▼

1. UDP-N-Aacetylgl ucosamine: α-6-D-Mannoside β-1,6,N-Acetylglucosaminyltransferase-V (GlcNAcT-V) that catalyzes β-1,6 branching in asparagine-linked oligosaccharides is associated with tumor metastasis. The increased amounts of such material in malignant cells are due to an increased activity of Gl

1. UDP-N-Aacetylgl ucosamine: α-6-D-Mannoside β-1,6,N-Acetylglucosaminyltransferase-V (GlcNAcT-V) that catalyzes β-1,6 branching in asparagine-linked oligosaccharides is associated with tumor metastasis. The increased amounts of such material in malignant cells are due to an increased activity of GlcNAcT-V, which catalyzes the lransfer of GltNAcfrom UDP-GlcNAc to an α-D-mannoside. An attempt was made to purify the enzyme from human hepatoma cell line, Hep3B. The purified GlcNAcT-V had Mr 67 kDa and 65 kDa on denaturated and natural conditions, respectively, with the _pI value of 5.9. To isolate the GlcNAcT-V gene from human hepatoma cells, PCR primers of GNN-1 and GNN-8 were designed and the amplified PCR product carrying the gene was cloned and sequenced. Nucleotide sequence analysis showed a 2220-bp's open reading frame encoding a 740 amino acid protein. This was almost same as the previously reported human sequences, except for some sequence differences in three amino acids. Three amino acid changes were as Iollows:³⁷⁵V→L, ⁵⁵⁵T→R and ⁵⁹²A→G.
2. When the GlcNAcT-V gene has been introduced into NIH3T3 cells using by pcDNA3-GlcNAcTV. the morphology of the transfected cells was changed. Although NIH3T3 cells formed the shape of flat oval, the transfected cells induced morphological changes of cells to round form. Cell-cell interaction of normal cells transduced the signal to be contacted on both edges of flat oval. The growth of the transfected cells was great slower than that of normal cells, and the normal cells were grown to uniform and straight direction, whereas the transfected cells were agglutinated to the cluster shape to grow and differentiate. SDS-PAGE analysis showed that slightly different protein patterns in the two cells were obseryed (32 and 40 kDa).
3. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates for GlcNAcT-V activity from hepatoma cells, the enzyme displayed optimum temperature and pH of 50℃ and 5.5, respectively, K_m values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K_m values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAcT-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1,423 pmol/h/mg) than that 1,066 pmol/h/mg) of HepG2. Treatment of hepatoma cells with retinoic acid and 1α,2,5-dihydroxyvitamin D₃ resulted in an increase in GlcNAcT-V activity, while treatment with DMSO and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid treated cells shows a changed GlcNAcT-V mRNA expression, expression of marker proteins such as AFP and albumin was not changed. This is the first demonstration of GlcNAcT-V activity in retinoic acid and 1α,2,5-dihydroxyvitamin D₃-treated hepatoma cell lines.
4. GlcNac-III. an another tumor-specific enzyme, was higher than those of GlcNAcT-V in hepatic carcinomca cells. In contrast, the two enzyme activities were assayed in highly metastatic colon cancer cells. GlcNAcT-V activities were much higher than those of GlcNAcT-III. The enzymes displayed different kinetic properties between hepatic and colon cancer cells, depending on their metastatic potentials. Hepatoma and colon cancer cells contained high activities. These data were supported by RT-PCR results, showing that expression of the GlcNAcT-V mRNAs increased in proportion to the enzymatic activities. Lectin blot analysis showed that oligosaccharides in many glycoproteins were observed in cancer cells. Thus. this is the first demonstration of GlcNAcT-III and V activities in human hepatoma and colon cell lines.
