| 研究生: |
陳炳宏 Chen, Ping-Hong |
|---|---|
| 論文名稱: |
1. 受Ha-ras調控之基因BNIP3之選殖、特性及功能性研究
2. 利用Real-Time PCR偵測膀胱癌、結腸癌檢體中之H-ras及K-ras基因氨基酸12之突變 1. Cloning, characterization and functional study of BNIP3, a Ha-ras up-regulated gene 2. Screening codon 12 mutation of H-ras and K-ras genes in bladder and colon cancers by Real-Time PCR |
| 指導教授: |
劉校生
Liu, Shiao-Sheng |
| 學位類別: |
碩士 Master |
| 系所名稱: |
醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology |
| 論文出版年: | 2003 |
| 畢業學年度: | 91 |
| 語文別: | 英文 |
| 論文頁數: | 85 |
| 中文關鍵詞: | 膀胱癌 |
| 外文關鍵詞: | Ras, Real-Time PCR, BNIP3 |
| 相關次數: | 點閱:97 下載:1 |
| 分享至: |
| 查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
Ras蛋白質在細胞內扮演一個訊息傳遞者的角色,調控細胞的增生、轉型、死亡。經由Ras蛋白質所誘導的細胞死亡機制被認為是一種防止細胞癌化的方法。Ras蛋白質可以在粒腺體上與Bcl-2蛋白質互相作用而調控細胞的生死。BNIP3 (Bcl-2 and Nineteen kDa Interacting Protein-3)屬於Bcl-2家族的BH3-domain only蛋白質。已知其可以與Bcl-2蛋白質結合,並會誘使細胞凋亡。本研究中顯示Ras和BNIP3蛋白質的表現在乳癌細胞株MCF7-ras、膀胱癌細胞株UB37, UB40及老鼠纖維母細胞株Bcl2-3A中均呈正相關。將可持續或誘導表現之BNIP3質體送入293細胞內大量表現時,造成該細胞之死亡。進一步分析發現這些細胞粒腺體膜通透性增加,但並不造成cytochrome C的釋放,而這些細胞是否死於細胞凋亡,仍未確定。此外也發現這些細胞中Ras蛋白質在粒腺體會與BNIP3蛋白質相互作用。綜述之,Ras可與Bcl-2及BNIP3;Bcl-2可與BNIP3相互結合,而三者之相互關係仍未可知,此外Ras與BNIP3正相關之作用機轉也有待進一步釐清。無論如何,Ras, Bcl-2和BNIP3可能在粒腺體上相互作用而進一步調節細胞的生死,這些假說均有待進一步之探討。
不同ras基因的點突變會改變其致癌能力。利用螢光探針及定量聚合酶儀器研發出一套快速篩檢臨床檢體中的K-ras及H-ras第12氨基酸突變的方法。藉由此方法在38個大腸癌檢體中偵測到8個檢體有K-ras codon 12突變,9個有H-ras codon 12突變;25個膀胱癌檢體中有8個H-ras codon 12突變且無K-ras codon 12突變。綜述之,本研究提供一個有別於傳統SSCP之方法用來快速、準確篩選Ras氨基酸12之突變,對於Ras相關之癌症診斷將有莫大之幫助。
Ras transduces multiple signals to modulate gene expression for cell proliferation, transformation and death. The cell death induced by Ras may play a critical role in preventing neoplasia formation. Mitochondria Ras interacts with Bcl-2 and regulates the death of cells. BNIP3 (Bcl-2 and Nineteen kDa Interacting Protein-3) belongs the BH3 domain only protein of Bcl-2 family. BNIP3 can bind with Bcl-2 and induces the death of mammalian cells. Our data showed that the expression of Ras correlated with the expression of BNIP3 in breast cancer cell MCF7-ras, mouse fibroblast NIH/3T3 cells Bcl2-3A, and bladder cancer cells, UB37, and UB40. Expression vector of BNIP3 was constructed, and overexpression of BNIP3 gene in 293 cells resulted in cell death and increase of mitochondria membrane permeability. Further study revealed that BNIP3 caused cell death is without release of cytochrome C. Our data also demonstrated that Ras interacted with BNIP3 on mitochondria. In conclusion, whether BNIP3 overexpression can induce the apoptosis of bladder cancer cell and whether the interaction among Ras, BNIP3 and Bcl-2 on mitochondria is involved in the cell death triggered by Ras-related BNIP3 remain unclear.
