跳到主要內容

臺灣博碩士論文加值系統

(44.212.99.208) 您好!臺灣時間:2024/04/17 17:12
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:林錩岳
研究生(外文):Chang-Yueh Lin
論文名稱:針對腎細胞癌表現上升之CD74基因的功能性研究
論文名稱(外文):Studies on the Function of CD74 up-regulated in Renal Cell Carcinoma
指導教授:林榮耀林榮耀引用關係
指導教授(外文):Jung-Yaw Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
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:2005
畢業學年度:93
語文別:英文
論文頁數:60
中文關鍵詞:腎細胞癌巨噬細胞遷移抑制因子致癌基因
外文關鍵詞:CD74RCCMIFp53Hmd2ERK
相關次數:
  • 被引用被引用:0
  • 點閱點閱:282
  • 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
腎細胞癌被認為是人類腎臟癌的主要型,它的最大特徵是具高度的致死率以及對化學性及放射性治療有高度的抗拒性。據估計,轉移性腎細胞癌病患可以存活五年以上者的機率乃低於百分之十。由於腎細胞癌發生早期缺乏明顯的病徵,以至於當患者發現致病時,通常癌細胞已惡化或轉移。因此癌症學者正積極致力於尋找其分子標誌及其探討治病機制。
CD74 一般會表現在抗原呈現細胞中,諸如: 巨噬細胞、B 細胞 以及 樹突細胞。一旦CD74 表現後,它會與第二型主要組織相容複合體 (MHC class II) 結合在一起並協助複合體在細胞內的運送、摺疊以及防止複合體與外來抗原不正常的結合。此外,研究發現CD74也會表現在MHC class II缺乏的細胞體及許多的肉瘤組織內,根據這些現象推論除了它的已知活性外CD74很有可能還具有未被發現的蛋白質功能。
巨噬細胞遷移抑制因子 (MIF) 是一種原發炎反應細胞素,其具有調控發炎及免疫反應的作用。最近的研究顯示,在纖維母細胞中的巨噬細胞遷移抑制因子會促進ERK1/2 MAP kinase的訊息活化、細胞增生及PGE2的產生,而巨噬細胞遷移抑制因子所造成的細胞活化乃是透過巨噬細胞遷移抑制因子與CD74鍵結而起始的。最近巨噬細胞遷移抑制因子被認為是一個會促進腫瘤生長的蛋白,然而,是否CD74也參與了巨噬細胞遷移抑制因子在促進腫瘤生長上的調控作用目

前仍然不清楚。
在唐賽文先生之前構築的腎細胞癌全長cDNA庫中發現,CD74基因的轉錄調控呈現過度表現之狀況。本研究發現,在許多臨床的配對採集樣本中,CD74基因之過度轉錄及轉譯狀況是普遍發生於這些腎細胞癌組織中的。針對CD74的功能性研究上,過度表現CD74會刺激ERK1/2 及 p38 訊息的活化,這可能是因為內生性分泌的巨噬細胞遷移抑制因子以自我活化的方式鍵結到細胞膜上的CD74所造成的結果。值得一提的是,腫瘤抑制基因p53之表現量會因為CD74的過度表現而被壓制,然而CD74過度表現卻會促使Hmd2表現量上升,這表示CD74調控可能是透過調控Hmd2表現量上升而促進p53降解最後造成細胞內p53蛋白量下降的結果,另外在HeLa 細胞中我們亦發現,CD74蛋白的胺端會被蛋白質水解脢斷裂並釋放出其N端片段 (NTF) 而移行入細胞核內。綜合實驗結果,我們初步認為過度表現CD74在腫瘤生成上的機制可能是透過MIF的合作性調節而活化ERK1/2 MAPK 及p38訊息,並造成Hmd2表現量的上升及p53的降解,然而CD74經水解後釋放出的N端片段 (NTF) 可能是CD74下游的作用子,而調控腫瘤生長相關的基因表現。
Renal cell carcinoma (RCC) was thought to be one of major types of human kidney cancers, and it is characterized by a high mortality rate and a high resistance to chemotherapeutic treatment as well as radiations. However, high occurrence of advanced and metstatic RCC often attributed to the absence of early warning signs and thus, the oncogenists have taken a lot of efforts to identify the molecular marker and investigate the mechanism of tumorigenesis of RCC.
