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系統識別號 U0026-0812200911170902
論文名稱(中文) 受Ras正向調控之基因BNIP3與RBBP7特性與功能性研究
論文名稱(英文) Characterization and Functional Studies of Two Ras Up-regulated Genes : BNIP3 and RBBP7
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 92
學期 2
出版年 93
研究生(中文) 許聿翹
研究生(英文) Yu-Chiao Hsu
電子信箱 felicite37@yahoo.com.tw
學號 s4691101
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 指導教授-劉校生
口試委員-洪文俊
口試委員-賴明德
口試委員-周楠華
中文關鍵字 細胞凋亡  腫瘤  轉移 
英文關鍵字 Ras  BNIP3  RBBP7  RECK  metastasis 
學科別分類
中文摘要   Ras 能將外在的訊息傳遞到細胞之中,進而引導許多不同的功能,在細胞中扮演一個很重要的角色。Ras相關下游不同的訊息傳導路徑以及其活化的相關基因可以調控細胞的存活、週期以及死亡。本研究主要探討兩個會受到ras誘導活化的基因,分別是 Bcl-2/E1B Nineteen kDa Interacting Protein-3 (BNIP3)和Retinoblastoma binding protein-7 (RBBP7)。BNIP3 是一個會促進細胞凋亡的蛋白質,並會和促進細胞存活的蛋白質Bcl-2結合,至於兩者結合的生物學意義仍在積極探討中。 本研究顯示當Ras 過度表現時會正向調控BNIP3 啟動子(promoter);且Ras和BNIP3會在粒腺體(mitochondria)中相結合。另一個基因RBBP7 最早被發現能與Rb蛋白質相結合。此外,RBBP7也參與在histone deacetylase (HDAC) complexes中;而許多報導更指出細胞 histone的調控和腫瘤的形成與轉移有關。也因此RBBP7 很可能在調控細胞週期與腫瘤形成過程中扮演重要角色。而我們證實了RBBP7是一個會被Ras正向調控的基因。RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) 是細胞膜上的醣蛋白,可以經由matrix metalloproteinases (MMPs)抑制腫瘤轉移和血管新生。最近有研究指出Ras活化會增強HDAC1結合在RECK 啟動子上的Sp1結合位置以抑制RECK表現量。本研究利用小鼠RECK啟動子luciferase assay和RBBP7 特異性的小片段干擾性RNA( siRNA)進一步發現當Ras過度表現時,細胞中的RBBP7和HDAC1會有明顯的結合,證實Ras過量表現會造成RBBP7大量表現並與HDAC1連結,一起結合至RECK 啟動子上的Sp1結合位置以抑制RECK表現量。總言之,當Ras蛋白質過量表現時,會活化各類功能特異的基因。本研究證實Ras與促使細胞死亡有的BNIP3密切關係;並可利用RBBP7去負向調控像RECK腫瘤抑制基因,.這些發現對於Ras過度表現活化下游相關的基因及其與腫瘤形成之關係都有莫大的助益。

英文摘要   Ras is an essential component in the transduction of extracellular signals that can cause multiple functions in the cell. Multiple signaling pathways downstream of Ras are responsible for survival, cell cycle progression, and cell death. We have identified two ras-upregulated genes, Bcl-2/E1B Nineteen kDa Interacting Protein-3 (BNIP3) and Retinoblastoma binding protein-7 (RBBP7), using PCR cDNA substration analysis and DNA microarray analysis, respectively. BNIP3 has been reported as an ras-upregulate gene and it is a pro-apoptotic protein that interacts with survival promoting protein Bcl-2. We demonstrated that Ras overexpression can up-regulate BNIP3 promoter and found that Ras binds to BNIP3 on the mitochondria by immunoprecipitation. As for the biological functions of this binding effect is still under investigation. Another gene, RBBP7 was first identified as a protein that binds to an Rb affinity column. It has been reported that RBBP7 involves many histone deacetylase (HDAC) complexes. Numerous studies support an important role for histone modification in tumor formation and in the metastatic spread of cancer. Therefore, RBBP7 may have an essential role in cell cycle control and tumorigenesis. We confirmed that RBBP7 was an ras-upregulate gene using the inducible Ras cell lines. RECK is a membrane –anchored glycoprotein that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor metastasis and angiogenesis. Recently, it is reported that Ras activation increased the binding between HDAC1 and the Sp1 site on RECK promoter. We discovered a strong interaction between RBBP7 and HDAC1 under Ras overexpression. Therefore, it is possible that Ras activation stimulates RBBP7 expression and recruits HDAC1 to the Sp1 site on the RECK promoter and represses it. We confirmed this speculation using mouse RECK promoter luciferase assays and RBBP7 RNA interference. In summary, we prove that Ras binds to BNIP3 in the mitochondria fraction and uses RBBP7 to down-regulate RECK.