Different mutation types of three major Ras proteins (H-Ras, K-Ras and N-Ras) may lead to diverse carcinogenesis. For rapid screen of amino acid 12 mutants of Ras in clinical samples, a method using fluorescent probes and Real-Time PCR was established. A total of 8 mutations of K-ras and 9 mutations of H-ras were detected in 38 colon cancer samples. In addition, a total of 8 mutations of H-ras and no mutation of K-ras were detected in 25 bladder cancer samples. Compare to traditional methodology (SSCP), here we demonstrated a rapid and more precise method to screen codon 12 Ras mutations.
1. Ayllon, V., and Rebollo, A. Ras-induced cellular events (review). Mol
Membr Biol. 17: 65-73, 2000.
2. Ayalew, T., Eric, D., Gordon, W., David, A. H., Matthew, E., and Thomas, C.
Primer on medical genomics. Part II: background principles and methods in
molecular genetics. Mayo Clin Proc. 77: 785-808, 2002.
3. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res. 49: 4682-
4289, 1989.
4. Breivik, J., Meling, G. I., Spurkland, A., Rognum, T. O., and
Gaudernack, G. K-ras mutation in colorectal cancer: relations to patient
age, sex and tumour location. Br J Cancer. 69: 367, 1994.
5. Behn, M., Thiede, C., Neubauer, A., Pankow, W., and Schuemann, M.
Facilitated detection of oncogene mutation from exfoliated tissue material
by a PNA-mediated ‘enriched PCR’ protocol. J Pathol. 190: 69-75, 2000.
6. Bruick, R. K. Expression of the gene encoding the proapoptotic protein
induced by hypoxia. Proc Natl Acad Sci U S A. 97: 9082-9087, 2000.
7. BDTM Tet-off and Tet-on gene expression systems user manual. BD
Biosciences Clontech U. S. A.
8. Chen, C. Y., and Faller, D. V. Direction of p21ras-generated signals
towards cell growth or apoptosis is determined by protein kinase C and Bcl-
2. Oncogene. 11: 1487-1498, 1995.
9. Chen, C. Y., and Faller, D. V. Phosphorylation of Bcl-2 protein and
association with p21 Ras in Ras-induced apoptosis. J Biol Chem. 271: 2376-
2379, 1996.
10. Chen, C. Y., Liou, J., Forman, L. W., and Faller, D. V. Differential
regulation of discrete apoptotic pathways by Ras. J Biol Chem. 273: 16700-
16709, 1998.
11. Chi, S., Kitanaka, C., Noguchi, K., Mochizuki, T., Nagashima, Y.,
Shirouzu, M., Fujita, H., Yoshida, M., Chen, W., Asai, A., Himeno, M.,
Yokoyama, S., and Kuchino, Y. Oncogenic Ras triggers cell suicide through
the activation of a caspase-independent cell death program in human cancer
cells. Oncogene. 18: 2281-2290, 1999.
12. Chen, G., Cizeau, J., Velde, C. V., Park, J. H., Bozek, G., Bolton, J.,
Shi, L., Dubik, D., and Greenberg, A. Nix and Nip3 form a subfamily of pro-
apoptotic mitochondria proteins. J Biol Chem. 274: 7-10, 1999.
13. Chen, C. Y., Juo, P., Liou, J. S., Li, C. Q., Yu, Q., Blenis, J., and
Faller, D. V. The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis. Cell Growth Differ. 12: 297-306, 2001.
14. Complete control inducible mammalian expression system. Stratagene U.S.A.
15. Denis, G. V., Yu, Q., Ma, P., Deeds, L., Faller, D. V., Chen, C. Y. Bcl-2,
via its BH4 domain, blocks apoptotic signaling mediated by mitochondrial
Ras. J Biol Chem. 278: 5775-5785, 2003.
16. Downward, J. Targeting Ras signaling pathway in cancer therapy. Nat Rev
Cancer. 3: 11-22,2003.
17. Fan, J., Banerjee, D., Scambrook, P. J., and Bertino, J. R. Modulation of
cytotoxicity of chemotherapeutic drug by activated H-ras. Biochem
Pharmacol. 53: 1203-1209, 1997.
18. Feig, L. A., and Buchsbaum, R. J. Cell signaling: life or death
decisions. Curr Biol. 12: R259-R261, 2002.
19. Ginzinger, D. G. Gene quantification using real-time quantitative PCR: An
emerging technology hits the mainstream. Exp Hematol. 30: 503–512, 2002.
20. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. Kinetic PCR
analysis: real-time monitoring of DNA amplification reactions.
Biotechnology. 11: 1026, 1993.
21. Hancock, J. H. Ras proteins: different signals from different locations.
Nat Rev Mol Cell Biol. 4: 373-384, 2003.