CD74 is expressed in antigen-presenting cells (APCs) such as macrophages, B-cells and dendritic cells. Once synthesized, CD74 is associated with major histocompatibility complex (MHC) class II and assists class II trafficking, class II folding and avoidance of abnormal-antigen binding. In addition, CD74 was found to be expressed in several types of carcinoma and class II negative cells inferring that CD74 may has a novel function besides to its class II chaperon activity.
By binding to CD74, MIF was demonstrated to elicit ERK1/2 MAP Kinase signaling, cell proliferation and PGE2 production. However, MIF has been described to be a tumor-promoting protein, and whether CD74 involves in MIF for promoting tumor growth is still needed to be studied
Transcriptional up-regulation of CD74 was found previously in full-length cDNA library of a RCC tissue by Mr. S. W. Tang in this laboratory. In this study, up-regulation of CD74 was generally found in mRNA level and protein level of several RCC tissue pairs. In functional study, overexpression of CD74 stimulated ERK1/2 and p38 signaling, which could be initiated by binding of MIFs to CD74 on the cell surface in an autocrine fashion. Surprisely, it was found that tumor suppressor p53 protein was decreased by expression of CD74, and it was accompanied by up-regulation of Hmd2. Also, the N-terminal proteolytically cleavage of CD74 carried out in HeLa cells, and the cleavage fragment (1a.a-58a.a, NTF) could translocate to nucleus. Taken together, we suggested that overexpression of CD74 led to the binding of MIF to CD74 on the cell surface in an autocrine manner, resulting in the activation of ERK1/2 and p38 signaling. In addition, overexpression of CD74 could also up-regulate Hmd2 and the down-regulate p53.
Abbreviations……………….………………………….………………….iii
摘要……………………..……………………………….…………….…..v
Abstract………………………………………………………...……..….vii
Introduction………………………………………………………….…….1
Materials and Methods……………………….……………………………5
1. Materials…………………….…………………………………………5
2. Construction of expression vectors…………………..……………………5
3. Plasmids maxi-preparation by ultracentrifugation……………………...…11
4. One step purification of MIFs from conditioned medium……………..……13
5. Transient transfection and protein expression………………………...…...14
6. Immunofluorescence and confocal microscopy…………………..…...…20
7. Co-immunoprecipitation assay………………………………..…….…21
8. RNAs extraction, reverse transcription and real-time PCR……….…………23
Results………………………………………………….……...…………26
1. RNA level of CD 74 was generally up-regulated in RCC tissues. ..…..…….. 26
2. CD74 protein was up-regulated in RCC tissue. ………………..……….....26
3. Cloning of CD74 and CD7417-232 cDNA into the expression vector. …………27
4. Transient transfection and protein expression in HeLa cells. ……………….27
5. CD74 was localized in ER, endosome, perinuclear region and plasma
membrane. ………………......………………………………………..28
6. Overexpression of CD74 induced the persistent activation of the ERK1/2
and p38 MAPK pathway. …...……………………………..………...…28
7. CD74 interacted with endogenous MIF secreted by HeLa cells. ……......……..30
8. Overexpression of CD74 induced a decrease of p53 in protein level. ……..…31
9. p53 down-regulated by CD74 may be through an increase of Hmd2. ………..32
10. The cytoplasmic domain of CD74 were proteolytically released in HeLa cells..32
11. Released cytsolic domain of CD74 may migrate to nucleus. …………..…..32
Discussion…………………………………………….…………..………35
1. Increase of CD74 in RCC tissues. …………………………….…..…...35
2. CD74 directly stimulates ERK1/2 and p38 MAPK signaling? ……..….……36
3. CD74, a new p53 negative regulator? …………..……………..…………37
Figures and Legends………………………………………………………40
References……………………………………………………………...…54
1.Parkin, D.M., F. Bray, J. Ferlay, and P. Pisani. 2001. Estimating the world cancer burden: Globocan 2000. Int J Cancer 94:153-156.
2.Linchan, W.M., Shipley, W. U. and Parkinson. 1993. Cancer of the kidney and ureter. Cancer: Principle and Practice of oncology.
3.Strubin, M., C. Berte, and B. Mach. 1986. Alternative splicing and alternative initiation of translation explain the four forms of the Ia antigen-associated invariant chain. Embo J 5:3483-3488.
