跳到主要內容

臺灣博碩士論文加值系統

(3.235.56.11) 您好!臺灣時間:2021/07/29 11:02
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:林坤陽
研究生(外文):Kun-Yang Lin
論文名稱:細胞普恩蛋白在腫瘤細胞侵犯時所扮演的角色
論文名稱(外文):The Roles of Cellular Prion Protein in Cancer Cell Invasion
指導教授:周德盈
指導教授(外文):Teh-Ying Chou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:67
中文關鍵詞:普恩蛋白腫瘤細胞侵犯能力扇狀偽足
外文關鍵詞:Prion proteintumorcell invasionlamellipodium
相關次數:
  • 被引用被引用:0
  • 點閱點閱:151
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
普恩蛋白( Prion protein )是一個透過GPI( glycosylphosphatidylinositol )結構固定在細胞膜上的膜蛋白,並且在哺乳動物中具有高度保留性。目前正常的普恩蛋白被發現與細胞的訊息傳遞、存活、分化和癌症有其相關性。先前實驗室的研究顯示,在原位的和侵犯的肺腺癌組織中,我們發現普恩蛋白的訊息RNA( messenger RNA )在侵犯的肺腺癌組織中表現量比原位的肺腺癌組織高。在肺腺癌細胞株CL1-5中阻斷普恩蛋白( Prion protein )的表現後,發現細胞的移動與侵犯能力發生缺陷。再者,我們在肺腺癌細胞珠CL1-5中,觀察到普恩蛋白與鏈狀肌動蛋白( Filament Actin, F-Actin )共同出現在傘狀偽足( Lamellipodium )的地方;在細胞侵犯的過程中,扇狀偽足的形成是一個相當重要的步驟。綜合先前的研究,我們認為普恩蛋白在癌細胞侵犯時可能扮演一個重要的角色。
此篇研究中,我們首先利用Swinholide A 破壞CL1-5中的鏈狀肌動蛋白,發現破壞鏈狀肌動蛋白後會導致普恩蛋白無法聚集在扇狀偽足處;此外,在CL1-5中,我們利用免疫共沉澱法發現普恩蛋白與肌動蛋白之間存在有交互作用,但在F-Actin共沉降法( co-sedimentation )中,普恩蛋白未出現與肌動蛋白有共同沉降的現象。我們發現在肺腺癌細胞株CL1-5中阻斷普恩蛋白表現後,出現扇狀偽足的細胞明顯減少,Rho family GTPase的成員- Rac1的活性則有下降的趨勢;我們亦觀察到在肺腺癌細胞株CL1-5中阻斷普恩蛋白表現後,出現壓力絲( Stress fiber )的細胞明顯增加。再者,我們將普恩蛋白或刪除signal peptide的普恩蛋白轉染至阻斷普恩蛋白表現的穩定細胞株中表現,皆可恢復細胞爬行、化學趨向與侵犯的能力;但無法接上GPI-anchor的普恩蛋白只能恢復細胞爬行和侵犯的能力。綜合此篇研究結果,我們觀察到破壞鏈狀肌動蛋白後,普恩蛋白即無法在扇狀偽足處聚集,並且發現普恩蛋白與肌動蛋白之間有交互作用;此外,普恩蛋白可能參與在調節Rac1活性的路徑之中,影響扇狀偽足的形成。另一方面,位在細胞膜和細胞質的普恩蛋白皆可能在腫瘤細胞的爬行、化學趨向和侵犯能力中扮演重要的角色,而位在細胞外的普恩蛋白則僅能影響腫瘤細胞的爬行和侵犯能力。
Prion protein ( PrP ) is a glycosylphosphatidylinositol(GPI) -anchored membrane protein that is highly conserved in mammalian species. The putative roles of cellular Prion protein (PrPc) are thought to be related to cell signaling, survival, and differentiation and cancer progression. In previous studies, we found higher expression of Prion mRNA in the invasive tumor cells than the in situ tumor cells. In lung adenocarcinoma cell line CL1-5, we demonstrated that Prion-knocked down cells exhibit migration and invasion defect. We showed that Prion protein co-localizes with F-actin in lamellipodia structure, which is one of the most important structures during cell invasion. Here, we investigated the possible role of PrPc in the process of cell invasion in lung cancers.
