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系統識別號 U0026-2508201023400600
論文名稱(中文) NDP Kinase A參與c-myc 轉錄調控
論文名稱(英文) NDP kinase A is involved in c-myc transcriptional regulation
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 詹凱惠
研究生(英文) Kai-Hui Chan
學號 t4697404
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-張玲
召集委員-陳玉玲
口試委員-蔣輯武
中文關鍵字 c-myc  核苷二磷酸激酶A (NDPK-A)  核苷二磷酸激酶B (NDPK-B)  轉錄 
英文關鍵字 c-myc  NDPK-A  NDPK-B  transcription 
學科別分類
中文摘要 Nucleoside diphosphate kinase (NDPK) 是一類廣泛存在的酵素,它通過乒乓機制將nucleoside diphosphates磷酸化為nucleoside triphosphates。該基因家族已知有8個成員,其中NDPK-A/NM23-H1和NDPK-B/NM23-H2研究較多。目前研究指出,雖然NDPK-A和NDPK-B的氨基酸序列同源性高達88%,NDPK-A與癌細胞轉移關係更密切。NDPK-B被證實為human c-myc transcription factor PuF,可結合至c-myc promoter上nuclease-hypersensitive III1 片段。c-myc 原致癌基因在正常細胞生理扮演關鍵性調控角色,其與細胞生長,繁殖,分化,凋亡有關。在大多數的腫瘤,常可發現c-myc表達失調。本實驗室先前發現NDPK-A也可結合至c-myc promoter in vitro並調節其活性in vivo。經由 EMSA 和 UV-crosslinking實驗分析,NDPK-A相較NDPK-B對c-myc的結合能力明顯較弱。於是我更進一步探討NDPK-A及NDPK-B如何分別及共同調控c-myc轉錄。藉由CAT assay分析,我發現NDPK-A對於c-myc調控不需要phosphotransferase活性。除了NDPK-AS120G/N82S,NDPKAWT和NDPKAS120G外生性的表現增加能抑制c-myc轉錄。相同地,NDPK-B也能抑制c-myc的轉錄。NDPK-A和NDPK-B的外生性表現同時增量則導致更強烈c-myc轉錄之抑制現象。相反地,利用shRNA降低內生性的NDPK-A或NDPK-B的表現則活化c-myc轉錄。在過氧壓力下,NDPK-A增進了細胞之存活,降低內生性的NDPK-A或NDPK-B的表現則增加細胞死亡。共同降低內生性NDPK-A與NDPK-B的表現使細胞存活率更低。雖然NDPK-A與NDPK-B對於細胞生長都不具影響,但兩者對於細胞的Wound healing 有相反的能力,本研究結果將癌症轉移所見到的NDPKs異常表現與c-myc 失調連結在一起。
英文摘要 Nucleoside diphosphate kinase (NDPK) is an ubiquitous enzyme, which catalyzes the transfer of the terminal phosphate from a nucleoside triphosphate to a nucleoside diphosphate through a ping-pong mechanism. At present, eight members of the human NDPK family have been identified. The most widely studied members are NDPK-A and NDPK-B which are encoded by NM23-H1 and NM23-H2 genes. Accumulating evidence indicates that NDPK-A, but not NDPK-B, is strongly correlated with tumor metastasis even though the two proteins share 88% identical amino acid residues. NDPK-B was identified as PuF, which is a transcriptional regulator of c-myc that binds to the nuclease-hypersensitive element III1 (NHE) III1 in the c-myc promoter. Our laboratory previously found that NDPK-A also specifically binds c-myc NHEIII1 in vitro and regulates the c-myc promoter activity in vivo. Compared to NDPK-B, however, NDPK-A displayed a significantly lower binding affinity toward c-myc DNA based on EMSA and UV-crosslinking. In my study, I have examined the individual and collaborative effects of NDPK-A and NDPK-B on c-myc transcriptional regulation in NB69 neuroblastoma cells and cervical cancer HeLa cells. Using the CAT assay, I found that the phosphotransferase activity or intra-molecular disulfide bond of NDPK-A was not essential for its regulation of c-myc transcription. Ectopic expression of wide-type NDPK-A (NDPKAWT) and metastasis-associated S120G mutation (NDPKAS120G), negatively regulated c-myc mediated transcription in a dose-dependent manner. Similarity, NDPK-B also displayed an inhibitory effect on c-myc transcription in both cell lines. When combined with NDPK-B, NDPK-A suppressed c-myc transcription more strongly than either protein alone. On the other hand, knockdown of endogenous NDPK-A and/or NDPK-B enhanced c-myc transcription. Under oxidative stress, knockdown of NDPK-A decreased cell viability by ~15% relative to the control cells. Knockdown of both NDPK-A and NDPK-B further decreased cell viability by ~50% after H2O2 treatment. Additionally, knockdown of NDPK-A increased the migration potential, whereas knockdown of NDPK-B reduced its migration capacity compared with the control. However, knockdown NDPK-A and/or NDPK-B did not significantly affect the proliferation of HeLa cells. These findings provide a link between metastasis-associated alterations of NDPK-A and c-myc deregulation.