4.Ong, G.L., D.M. Goldenberg, H.J. Hansen, and M.J. Mattes. 1999. Cell surface expression and metabolism of major histocompatibility complex class II invariant chain (CD74) by diverse cell lines. Immunology 98:296-302.
5.Wistow, G.J., M.P. Shaughnessy, D.C. Lee, J. Hodin, and P.S. Zelenka. 1993. A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc Natl Acad Sci U S A 90:1272-1275.
6.Henne, C., F. Schwenk, N. Koch, and P. Moller. 1995. Surface expression of the invariant chain (CD74) is independent of concomitant expression of major histocompatibility complex class II antigens. Immunology 84:177-182.
7.Leng, L., C.N. Metz, Y. Fang, J. Xu, S. Donnelly, J. Baugh, T. Delohery, Y. Chen, R.A. Mitchell, and R. Bucala. 2003. MIF signal transduction initiated by binding to CD74. J Exp Med 197:1467-1476.
8.Chesney, J., C. Metz, M. Bacher, T. Peng, A. Meinhardt, and R. Bucala. 1999. An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma. Mol Med 5:181-191.
9.Beswick, E.J., D.A. Bland, G. Suarez, C.A. Barrera, X. Fan, and V.E. Reyes. 2005. Helicobacter pylori Binds to CD74 on Gastric Epithelial Cells and Stimulates Interleukin-8 Production. Infect Immun 73:2736-2743.
10.Matza, D., A. Kerem, H. Medvedovsky, F. Lantner, and I. Shachar. 2002. Invariant chain-induced B cell differentiation requires intramembrane proteolytic release of the cytosolic domain. Immunity 17:549-560.
11.Matza, D., F. Lantner, Y. Bogoch, L. Flaishon, R. Hershkoviz, and I. Shachar. 2002. Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity. Proc Natl Acad Sci U S A 99:3018-3023.
12.Hua, Z.X., K.E. Tanaka, H.D. Tazelaar, J. Myers, G.S. Markowitz, and A.C. Borczuk. 1998. Immunoreactivity for LN2 and LN3 distinguishes small cell carcinomas from non-small cell carcinomas in the lung. Hum Pathol 29:1441-1446.
13.Young, A.N., M.B. Amin, C.S. Moreno, S.D. Lim, C. Cohen, J.A. Petros, F.F. Marshall, and A.S. Neish. 2001. Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers. Am J Pathol 158:1639-1651.
14.Ishigami, S., S. Natsugoe, K. Tokuda, A. Nakajo, H. Iwashige, K. Aridome, S. Hokita, and T. Aikou. 2001. Invariant chain expression in gastric cancer. Cancer Lett 168:87-91.
15.Moller, P., T. Mattfeldt, C. Gross, P. Schlosshauer, A. Koch, K. Koretz, G. Moldenhauer, M. Kaufmann, and H.F. Otto. 1989. Expression of HLA-A, -B, -C, -DR, -DP, -DQ, and of HLA-D-associated invariant chain (Ii) in non-neoplastic mammary epithelium, fibroadenoma, adenoma, and carcinoma of the breast. Am J Pathol 135:73-83.
16.Lu, Y., G.D. Ussery, M. Jacim, M. Tschickardt, J.M. Boss, and G. Blanck. 1994. Retinoblastoma protein regulation of surface CD74 (invariant chain) expression in breast carcinoma cells. Mol Immunol 31:1365-1368.
17.Zhao, Y., D. Boczkowski, S.K. Nair, and E. Gilboa. 2003. Inhibition of invariant chain expression in dendritic cells presenting endogenous antigens stimulates CD4+ T-cell responses and tumor immunity. Blood 102:4137-4142.
18.Bernhagen, J., M. Bacher, T. Calandra, C.N. Metz, S.B. Doty, T. Donnelly, and R. Bucala. 1996. An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction. J Exp Med 183:277-282.
19.Bernhagen, J., T. Calandra, R.A. Mitchell, S.B. Martin, K.J. Tracey, W. Voelter, K.R. Manogue, A. Cerami, and R. Bucala. 1993. MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia. Nature 365:756-759.
20.Mikulowska, A., C.N. Metz, R. Bucala, and R. Holmdahl. 1997. Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice. J Immunol 158:5514-5517.
21.Mitchell, R.A., C.N. Metz, T. Peng, and R. Bucala. 1999. Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action. J Biol Chem 274:18100-18106.