5. Cloning and expression of cDNA for a human Siaα2,3Galβ1,4GlcNAc α2,8-sialyltransferase (hST8Sia III) and hST8Sia V genomic DNA: The cDNA encoding human hST8Sia III was isolated from human brain eDNA library with PCR-amplified DNA probe and by 5' rapid amplification of cDNA ends of mRNA. Comparative analysis of the predicted protein-coding region between our cloned hST8Sia III and mST8Sia III showed 92 and 96% identities in the nucleotide and the amino acid sequence. respectively. The soluble hST8Sia III protein expressed in COS-7 cells showed an extremely high catalytic activity of transferring sialic acid through α2,8-linkage to intact fetuin glycoprotein, whereas the transferring activity was completely undetectable toward either α2,6-sialylated glycoprotein or asialo-glycqprotein acceptors. Northern analysis of hST8Sia III showed that the transcript corresponding to 11 kb was expressed in both human fetal and adult brain, while the expression of 5.5 kb transcript was restricted to fetal liver, indicating that the expression of hST8Sia III is developmentally and tissue-specifically regulated.
6. Isolation and structure analysis of hST8Sia III genomic DNA for cancer specific-transcrional regulation: To elucidate the mechanism for transcriptional regulation of hST8Sia III. hST8Sia III genomic gene was isloated from human genomic DNA library and its 5'-flanking region was analyzed. The gene was found to span about 8 kb and to be composed of only four exons. Characterization of 5'-flanking region indicated that hST8Sia III promoter is embeded in a GC-rich domain and contains no TATA or CAAT box but has putative binding sites for transcription factors Sp 1 and AP-2. hST8Sia V genomic gene was isloated from human genomic DNA library and its intron/exon structure was analyzed. The gene was found to span about 45 kb and to be composed of five exons.
7. Developmental patterns of Galβ1,3(4)GlcNAc α2,3-sialyltransferase (ST3Gal III) expression in the mouse: in situ hybridization using DIG-labeled RNA probes:The appearance and differential distribution of ST3GalIII mRNA during the mouse embryogenesis [embryonic (E) days E9,. Ell, E13, E15J was investigated by in situ hybridization with DIG-labeled RNA probes. On E9, all tissues were positive for ST3GalIII mRNA expression. whereas ST3Gal III mRNA on E11 was not detected throughout all tissues. On E13, ST3Gal III mRNA was expressed in different manner in various tissues. In this stage, ST3Gal III mRNA was positive only in the liver, pancreas and bladder. On E15. specific signal was detected in the liver, lung and forebrain.
8. Modulation of GlcNAc-T-V activity and cancer metastasis by Oriental medicines such as Trichosanthes kirilowii Maxim(TKM) and Scutellaria baicalensis Georgi (SG) extracts: To examine the above mentioned cellular mechanism. the activity of GlcNAcT-V on several tumor cells such as Melanoma B-16, was examined after incubation with IL-1β, TKM and SG extracts severely inhibited the [³H] thymidine uptake of the cancer cell and TKM and SG extracts appears to inhibit the proliferation. In addition. TKM and se extracts severely decreased viability and inhibited the proliferation of Melanoma B-16. TKM and SG extracts-treated cell lines showed low levels of secretion of GlcNAcT-V mRNA transcription as elucidated by RT-PCR. Thus, with together lower GlcNAcT-V activity levels in the medium, TKM and SG extracts was highly effective for lung cancer metastasis treatment and it was concluded that the medicine can be used as a potent anti-lung cancer metastasis medicine.

목차 Contents

• 표 지 ... 1
• 제 출 문 ... 3
• 요 약 문 ... 4
• 영 문 요 약 문 ... 9
• 목 차 ... 11
• 제 1 장 서론 ... 12
• 제 1 절 연구개발 목표 ... 12
• 제 2 절 연구개발의 목적 및 필요성 ... 12
• 제 2 장 국내·외 기술개발 현황 ... 15
• 제 3 장 연구개발수행 내용 및 결과 ... 17
• 제 1 절: 총괄과제 및 제 1 세부 과제: 인체암전이 및 악성화관련 당쇄전이유전자를 이용한 간암 및 폐암의 새로운 치료기술개발 ... 17
• 제 2 절 위탁연구과제: 암전이 및 악성화저해 한약제재 탐색 및 개발 ... 69
• 제 4 장 연구개발목표 달성도 및 대외기여도 ... 106
• 제 1 절 목표달성도 ... 106
• 제 2 절 대외기여도 ... 107
• 제 5 장 연구개발결과의 활용성과 및 계획 ... 108
• 제 6 장 기타 중요변경사항 ... 110
• 제 7 장 참고문헌 ... 111
• 최종보고서 요약 (초록) ... 114
• 끝페이지 ... 114