Actin Binding Protein SM22α(Transgelin)의 방사선 및 Stress 저항성 기능 연구

다국어 초록 (Multilingual Abstract)

Transgelin, which is expressed abundantly in smooth muscles and thus has been referred to as a smooth muscle protein 22KDa(SM22α), may be hypothesized to affect the structure and function of the actin filament. However, little is known regarding the direct involvement of SM22α in a cell proliferation and resistance. In this study, we report on the significant induction of SM22α by cytotoxic drugs such as luteolin, a natural flavonoid, in HepG2 cell. SM22α-overexpression inhibited the activation of IGF-1Rβ/AKT and ERK, consequently suppressing cell proliferation. On the other hand, SM22α-overexpressing cells became resistant to apoptotic cell death caused by a variety of cytotoxic agents including γ-radiation. On the contrary, siRNA application to suppress SM22α expression sensitizes the cells to the effect of damaging agents. Interestingly, as a result of real time PCR analysis, SM22α affects the induction of metallothionein(MT) isoforms, especially MT1G and MT1F, which are also induced by luteolin in HepG2 cells. We showed that MT1G overexpression also confers chemo- or radio-resistance to the cells. Increased expression of MT isoforms caused by SM22α overexpression did not result from the demethylation of hypermethylation of the promoter regions which are shown in the MT gene families of hepatocellular carcinoma cells. We confirmed that SM22α may increase the MT isoforms including MT1G at a transcriptional level by using a reporter assay. Until now, the gene or protein that could influence MT expression in human tumors was not yet understood. In this aspect, SM22α is the first protein shown to regulate gene expression of the MT isoforms. SM22α/MT/p53 signal may play an important role in protection against damage agent-induced cell death. Here we also report on the significant induction of SM22α by hypoxia and cytotoxic agents in lung carcinoma cell A549 and H1299 cells. Little is known regarding hypoxic regulation of SM22α gene, and its direct involvement of resistance or tolerance in cancer cell. SM22α is new important hypoxia inducible transcription factor in lung carcinoma cell. Cells with SM22α overexpression, which did not transform cells into senescence state, became resistant or tolerant to cell death caused by a variety of DNA damaging agents such as ionizing radiation, anti-cancer drug and toxic chemicals. SM22α was induced by hypoxia per se rather than by the secondary effects of hypoxia. HIF-1α, the primary transcription factor that is responsible for multiple gene activation under hypoxia, does not have a role in SM22α expression. Moreover, SM22α was also not involved in the HIF-1α induction or stabilization. In this study, SM22α overexpression markedly phosphorylated IGF-IRβ and activation of the downstream molecule AKT in two human lung adenocarcinoma cell lines (A549 and H1299). These results indicate that Radio- and chemo-protective effect by SM22αinduction in hypoxic A549 cells may be performed by SM22α/P-IGF-IRβ/P-AKT signal. SM22α-overexpression significantly induce the activation of IGF-1R/AKT via direct interaction with IGF-1Rβ. Thus, this study not only provides the first demonstration of severe hypoxic regulation of SM22α but also suggests the participation of novel mechanisms about resistance to ionizing radiation and anticancer drugs caused by hypoxia. SM22α is a new protein shown to regulate gene activation of the IGF-1Rβ in lung carcinoma cells.
The expression of down-stream genes regulated by SM22α is different in hepatocellular carcinoma HepG2 cells and lung carcinoma A549 cells. Because SM22α was translocated to the nucleus through Lysine methylation at the N-terminal domain of human SM22α in HepG2 cells.
Resistance or tolerance to chemotherapy and radiotherapy is a major obstacle in treating malignant cancer. Based on our studies, SM22α may be able to mediate the activation of genes essential for cell survival against cytotoxic drugs and thus assist selection for resistance or tolerance. In conclusion, SM22α is a protein newly identified as playing a crucial role in blocking cell proliferation and in resistance or tolerance to overcome detrimental effect of damaging agent such as ionizing radiation and anticancer drugs. This study suggests novel mechanisms for resistance or tolerance to damaging agents.