22.Hudson, J.D., M.A. Shoaibi, R. Maestro, A. Carnero, G.J. Hannon, and D.H. Beach. 1999. A proinflammatory cytokine inhibits p53 tumor suppressor activity. J Exp Med 190:1375-1382.
23.Lavelle, F. 2000. [MIF and P53: 2 major partners at the inflammation and cancer crossroad]. Bull Cancer 87:133-134.
24.Shimizu, T., R. Abe, H. Nakamura, A. Ohkawara, M. Suzuki, and J. Nishihira. 1999. High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis. Biochem Biophys Res Commun 264:751-758.
25.Baugh, J.A., and R. Bucala. 2002. Macrophage migration inhibitory factor. Crit Care Med 30:S27-S35.
26.Blocki, F.A., L.B. Ellis, and L.P. Wackett. 1993. MIF protein are theta-class glutathione S-transferase homologs. Protein Sci 2:2095-2102.
27.Bernhagen, J., R.A. Mitchell, T. Calandra, W. Voelter, A. Cerami, and R. Bucala. 1994. Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF). Biochemistry 33:14144-14155.
28.Pieters, J., H. Horstmann, O. Bakke, G. Griffiths, and J. Lipp. 1991. Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain. J Cell Biol 115:1213-1223.
29.Troppmair, J., J.T. Bruder, H. Munoz, P.A. Lloyd, J. Kyriakis, P. Banerjee, J. Avruch, and U.R. Rapp. 1994. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J Biol Chem 269:7030-7035.
30.deFazio, A., Y.E. Chiew, R.L. Sini, P.W. Janes, and R.L. Sutherland. 2000. Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines. Int J Cancer 87:487-498.
31.Metz, C.N.B., R. 2001. San Diego. 703-716 pp.
32.Mitchell, R.A., H. Liao, J. Chesney, G. Fingerle-Rowson, J. Baugh, J. David, and R. Bucala. 2002. Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response. Proc Natl Acad Sci U S A 99:345-350.
33.Sampey, A.V., P.H. Hall, R.A. Mitchell, C.N. Metz, and E.F. Morand. 2001. Regulation of synoviocyte phospholipase A2 and cyclooxygenase 2 by macrophage migration inhibitory factor. Arthritis Rheum 44:1273-1280.
34.Struckmann, K., P. Schraml, R. Simon, K. Elmenhorst, M. Mirlacher, J. Kononen, and H. Moch. 2004. Impaired expression of the cell cycle regulator BTG2 is common in clear cell renal cell carcinoma. Cancer Res 64:1632-1638.
35.Kastan, M.B., O. Onyekwere, D. Sidransky, B. Vogelstein, and R.W. Craig. 1991. Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51:6304-6311.
36.Minn, A.J., L.H. Boise, and C.B. Thompson. 1996. Expression of Bcl-xL and loss of p53 can cooperate to overcome a cell cycle checkpoint induced by mitotic spindle damage. Genes Dev 10:2621-2631.
37.Graeber, T.G., C. Osmanian, T. Jacks, D.E. Housman, C.J. Koch, S.W. Lowe, and A.J. Giaccia. 1996. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379:88-91.
38.Andera, L., and B. Wasylyk. 1997. Transcription abnormalities potentiate apoptosis of normal human fibroblasts. Mol Med 3:852-863.
39.Shachar, I., and R.A. Flavell. 1996. Requirement for invariant chain in B cell maturation and function. Science 274:106-108.
40.Koch, N., and A.W. Harris. 1984. Differential expression of the invariant chain in mouse tumor cells: relationship to B lymphoid development. J Immunol 132:12-15.
41.Collins, T., A.J. Korman, C.T. Wake, J.M. Boss, D.J. Kappes, W. Fiers, K.A. Ault, M.A. Gimbrone, Jr., J.L. Strominger, and J.S. Pober. 1984. Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts. Proc Natl Acad Sci U S A 81:4917-4921.
42.Benacerraf, B. 1981. Role of MHC gene products in immune regulation. Science 212:1229-1238.
43.Claesson, L., D. Larhammar, L. Rask, and P.A. Peterson. 1983. cDNA clone for the human invariant gamma chain of class II histocompatibility antigens and its implications for the protein structure. Proc Natl Acad Sci U S A 80:7395-7399.
44.Long, E.O. 1989. Intracellular traffic and antigen processing. Immunol Today 10:232-234.