22. Hingorani, S. R., and Tuveson, D. A. Ras redux: rethinking how and where
Ras acts. Curr Opin Genet Dev.13: 6-13, 2003.
23. Johne, A., Roots, I., and Brockmoller, J. A single nucleotide polymorphism
in the human H-ras proto-oncogene determines the risk of urinary bladder
cancer. Cancer Epidemiol Biomarkers Prev. 12: 68-70, 2003.
24. Kruglyak, L. and Nickerson, D. A. Variation is the spice of life. Nat
Genet. 27: 234-236, 2001.
25. Khokhlatchev, A., Rabizadeh, S., Xavier, R., Nedwidek, M., Chen, T.,
Zhang, X. F., Seed, B., and Avruch, J. Identification of a novel Ras-
regulated proapoptotic pathway. Curr Biol. 12: 253-265, 2002.
26. Kim, J-Y., Cho, J-J., Ha, J., and Park, J-H. The Carboxy terminal C-tail
of BNIP3 is crucial in induction of mitochondrial permeability transition
in isolated mitochondria. Arch Biochem Biophys. 398: 147-152, 2002.
27. 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. J Biol Chem. 99: 12825-12830, 2002.
28. Liu, H. S., Chen, C. Y., Lee, C. H., and Chou, Y. I. Selective induction
of oncogenic Ha-ras-activation apoptosis in NIH/3T3 cells. Br J Cancer.
77: 1777-1786, 1998.
29. Liou, J. S., Chen, C. Y., Chen, J. S., and Faller, D. V. Oncogenic ras
mediates apoptosis in response to protein kinase C inhibition through the
generation of reactive oxygen species. J Biol Chem. 275: 39001-11, 2000.
30. Lyon, E. Mutation detection using fluorescent hybridization probes and
melting curve analysis. Expert Rev Mol Diagn. 1: 92-101, 2001.
31. 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: 23915-23921,
2003.
32. McCoy, M. S., Toole, J. J., Cunningham, J. M, Chang, E. H., Lowy, D. R.,
and Weinberg, R. A. Characterization of a human colon/lung carcinoma.
Nature. 302: 79-81, 1983.
33. Millan, O., Ballester, A., Castrillo, A., Oliva, J. L., Traves, P. G.,
Rojas, J. M., and Bosca, L. H-Ras-specific activation of NF-kappaB
protects NIH 3T3 cells against stimulus-dependent apoptosis. Oncogene.
22: 477-483, 2003.
34. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T.
Detection of polymorphisms of human DNA by gel electrophoresis as single-
strand conformation polymorphisms. Proc Natl Acad Sci U S A. 86: 2766-
2770, 1989.
35. Olderoy, G., Daehlin, L., and Ogreid, D. Low-frequency mutation of Ha-ras
and Ki-ras oncogenes in transitional cell carcinoma of the bladder.
Anticancer Res. 18: 2675, 1998.
36. Rebollo, A., Perez-Sala, D., and Martinez-A, C. Bcl-2 differentially
targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived
cells: implications in prevention of apoptosis. Oncogene. 18: 4930-4939,
1999.
37. Ray, R., Chen, G., Velde, C. V., Cizeau, J., Park, J. H., Reed, J. C.,
Gietz, R. D., and Greenberg, A. H. BNIP3 heterodimerizes with Bcl-2/Bcl-XL
and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at
both mitochondria and nonmitochondrial sites. J Biol Chem. 275: 1439-
1448, 2000.
38. Shields, J. M., Pruitt, K., McFall, A., Shaub, A., and Der, C. J.
Understanding Ras: 'it ain't over 'til it's over'. Trends in Cell Biol.
10: 147-154, 2000.
39. Strausberg, R. L., Simpson, A. J. G., and Wooster, R. Sequence-based
cancer genomeics: progress, lessons, and opportunities. Nat Rev Genetics.
4: 409-418, 2003.
40. Timofeeva, O. A, Gorshkova, E. V., Levashova, Z. B., Kobzev, V. F.,
Filipenko, M. L., Kaledin, V. I., and Merkulova, T. I. Pulmonary
carcinogenesis susceptibility-associated single-nucleotide polymorphisms
in K-ras intron 2 affect the binding of factor Gata-6 but not gene
expression. Mol Biol (Mosk). 36: 817-24, 2002.
41. The CANCER GENOME ANATOMY PROJECT (CGAP) http://cgap.nci.nih.gov/
42. Vande, V. 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: 5454-5468, 2000.
43. 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: 144-152, 2000.