論文目次 Abstract i
中文摘要 ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Figures ix
Abbreviations xi

Chapter 1 Introduction 1
1.1 BNIP3 2
1.1.1 BNIP3 and apoptosis 2
1.1.2 BNIP3 promoter 4

1.2 RBBP7 5
1.2.1 RBBP7 and HDACs 5
1.2.2 RECK and its role on tumor metastasis 6
1.2.3 RBBP7, RECK and tumor metastasis 7

Chapter 2 Materials and methods 9
2.1 Cell lines 9


2.3 Antibodies 10

2.4 BNIP3 Promoter cloning and analysis 11
2.4.1 Cloning of human BNIP3 promoter 11
2.4.2 Transfection 11
2.4.3 Normoxic and hypoxic conditions 12
2.4.4 Induction of Ras expression 12

2.5 Ras and BNIP3 interaction assay 13
2.5.1 Calsium-phosphate transfection 13
2.5.2 Separation of mitochondria and cytosol protein 13
2.5.3 Co-immunoprecipitation 14

2.6 Cloning of full-length human RBBP7 14

2.7 Real-time PCR quantification assay 15

2.8 Northern blotting 16

2.9 RECK promoter activity assays 17
2.9.1 Transfection and induction of Ras 17
2.9.2 RBBP7-specific siRNA 17
2.9.3 Treatment of HDAC inhibitor TSA 18
2.9.4 Statistical analysis 18

2.10 RT-PCR 18

2.11 Co-immunoprecipitation 19

2.12 DNA affinity precipitation assay (DAPA) 20
2.12.1 Nuclear protein extraction 20
2.12.2 DNA affinity precipitation assay 20