목차 (Table of Contents)

• 목 차
• 목 차 ⅰ
• LIST OF FIGURES ⅳ
• LIST OF TABLES ⅵ
• ABBREVIATIONS ⅶ
• I. 서 론 1
• II. 재료 및 방법 21
• 1. 세포 배양 21
• 2. 무산소 혹은 저산소 환경에서 세포 배양 21
• 3. RNA 추출 22
• 4. 역전사 중합효소 연쇄반응(RT-PCR) 22
• 5. 실시간 중합효소 연쇄반응(real-time PCR) 분석 23
• 6. Sense SM22α 발현벡터와 Sense MT1G 발현벡터의
• 제작 및 형질도입 25
• 7. Western blotting 29
• 8. Cisplatin, Menadione, H2O2 처리 및 감마방사선 조사 30
• 9. 유성세포 분석을 이용한 세포사멸, 세포주기 분석 30
• 10. β-galactosidase 활성 염색법을 이용한 세포 노화 (Senescence) 측정 31
• 11. SM22α-siRNA의 제작 및 형질도입 31
• 12. In Situ Hybridization 32
• 13. Methylation-specific PCR(MSP) assay 34
• 14. Luciferase Reporter assay 37
• 15. IGF-IR 억제제인 AG1024의 처리 38
• 16. 세포성장률의 측정 및 생존세포 식별을 위한 Crystal Violet 염색법 38
• 17. 면역 침강법(Immunoprecipitation) 38
• 18. Colony forming assay 39
• 19. 항원의 세포내 발현위치 확인을 위한 세포내 형광염색
• 40
• Ⅲ. 결 과 41
• 1. 간암세포주에서 SM22α 기능 연구 41
• 1-1. SM22α의 과발현에 의한 세포증식 억제와 저항성 기작 41
• 1-2. IGF-1Rβ/AKT와 ERK 신호기작의 억제자로 작용하는 SM22α 47
• 1-3. MT isoforms 유도자 기능을 하는 SM22α 51
• 1-4. 전사 수준에서 조절되며, 여러 세포독성물질에
• 대한 방어기작을 갖는 MT1G 54
• 1-5. SM22α 단백질의 신호전달 모식도 58
• 2. 폐암세포주에서의 SM22α 기능 연구 61
• 2-1. 무산소와 1% 저산소에 의한 SM22α의 발현 유도 61
• 2-2. 다양한 스트레스원에 의한 SM22α의 발현 유도 64
• 2-3. SM22α 형질 도입 세포의 선별, 노화 연관성과 스
• 트레스원에 대한 민감도 분석 64
• 2-4. SM22α의 발현억제와 스트레스원에 대한 민감도 분석 69
• 2-5. 폐암 세포주에서 SM22α 발현과 AKT 인산화 조절 72
• 2-6. 폐암 세포주에서 SM22α 발현과 IGF-1R 인산화 조절 73
• 2-7. IGF-IR 억제제 처리와 신호기작 78
• 2-8. SM22α와 IGF-1Rβ의 직접적인 상호작용 여부 분석 78
• 2-9. SM22α 발현억제에 따른 IGF-1Rβ과의 상호작용
• 억제 82
• 2-10. SM22α의 점 돌연변이(point mutation) 85
• 2-11. ERK 신호기작 억제로 세포증식을 억제하는 SM22α
• 88
• 2-12. SM22α의 발현억제에 따른 세포증식의 감소 88
• 2-13. SM22α 단백질의 신호전달 모식도 92
• 3. 간암세포주 HepG2와 폐암세포주 A549에서 서로 다른 SM22α 관련 신호기작 원인 규명 연구 94
• IV. 논 의 99
• Ⅴ. 결 론 106
• VI. 참 고 문 헌 108
• ABSTRACT 134