초록 ▼

1. 연구개발목표 및 내용
HSP27에 기인한 방사선 내성을 극복하는 Peptide 발굴
- 방사선 내성을 유도하는 단백질의 각종 암종에서의 발현 규명
- 내성 단백질 결합 단백질의 결합 부위 규명
- 내성 단백질 결합 peptide 발굴 및 유용성 연구
2. 연구결과
1. 다양한 암종 (폐암, 자궁암, 유방암)에서 내성 단백질 발현 양상 규명
2. 세포주를 이용한 방사선 내성정도 및 내성 단백질 발현도 측정
3. 내성 유도 단백질 결합 단백질의 유전자 sequence 분석
4. 내성

1. 연구개발목표 및 내용
HSP27에 기인한 방사선 내성을 극복하는 Peptide 발굴
- 방사선 내성을 유도하는 단백질의 각종 암종에서의 발현 규명
- 내성 단백질 결합 단백질의 결합 부위 규명
- 내성 단백질 결합 peptide 발굴 및 유용성 연구
2. 연구결과
1. 다양한 암종 (폐암, 자궁암, 유방암)에서 내성 단백질 발현 양상 규명
2. 세포주를 이용한 방사선 내성정도 및 내성 단백질 발현도 측정
3. 내성 유도 단백질 결합 단백질의 유전자 sequence 분석
4. 내성유도 단백질의 단백질 결합에 따른 방사선 내성도 측정
5. 내성 단백질 결합 단백 결합부위에 대한 peptide 규명
6. Peptide의 방사선 내성 극복 유용성 연구 (in vitro 및 in vivo 실험)
3. 기대효과 및 활용방안
${\bullet}$ 새로운 타겟을 이용한 방사선 내성 진단 및 치료제 개발.
${\bullet}$ HSP27의 경우 HSP27 단일 단백질이 관여하는 기전이 다양하므로 한가지 단백질 발현을 예측 또는 제어하므로써 암의 다양한 성질은 비교적 쉽게 예측 및 치료가능.
${\bullet}$ 현재 oncogene을 target으로 하는 peptide 치료제는 새로운 방사선 치료증진제 시장으로 대두되고 있으면 HSP27을 target으로 하는 peptide 개발은 전세계적으로 전혀 이루어지고 있지 않은 실정이므로 새로운 단백질을 대상으로한 peptide 개발
${\bullet}$ HSP27의 상호작용 연구를 통한 효율적 방사선치료효율증진기술의 개발은 HSP27 단백을 target으로여 천연물유래 항암제의 개발, 방사선 및 항암제 치료 내성극복 기전연구를 통한 치료효율의 증대, 혈액을 통한 특정암 (특히 폐암)의 진단 등에 관한 연구에 활용될 예정이며, 이에 따라 천연물신약 관련 산업에서 새로운 작용기전을 가진 항암제, 항암치료 보조제의 개발 및 암진단 산업과 관련하여 유전자 칩, 진단키트 등의 개발에 응용될 수 있을 것이다. 암은 현재 한국인 사망원인 1위의 질병이며 전 세계적으로 매년 약 3천만명씩 암발생 인구가 증가할 것으로 예상된다. 따라서 항암제, 치료보조제 및 암진단과 관련한 수요가 폭발적으로 늘어날 것으로 예상되며, 실제로 항암제시장 성장률은 15%로서 의약품 시장 평균 성장률을 능가할 뿐만 아니라 모든 약효군 중에서 가장 높은 성장률을 보이고 있다. 또한 시장장규모는 현재 300억 달러대에서 2008년경에는 600억 달러 안팎으로 2배의 급성장이 가능할 것으로 예상된다. 국내 항암제시장의 경우 현재 3000억원대 규모로 연평균 10%대의 성장률을 보이고있어 역시 급성장이 예상됨.

Abstract ▼

Cell culture. Human non-small cell lung cancer cell lines, NCI-H23,-H358,-H460, -H596 and -H1299 cells, were grown in RPMI 1640 supplemented with 10% FBS, glutamine, HEPES, and antibiotics at 37C in a 5% CO2 humidified incubator. L929 (murine fibroblast) cells were cultured in Dulbecco`s minimal ess