論文目次 1. Introduction
1.1 NM23/Nucleoside Diphosphate Kinase in Human Cancers ……………………. 7
1.2 NDPK-A and NDPK-B in gene regulation …………………………………….... 8
1.3 Oligomeric structure of NDPK-A and NDPK-B ……………………………....... 9
1.4 The c-myc concogene ……..………………………………….………...……..…. 10
1.5 Hypothesis …………..……………………………………..………………..…… 11

2. Materials and methods
2.1 Cell lines, plasmids and transfection …..……………..………………..………..…. 12
2.2 Establish stable knockdown cell lines ………………..……………..…………..…. 12
2.3 Site – directed mutagenesis PCR ……………………..…..……………………..…. 13
2.4 Reverse-transcription polymerase chain reaction (RT-PCR) ……..……………....…. 14
2.5 Quantitative Real-time Reverse Transcription-PCR ………..………………......…. 14
2.6 CAT and β-GAL assays …………..………………………………………….……. 15
2.7 Protein Isolation and Western Blot Analysis …………………………………….….. 15
2.8 Survival assay …………………………………………………………………..….. 16
2.9 Proliferation assays ……………………………………………………………..….. 16
2.10 In vitro cell migration assay …………………………………………………...…. 17
2.10 Statistical analysis ……………………………………………...…………….…... 17

3. Experimental results
3.1 Overexpression of wide type, S120G mutant and H118F mutant of NDPK-A suppress
the c-myc promoter activity ……...……………………………..…………........…. 18
3.2 Suppression of NDPK-A in c-myc transcription requires NHEIII1 and upstream cis-
elements in NB69 cells ……………………………….….......…………………...…. 19
3.3 Intra-molecular disulfide bond is not required for the suppression effect of NDPK-A on the c-myc promoter activity ……...………………………………………………...… 20
3.4 Altered ratios of NDPK-A and NDPK-B affect c-myc transcription …..…………..... 21
3.5 Knockdown of NDPK-A and/ or NDPK-B expression decreases cell viability under
H2O2 –induced oxidative stress ……...…………………………………………..… 22
3.6 Knockdown NDPK-A and/or NDPK-B expression displays different migration
potential in HeLa cells …….......…………………….………………………….... 23
3.7 Knockdown NDPK-A and/or NDPK-B does not affect the proliferative capacities of
HeLa cells ……………………………………………………………..……...….… 24

4. Discussion …………….………………………….……………….… 25
5. Conclusion ……………………………………….…………………. 30
6. References …………………………………………..……………… 31
7. Table …………………………………………………..……………… 36
8. Figures ………………………………………………………………... 37
9. Abbreviation list .…………………………………………………... 49
10 Appendix ...…………………………………………………………... 50
參考文獻 1. Steeg, P.S., et al. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 80, 200-204 (1988).
2. Easty, D.J., et al. Expression of NM23 in human melanoma progression and metastasis. Br J Cancer 74, 109-114 (1996).
3. Jin, L., et al. Nm23-H1 regulates the proliferation and differentiation of the human chronic myeloid leukemia K562 cell line: a functional proteomics study. Life Sci 84, 458-467 (2009).