In this study, we found that disruption of F-actin using the actin-dissembling agent, Swinholide A, prevents lamellipodia localization of Prion protein . We further demonstrated that Prion protein interacts with actin in vivo in a co-immunoprecipitation assay, but does not co-cosediment with F-actin. We showed that Prion silencing decreases numbers of lamellipodia and diminishes Rac1 activity in human lung adenocarcinoma cells, meanwhile , Prion silencing increases the production of stress fiber. We found that expression of Prion protein ( Membrane ) or Prion△SP ( cytoplasm ) restores migration, chemotaxis and invasion ability of PRNPi-2 cells, while expression of Prion△GPI ( extracellular ) only restores migration and invasion ability of PRNPi-2 cells.
Taken together, We found that disruption of F-actin prevents lamellipodia localization of Prion protein and Prion protein interacts with actin. In addition, Prion protein may be involves in Rac1 regulation pathway and affects lamellipodium formation. On the other hand, membrane and cytoplasmic Prion proteins are important for tumor cell migration, chemotaxis and invasion; extracellular Prion protein may only affect tumor cell migration and invasion.
中文摘要.........................................................4
英文摘要.........................................................5
壹、 緒論........................................................6
一、 肺癌
二、 腫瘤細胞的侵犯與轉移
三、 普恩蛋白( Prion protein )
四、 普恩蛋白與腫瘤之關係
五、 Rho family GTPases與肌動蛋白( Actin )的結構
貳、 實驗材料與方法..................................12
一、 實驗材料
二、 實驗方法
參、 結果......................................................24
一、普恩蛋白與肌動蛋白的結構和動力學之關連性
二、普恩蛋白與肌動蛋白之間的交互作用
三、阻斷普恩蛋白的表現導致肌動蛋白結構發生重排( rearrangement )
四、在PRNPi-2細胞株中大量表現普恩蛋白可以恢復扇狀偽足的形成
五、阻斷普恩蛋白的表現導致Rac1活性下降
六、建構表現於細胞中不同位置的普恩蛋白建構體
七、不同普恩蛋白建構體在細胞中的位置
八、表現不同的PrP建構體皆能恢復PRNPi-2細胞株的爬行能力
九、表現的PrP或PrP△SP建構體皆能恢復PRNPi-2細胞株的化學趨向
能力,PrP△GPI則無法恢復
十、表現不同的PrP建構體皆能恢復PRNPi-2細胞株的侵犯能力
十一、表現不同的PrP建構體皆能恢復PRNPi-2細胞株之扇狀偽足的形成
十二、阻斷普恩蛋白的表現和表現不同的PrP建構體皆不會影響細胞的生長速

肆、 討論......................................................31
一、普恩蛋白與肌動蛋白之關聯性
二、普恩蛋白與扇狀偽足和壓力絲之關聯性
三、可能參與在普恩蛋白影響扇狀偽足形成的訊號傳遞路徑
四、普恩蛋白的位置與細胞侵犯能力之關聯性
五、不同普恩蛋白的建構體及扇狀偽足之形成與細胞侵犯能力之關聯性
六、表現PrP△GPI無法恢復PRNPi-2細胞的化學趨向能力之可能因素
伍、 參考文獻..............................................36
陸、 圖表......................................................40
附錄...............................................................61
1. Bilello, K.S., Murin, S. & Matthay, R.A. Epidemiology, etiology, and prevention of lung cancer. Clin Chest Med 23, 1-25 (2002).