45.Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S.L. Schmid, V. Quaranta, and P.A. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature 348:600-605.
46.Simonsen, A., F. Momburg, J. Drexler, G.J. Hammerling, and O. Bakke. 1993. Intracellular distribution of the MHC class II molecules and the associated invariant chain (Ii) in different cell lines. Int Immunol 5:903-917.
47.Badve, S., C. Deshpande, Z. Hua, and L. Logdberg. 2002. Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus. J Histochem Cytochem 50:473-482.
48.Moch, H., J.C. Presti, Jr., G. Sauter, N. Buchholz, P. Jordan, M.J. Mihatsch, and F.M. Waldman. 1996. Genetic aberrations detected by comparative genomic hybridization are associated with clinical outcome in renal cell carcinoma. Cancer Res 56:27-30.
49.Saito, T., M. Kimura, T. Kawasaki, S. Sato, and Y. Tomita. 1997. MHC class II antigen-associated invariant chain on renal cell cancer may contribute to the anti-tumor immune response of the host. Cancer Lett 115:121-127.
50.Jiang, Z., M. Xu, L. Savas, P. LeClair, and B.F. Banner. 1999. Invariant chain expression in colon neoplasms. Virchows Arch 435:32-36.
51.McKean, D.J., A.J. Infante, A. Nilson, M. Kimoto, C.G. Fathman, E. Walker, and N. Warner. 1981. Major histocompatibility complex-restricted antigen presentation to antigen-reactive T cells by B lymphocyte tumor cells. J Exp Med 154:1419-1431.
52.Takiguchi, M., J.P. Ting, S.C. Buessow, C. Boyer, Y. Gillespie, and J.A. Frelinger. 1985. Response of glioma cells to interferon-gamma: increase in class II RNA, protein and mixed lymphocyte reaction-stimulating ability. Eur J Immunol 15:809-814.
53.Santos, L.L., D. Lacey, Y. Yang, M. Leech, and E.F. Morand. 2004. Activation of synovial cell p38 MAP kinase by macrophage migration inhibitory factor. J Rheumatol 31:1038-1043.
54.Wu, L., and A.J. Levine. 1997. Differential regulation of the p21/WAF-1 and mdm2 genes after high-dose UV irradiation: p53-dependent and p53-independent regulation of the mdm2 gene. Mol Med 3:441-451.
55.Levine, A.J. 1997. p53, the cellular gatekeeper for growth and division. Cell 88:323-331.
56.Gunther, T., R. Schneider-Stock, C. Hackel, H.U. Kasper, M. Pross, A. Hackelsberger, H. Lippert, and A. Roessner. 2000. Mdm2 gene amplification in gastric cancer correlation with expression of Mdm2 protein and p53 alterations. Mod Pathol 13:621-626.
57.Ladanyi, M., C. Cha, R. Lewis, S.C. Jhanwar, A.G. Huvos, and J.H. Healey. 1993. MDM2 gene amplification in metastatic osteosarcoma. Cancer Res 53:16-18.
58.Oliner, J.D., K.W. Kinzler, P.S. Meltzer, D.L. George, and B. Vogelstein. 1992. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358:80-83.
59.Yaginuma, Y., and H. Westphal. 1991. Analysis of the p53 gene in human uterine carcinoma cell lines. Cancer Res 51:6506-6509.
60.Wasylyk, C., R. Salvi, M. Argentini, C. Dureuil, I. Delumeau, J. Abecassis, L. Debussche, and B. Wasylyk. 1999. p53 mediated death of cells overexpressing MDM2 by an inhibitor of MDM2 interaction with p53. Oncogene 18:1921-1934.
61.Marston, N.J., J.R. Jenkins, and K.H. Vousden. 1995. Oligomerisation of full length p53 contributes to the interaction with mdm2 but not HPV E6. Oncogene 10:1709-1715.
62.Zou, Z., B. Chung, T. Nguyen, S. Mentone, B. Thomson, and D. Biemesderfer. 2004. Linking receptor-mediated endocytosis and cell signaling: evidence for regulated intramembrane proteolysis of megalin in proximal tubule. J Biol Chem 279:34302-34310.
63.Hartmann, D., J. Tournoy, P. Saftig, W. Annaert, and B. De Strooper. 2001. Implication of APP secretases in notch signaling. J Mol Neurosci 17:171-181.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top