4. Cipollini, G., et al. Down-regulation of the nm23.h1 gene inhibits cell proliferation. Int J Cancer 73, 297-302 (1997).
5. Postel, E.H., et al. Targeted deletion of Nm23/nucleoside diphosphate kinase A and B reveals their requirement for definitive erythropoiesis in the mouse embryo. Dev Dyn 238, 775-787 (2009).
6. Bilitou, A., Watson, J., Gartner, A. & Ohnuma, S. The NM23 family in development. Mol Cell Biochem 329, 17-33 (2009).
7. Almgren, M.A., Henriksson, K.C., Fujimoto, J. & Chang, C.L. Nucleoside diphosphate kinase A/nm23-H1 promotes metastasis of NB69-derived human neuroblastoma. Mol Cancer Res 2, 387-394 (2004).
8. Caligo, M.A., Cipollini, G., Petrini, M., Valentini, P. & Bevilacqua, G. Down regulation of NM23.H1, NM23.H2 and c-myc genes during differentiation induced by 1,25 dihydroxyvitamin D3. Leuk Res 20, 161-167 (1996).
9. Lombardi, D., Lacombe, M.L. & Paggi, M.G. nm23: unraveling its biological function in cell differentiation. J Cell Physiol 182, 144-149 (2000).
10. Choudhuri, T., et al. Nm23-H1 can induce cell cycle arrest and apoptosis in B cells. Cancer Biol Ther 9(2010).
11. Tee, Y.T., Chen, G.D., Lin, L.Y., Ko, J.L. & Wang, P.H. Nm23-H1: a metastasis-associated gene. Taiwan J Obstet Gynecol 45, 107-113 (2006).
12. Caligo, M.A., et al. NM23 gene expression in human breast carcinomas: loss of correlation with cell proliferation in the advanced phase of tumor progression. Int J Cancer 74, 102-111 (1997).
13. Kapitanovic, S., et al. nm23-H1 expression and loss of heterozygosity in colon adenocarcinoma. J Clin Pathol 57, 1312-1318 (2004).
14. Lacombe, M.L., Milon, L., Munier, A., Mehus, J.G. & Lambeth, D.O. The human Nm23/nucleoside diphosphate kinases. J Bioenerg Biomembr 32, 247-258 (2000).
15. Zou, M., Shi, Y., al-Sedairy, S. & Farid, N.R. High levels of Nm23 gene expression in advanced stage of thyroid carcinomas. Br J Cancer 68, 385-388 (1993).
16. Okabe-Kado, J., et al. Clinical significance of serum NM23-H1 protein in neuroblastoma. Cancer Sci 96, 653-660 (2005).
17. Postel, E.H. Cleavage of DNA by human NM23-H2/nucleoside diphosphate kinase involves formation of a covalent protein-DNA complex. J Biol Chem 274, 22821-22829 (1999).
18. Postel, E.H. & Ferrone, C.A. Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions. J Biol Chem 269, 8627-8630 (1994).
19. Ma, D., et al. NM23-H1 and NM23-H2 repress transcriptional activities of nuclease-hypersensitive elements in the platelet-derived growth factor-A promoter. J Biol Chem 277, 1560-1567 (2002).
20. Postel, E.H. Multiple biochemical activities of NM23/NDP kinase in gene regulation. J Bioenerg Biomembr 35, 31-40 (2003).
21. Heo, Y.J., Kim, S.Y., Kim, E. & Lee, K.J. Quaternary structural analysis of nucleoside diphosphate kinases using capillary electrophoresis. J Chromatogr A 781, 251-261 (1997).
22. Cervoni, L., et al. DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells. J Cell Biochem 98, 421-428 (2006).
23. MacDonald, N.J., Freije, J.M., Stracke, M.L., Manrow, R.E. & Steeg, P.S. Site-directed mutagenesis of nm23-H1. Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells. J Biol Chem 271, 25107-25116 (1996).