Cell culture. Human non-small cell lung cancer cell lines, NCI-H23,-H358,-H460, -H596 and -H1299 cells, were grown in RPMI 1640 supplemented with 10% FBS, glutamine, HEPES, and antibiotics at 37C in a 5% CO2 humidified incubator. L929 (murine fibroblast) cells were cultured in Dulbecco`s minimal essential medium (DMEM) (GIBCO, Gaithersburg, MD) supplemented with 10% FBS, glutamine, HEPES, and antibiotics at 37C in a 5% CO2 humidified incubator.
Chemicals and reagents. Anti-HSP27, anti-actin, anti-PKC and anti-GST were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytochrome C (Pharmingen, San Diego, CA, USA) was also used. Cisplatin was purchased from Calbiochem (La Jolla, CA, USA). Anti-biotin, streptavidin-linked agarose beads, cisplatin and taxol were from Sigma (St. Louis, MO, USA).
Plasmids. Wild type PKC (GenBankTM accession number Ay545076), the catalytic domain (amino acids 334-674,CAT), and the dominant-negative catalytic domain (amino acids 334-674, CAT-KR) were cloned into pcDNA3 that contains a C-terminal HA tag. To construct tile PKC-deletion mutants, each DNA fragment was amplified using mutagenic primers containing an EcoRI site by PCR. The amplified PKCmutants were also cloned into pcDNA3 that contains a GST tag. Wild type mouse HSP25 (GenBankTM accession number XM124655) was cloned into pcDNA4HisMaxC (Invitrogen, Carlsbad, CA, USA), which contains an N-terminal His tag (23).
Irradiation. Cells were plated in 60-mm dishes and incubated at 37C under humidified 5% CO2 ill culture medium until 70-80% confluent. Cells were then exposed to -rays with 137Cs -ray source (Atomic Energy of Canada, Ltd., Canada) with a dose rate of 3.81Gy/min.
Cell transfection. Pre-designed siRNA for human HSP27 (5`-GUUCAAAGCAACCACCUGUtt-3`) was purchased from Ambion, Inc. (Ambion, Austin, TX, USA) and negative control siRNA (5`-UAGCGACUAAACACAUCAA-3`) was purchased from Dharmacon, Inc. (Dharmacon, Lafayette, Colorado, USA).
Colony-forming assay. Clonogenicity was examined by the colony-forming assay as described previously. Cells were seeded into 60-mm dishes at a density to produce about 500 colonies per dish in the control and were incubated for 714 days. Colonies were fixed with a mixture of 75% methanol and 25% acetic acid, and stained with 0.4% tryphan blue. The number of colonies consisting of 50 or more was scored.
MTT assay. After cisplatin treatment, the cells were assayed for their growth activity, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, MO, USA] test. The MTT test was performed as described previously.
Flow cytometry. Cells were cultured, harvested at the indicated times, stained with propidium iodide (PI, 1g/ml) according to the manufacture`s protocol, and then analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin, Lakes, NJ, USA).
Immnunoblotting For polyacrylamide gel electrophoresis (PAGE) and Western blotting, cells were solubilized with lysis buffer [120mM NaCl, 40mM Tris (pH 8.0), 0.1% NP40]. Immunoblotting was performed as described previously.
Immunoprecipitation. Cells $(1\times107)$ were lysed in immunoprecipitation buffer [50mM HEPES (pH 7.6), 150mM NaCl, 5mM EDTA, 0.1% NP-40]. After centrifugation (10min at 15,000 g) to remove particulate material, the supernatant was incubated with antibodies (1:100) with constant agitation at 4C. The immunocomplexes were precipitated with protein ASepharose (Sigma) and analyzed by immunoblotting.
PKC assay. Cellular proteins were extracted by PKC extraction buffer [50mM HEPES (pH 7.5), 150mM NaCl, 0.1% Tureen 20, 1mM EDTA, 2.5mM EGTA and 10% glycerol], containing protease inhibitors (10mg/ml each of aprotinin and leupeptin, and 0.1mM phenylmethylsufonyl fluoride) and phosphatase inhibitors (1mM NaF, 0.1mM Na3VO4 and 10mM b-glycerophosphate).
Immunofluorescence analysis. Cells transfected with FITC-peptides were analyzed by a confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).
Immunohistochemistry. Tissue microarray of lung tissues was used for the immunostainning of HSP27 Tissue microarray of human lung cancers was purchased from US biomax Inc. (MD, USA). Tumor slides were deparaffinized and rehydrated using xylene and alcohol, and for immunoperoxidase labeling, endogenous peroxidase was blocked with 0.3% H2O2 in absolute methanol for 15min at room temperature.
Histidine pull-down assay. After transfection with Histidine-tagged DNA for 48hrs, cells were harvested, and whole cell extracts were prepared as described above. Cell extracts were mixed and incubated with nickel-nitrilotriacetic acid-agarose beads for 30min at 4C in the presence of 10mM imidazole. After washing the resin with buffer containing 10mM imidazole, proteins were recovered by suspending in Laemmli sample buffer and were then subjected to SDS-PAGE and Western blot analysis using anti-GST antibodies.
Biotinylated peptides binding assay. Biotin-labelled peptides (Peptron, Daejeon, Korea) were transfected into cells by using LipofectamineTM 2000 (Invitrogen). After incubation for 24hrs, cells were harvested, and whole cell extracts were prepared as described above.
Tumor xenografts in nude mice. A single cell suspension ($3\times106$ cells) with a viability of 95% was subcutaneously injected into the hind leg of 5-week-old BALB/c athymic nude mice (Charles River, Japan).

목차 Contents

• 표지...1
• 제출문...2
• 최종연구보고서 초록...3
• 요약문...4
• SUMMARY...10
• CONTENTS...22
• 목차...23
• 제 1 장 연구개발과제의 개요...24
• 제 2 장 국내외 기술개발 현황...26
• 제 3 장 연구개발수행 내용 및 결과...28
• 제 4 장 목표달성도 및 관련분야에의 기여도...55
• 제 5 장 연구개발결과의 활용계획...58
• 제 6 장 연구개발과정에서의 수집한 해외과학기술정보...59
• 제 7 장 Reference...60