2. Steeg, P.S. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 12, 895-904 (2006).
3. Woodhouse, E.C., Chuaqui, R.F. & Liotta, L.A. General mechanisms of metastasis. Cancer 80, 1529-1537 (1997).
4. Bashyam, M.D. Understanding cancer metastasis: an urgent need for using differential gene expression analysis. Cancer 94, 1821-1829 (2002).
5. Chambers, A.F., Groom, A.C. & MacDonald, I.C. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2, 563-572 (2002).
6. Thiery, J.P. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2, 442-454 (2002).
7. Guarino, M. Epithelial-mesenchymal transition and tumour invasion. Int J Biochem Cell Biol 39, 2153-2160 (2007).
8. Kawamata, H. et al. Active-MMP2 in cancer cell nests of oral cancer patients: correlation with lymph node metastasis. Int J Oncol 13, 699-704 (1998).
9. van Kempen, L.C. & Coussens, L.M. MMP9 potentiates pulmonary metastasis formation. Cancer Cell 2, 251-252 (2002).
10. Frixen, U.H. et al. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol 113, 173-185 (1991).
11. Danen, E.H. et al. E-cadherin expression in human melanoma. Melanoma Res 6, 127-131 (1996).
12. Bergers, G. & Benjamin, L.E. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3, 401-410 (2003).
13. Dvorak, H.F., Brown, L.F., Detmar, M. & Dvorak, A.M. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 146, 1029-1039 (1995).
14. Mountain, C.F. New prognostic factors in lung cancer. Biologic prophets of cancer cell aggression. Chest 108, 246-254 (1995).
15. Kozlow, W. & Guise, T.A. Breast cancer metastasis to bone: mechanisms of osteolysis and implications for therapy. J Mammary Gland Biol Neoplasia 10, 169-180 (2005).
16. Taylor, D.R. & Hooper, N.M. The prion protein and lipid rafts. Mol Membr Biol 23, 89-99 (2006).
17. Soto, C. Endoplasmic reticulum stress, PrP trafficking, and neurodegeneration. Dev Cell 15, 339-341 (2008).
18. Chakrabarti, O., Ashok, A. & Hegde, R.S. Prion protein biosynthesis and its emerging role in neurodegeneration. Trends Biochem Sci 34, 287-295 (2009).
19. Prusiner, S.B. Prions. Sci Am 251, 50-59 (1984).
20. Martins, V.R., Mercadante, A.F., Cabral, A.L., Freitas, A.R. & Castro, R.M. Insights into the physiological function of cellular prion protein. Braz J Med Biol Res 34, 585-595 (2001).
21. Pradines, E. et al. CREB-dependent gene regulation by prion protein: impact on MMP-9 and beta-dystroglycan. Cell Signal 20, 2050-2058 (2008).
22. Milhavet, O. & Lehmann, S. Oxidative stress and the prion protein in transmissible spongiform encephalopathies. Brain Res Brain Res Rev 38, 328-339 (2002).
23. Brown, D.R. & Besinger, A. Prion protein expression and superoxide dismutase activity. Biochem J 334 ( Pt 2), 423-429 (1998).
24. Weise, J. et al. Deletion of cellular prion protein results in reduced Akt activation, enhanced postischemic caspase-3 activation, and exacerbation of ischemic brain injury. Stroke 37, 1296-1300 (2006).
25. Kurschner, C. & Morgan, J.I. Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion protein. Brain Res Mol Brain Res 37, 249-258 (1996).
26. Du, J. et al. Overexpression and significance of prion protein in gastric cancer and multidrug-resistant gastric carcinoma cell line SGC7901/ADR. Int J Cancer 113, 213-220 (2005).
27. Liang, J. et al. Overexpression of PrPC and its antiapoptosis function in gastric cancer. Tumour Biol 27, 84-91 (2006).
28. Liang, J. et al. Cellular prion protein promotes proliferation and G1/S transition of human gastric cancer cells SGC7901 and AGS. FASEB J 21, 2247-2256 (2007).