Chapter 3 Results 22
3.1 Cloning of human BNIP3 promoter 22

3.2 Ras overexpression activated BNIP3 promoter 23

3.3 BNIP3 associated with Ras in the mitochondria fraction 23

3.4 Construction and expression of human RBBP7 gene 24

3.5 RBBP7 is an ras-upregulated gene 24

3.6 Ras suppressed RECK promoter expression through HDAC and RBBP7 25

3.7 RECK mRNA was reduced when Ras or RBBP7 overexpressed 26

3.8 Association of RBBP7 with HDAC1 27

3.9 RBBP7 and HDAC1 bound on the Sp1 site of RECK promoter 27

Chapter 4 Discussion 29

References 33
參考文獻 Ahringer, J.: NuRD and SIN3 histone deacetylase complexes in development. Trends Genet 16: 351-6, 2000
Aoyagi, M., Higashino, F., Yasuda, M., Takahashi, A., Sawada, Y., Totsuka, Y., Kohgo, T., Sano, H., Kobayashi, M., and Shindoh, M.: Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins. Oncogene 22: 6919-27, 2003
Bos, J. L.: ras oncogenes in human cancer: a review. Cancer Res 49: 4682-9, 1989
Boyd: Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79: 1121, 1994
Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., and Der, C. J.: Increasing complexity of Ras signaling. Oncogene 17: 1395-413, 1998
Chang, H. C., Liu, L. T., and Hung, W. C.: Involvement of histone deacetylation in ras-induced down-regulation of the metastasis suppressor RECK. Cell Signal 16: 675-9, 2004
Chen, G., Ray, R., Dubik, D., Shi, L., Cizeau, J., Bleackley, R. C., Saxena, S., Gietz, R. D., and Greenberg, A. H.: The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med 186: 1975-83, 1997
Choy, E., Chiu, V. K., Silletti, J., Feoktistov, M., Morimoto, T., Michaelson, D., Ivanov, I. E., and Philips, M. R.: Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98: 69-80, 1999
Daido, S., Kanzawa, T., Yamamoto, A., Takeuchi, H., Kondo, Y., and Kondo, S.: Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells. Cancer Res 64: 4286-93, 2004
Gross, A., McDonnell, J. M., and Korsmeyer, S. J.: BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13: 1899-911, 1999
Huang, D. C. and Strasser, A.: BH3-Only proteins-essential initiators of apoptotic cell death. Cell 103: 839-42, 2000
Imazu, T., Shimizu, S., Tagami, S., Matsushima, M., Nakamura, Y., Miki, T., Okuyama, A., and Tsujimoto, Y.: Bcl-2/E1B 19 kDa-interacting protein 3-like protein (Bnip3L) interacts with bcl-2/Bcl-xL and induces apoptosis by altering mitochondrial membrane permeability. Oncogene 18: 4523-9, 1999
Kim, M. S., Kwon, H. J., Lee, Y. M., Baek, J. H., Jang, J. E., Lee, S. W., Moon, E. J., Kim, H. S., Lee, S. K., Chung, H. Y., Kim, C. W., and Kim, K. W.: Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes. Nat Med 7: 437-43, 2001
Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M.: A ras-related gene with transformation suppressor activity. Cell 56: 77-84, 1989
Klochendler-Yeivin, A., Muchardt, C., and Yaniv, M.: SWI/SNF chromatin remodeling and cancer. Curr Opin Genet Dev 12: 73-9, 2002
Kothari, S., Cizeau, J., McMillan-Ward, E., Israels, S. J., Bailes, M., Ens, K., Kirshenbaum, L. A., and Gibson, S. B.: BNIP3 plays a role in hypoxic cell death in human epithelial cells that is inhibited by growth factors EGF and IGF. Oncogene 22: 4734-44, 2003
Kouzarides, T.: Histone acetylases and deacetylases in cell proliferation. Curr Opin Genet Dev 9: 40-8, 1999
Kubasiak, L. A., Hernandez, O. M., Bishopric, N. H., and Webster, K. A.: Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci U S A 99: 12825-30, 2002
Lamy, L., Ticchioni, M., Rouquette-Jazdanian, A. K., Samson, M., Deckert, M., Greenberg, A. H., and Bernard, A.: CD47 and the 19 kDa interacting protein-3 (BNIP3) in T cell apoptosis. J Biol Chem 278: 23915-21, 2003
Liu, L. T., Chang, H. C., Chiang, L. C., and Hung, W. C.: Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer Res 63: 3069-72, 2003
Mazumdar, A., Wang, R. A., Mishra, S. K., Adam, L., Bagheri-Yarmand, R., Mandal, M., Vadlamudi, R. K., and Kumar, R.: Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor. Nat Cell Biol 3: 30-7, 2001
Niv, H., Gutman, O., Kloog, Y., and Henis, Y. I.: Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells. J Cell Biol 157: 865-72, 2002
Ray, R., Chen, G., Vande Velde, C., Cizeau, J., Park, J. H., Reed, J. C., Gietz, R. D., and Greenberg, A. H.: BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem 275: 1439-48, 2000
Reed, J. C.: Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr Opin Oncol 7: 541-6, 1995
Regula, K. M., Ens, K., and Kirshenbaum, L. A.: Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res 91: 226-31, 2002
Sasahara, R. M., Takahashi, C., and Noda, M.: Involvement of the Sp1 site in ras-mediated downregulation of the RECK metastasis suppressor gene. Biochem Biophys Res Commun 264: 668-75, 1999
Shimizu, S. and Tsujimoto, Y.: Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity. Proc Natl Acad Sci U S A 97: 577-82, 2000
Strasser, A., Puthalakath, H., Bouillet, P., Huang, D. C., O'Connor, L., O'Reilly, L. A., Cullen, L., Cory, S., and Adams, J. M.: The role of bim, a proapoptotic BH3-only member of the Bcl-2 family in cell-death control. Ann N Y Acad Sci 917: 541-8, 2000
Takahashi, C., Sheng, Z., Horan, T. P., Kitayama, H., Maki, M., Hitomi, K., Kitaura, Y., Takai, S., Sasahara, R. M., Horimoto, A., Ikawa, Y., Ratzkin, B. J., Arakawa, T., and Noda, M.: Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK. Proc Natl Acad Sci U S A 95: 13221-6, 1998
Toh, Y., Kuninaka, S., Endo, K., Oshiro, T., Ikeda, Y., Nakashima, H., Baba, H., Kohnoe, S., Okamura, T., Nicolson, G. L., and Sugimachi, K.: Molecular analysis of a candidate metastasis-associated gene, MTA1: possible interaction with histone deacetylase 1. J Exp Clin Cancer Res 19: 105-11, 2000
Toh, Y., Kuwano, H., Mori, M., Nicolson, G. L., and Sugimachi, K.: Overexpression of metastasis-associated MTA1 mRNA in invasive oesophageal carcinomas. Br J Cancer 79: 1723-6, 1999
Toh, Y., Oki, E., Oda, S., Tokunaga, E., Ohno, S., Maehara, Y., Nicolson, G. L., and Sugimachi, K.: Overexpression of the MTA1 gene in gastrointestinal carcinomas: correlation with invasion and metastasis. Int J Cancer 74: 459-63, 1997
Tsujimoto, Y. and Shimizu, S.: Bcl-2 family: life-or-death switch. FEBS Lett 466: 6-10, 2000
Vande Velde, C., Cizeau, J., Dubik, D., Alimonti, J., Brown, T., Israels, S., Hakem, R., and Greenberg, A. H.: BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol 20: 5454-68, 2000
Vermaak, D., Wade, P. A., Jones, P. L., Shi, Y. B., and Wolffe, A. P.: Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte. Mol Cell Biol 19: 5847-60, 1999
Verreault, A., Kaufman, P. D., Kobayashi, R., and Stillman, B.: Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase. Curr Biol 8: 96-108, 1998
Yao, Y. L. and Yang, W. M.: The metastasis-associated proteins 1 and 2 form distinct protein complexes with histone deacetylase activity. J Biol Chem 278: 42560-8, 2003
Yasuda, M., Theodorakis, P., Subramanian, T., and Chinnadurai, G.: Adenovirus E1B-19K/BCL-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence. J Biol Chem 273: 12415-21, 1998
Zhang, H. S., Gavin, M., Dahiya, A., Postigo, A. A., Ma, D., Luo, R. X., Harbour, J. W., and Dean, D. C.: Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF. Cell 101: 79-89, 2000
Zuber, J., Tchernitsa, O. I., Hinzmann, B., Schmitz, A. C., Grips, M., Hellriegel, M., Sers, C., Rosenthal, A., and Schafer, R.: A genome-wide survey of RAS transformation targets. Nat Genet 24: 144-52, 2000


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