29. Roucou, X. et al. Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells. Cell Death Differ 12, 783-795 (2005).
30. Pan, Y. et al. Cellular prion protein promotes invasion and metastasis of gastric cancer. FASEB J 20, 1886-1888 (2006).
31. Liang, J. et al. Hypoxia induced overexpression of PrP(C) in gastric cancer cell lines. Cancer Biol Ther 6, 769-774 (2007).
32. Liang, J. et al. Inhibition of PI3K/Akt partially leads to the inhibition of PrP(C)-induced drug resistance in gastric cancer cells. FEBS J 276, 685-694 (2009).
33. Li, Q.Q. et al. The role of P-glycoprotein/cellular prion protein interaction in multidrug-resistant breast cancer cells treated with paclitaxel. Cell Mol Life Sci 66, 504-515 (2009).
34. Raftopoulou, M. & Hall, A. Cell migration: Rho GTPases lead the way. Dev Biol 265, 23-32 (2004).
35. Burridge, K. & Wennerberg, K. Rho and Rac take center stage. Cell 116, 167-179 (2004).
36. Chhabra, E.S. & Higgs, H.N. The many faces of actin: matching assembly factors with cellular structures. Nat Cell Biol 9, 1110-1121 (2007).
37. Kustermans, G. et al. Actin cytoskeleton differentially modulates NF-kappaB-mediated IL-8 expression in myelomonocytic cells. Biochem Pharmacol 76, 1214-1228 (2008).
38. Keshet, G.I., Bar-Peled, O., Yaffe, D., Nudel, U. & Gabizon, R. The cellular prion protein colocalizes with the dystroglycan complex in the brain. J Neurochem 75, 1889-1897 (2000).
39. Morel, E. et al. The cellular prion protein PrP(c) is involved in the proliferation of epithelial cells and in the distribution of junction-associated proteins. PLoS One 3, e3000 (2008).
40. Shintani, Y., Wheelock, M.J. & Johnson, K.R. Phosphoinositide-3 kinase-Rac1-c-Jun NH2-terminal kinase signaling mediates collagen I-induced cell scattering and up-regulation of N-cadherin expression in mouse mammary epithelial cells. Mol Biol Cell 17, 2963-2975 (2006).
41. Ma, J., Wollmann, R. & Lindquist, S. Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 298, 1781-1785 (2002).
42. Mironov, A., Jr. et al. Cytosolic prion protein in neurons. J Neurosci 23, 7183-7193 (2003).
43. Roucou, X., Guo, Q., Zhang, Y., Goodyer, C.G. & LeBlanc, A.C. Cytosolic prion protein is not toxic and protects against Bax-mediated cell death in human primary neurons. J Biol Chem 278, 40877-40881 (2003).
44. Grenier, C., Bissonnette, C., Volkov, L. & Roucou, X. Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells. J Neurochem 97, 1456-1466 (2006).
45. Norstrom, E.M., Ciaccio, M.F., Rassbach, B., Wollmann, R. & Mastrianni, J.A. Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation. J Virol 81, 2831-2837 (2007).
46. Lin, D.T., Jodoin, J., Baril, M., Goodyer, C.G. & Leblanc, A.C. Cytosolic prion protein is the predominant anti-Bax prion protein form: exclusion of transmembrane and secreted prion protein forms in the anti-Bax function. Biochim Biophys Acta 1783, 2001-2012 (2008).
47. Wang, X. et al. Cytoplasmic prion protein induces forebrain neurotoxicity. Biochim Biophys Acta 1792, 555-563 (2009).
48. Campana, V. et al. Characterization of the properties and trafficking of an anchorless form of the prion protein. J Biol Chem 282, 22747-22756 (2007).
49. Ashok, A. & Hegde, R.S. Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation. Mol Biol Cell 19, 3463-3476 (2008).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top