24. Hemmerich, S. & Pecht, I. Oligomeric structure and autophosphorylation of nucleoside diphosphate kinase from rat mucosal mast cells. Biochemistry 31, 4580-4587 (1992).
25. Chang, C.L., et al. A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction. Oncogene 12, 659-667 (1996).
26. Lascu, I., et al. A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding. J Biol Chem 272, 15599-15602 (1997).
27. Song, E.J., et al. Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Biochemistry 39, 10090-10097 (2000).
28. Lascu, L., Giartosio, A., Ransac, S. & Erent, M. Quaternary structure of nucleoside diphosphate kinases. J Bioenerg Biomembr 32, 227-236 (2000).
29. Backer, J.M., et al. Chromosomal localization and nucleoside diphosphate kinase activity of human metastasis-suppressor genes NM23-1 and NM23-2. Oncogene 8, 497-502 (1993).
30. Webb, P.A., Perisic, O., Mendola, C.E., Backer, J.M. & Williams, R.L. The crystal structure of a human nucleoside diphosphate kinase, NM23-H2. J Mol Biol 251, 574-587 (1995).
31. Postel, E.H., Weiss, V.H., Beneken, J. & Kirtane, A. Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element. Proc Natl Acad Sci U S A 93, 6892-6897 (1996).
33. Pelengaris, S. & Khan, M. The many faces of c-MYC. Arch Biochem Biophys 416, 129-136 (2003).
34. Meyer, N. & Penn, L.Z. Reflecting on 25 years with MYC. Nat Rev Cancer 8, 976-990 (2008).
35. Levens, D.L. Reconstructing MYC. Genes Dev 17, 1071-1077 (2003).
36. Brooks, T.A. & Hurley, L.H. The role of supercoiling in transcriptional control of MYC and its importance in molecular therapeutics. Nat Rev Cancer 9, 849-861 (2009).
37. Gonzalez, V. & Hurley, L.H. The c-MYC NHE III(1): function and regulation. Annu Rev Pharmacol Toxicol 50, 111-129 (2010).
38. Cashman, D.J., et al. Molecular modeling and biophysical analysis of the c-MYC NHE-III1 silencer element. J Mol Model 14, 93-101 (2008).
39. Wierstra, I. & Alves, J. The c-myc promoter: still MysterY and challenge. Adv Cancer Res 99, 113-333 (2008).
40. Postel, E.H., Berberich, S.J., Rooney, J.W. & Kaetzel, D.M. Human NM23/nucleoside diphosphate kinase regulates gene expression through DNA binding to nuclease-hypersensitive transcriptional elements. J Bioenerg Biomembr 32, 277-284 (2000).
41. Min, K., et al. Crystal structure of human nucleoside diphosphate kinase A, a metastasis suppressor. Proteins 46, 340-342 (2002).
42. Arnaud-Dabernat, S., et al. Nm23-M2/NDP kinase B induces endogenous c-myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death. Exp Cell Res 301, 293-304 (2004).
43. Dexheimer, T.S., et al. NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III1. Mol Cancer Ther (2009).
44. Michelotti, E.F., et al. Nm23/PuF does not directly stimulate transcription through the CT element in vivo. J Biol Chem 272, 22526-22530 (1997).
45. Hagen, G., Muller, S., Beato, M. & Suske, G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13, 3843-3851 (1994).
46. Majello, B., De Luca, P., Suske, G. & Lania, L. Differential transcriptional regulation of c-myc promoter through the same DNA binding sites targeted by Sp1-like proteins. Oncogene 10, 1841-1848 (1995).
47. Yu, B., Datta, P.K. & Bagchi, S. Stability of the Sp3-DNA complex is promoter-specific: Sp3 efficiently competes with Sp1 for binding to promoters containing multiple Sp-sites. Nucleic Acids Res 31, 5368-5376 (2003).
48. Geltinger, C., Hortnagel, K. & Polack, A. TATA box and Sp1 sites mediate the activation of c-myc promoter P1 by immunoglobulin kappa enhancers. Gene Expr 6, 113-127 (1996).
49. Kim, Y.I., Park, S., Jeoung, D.I. & Lee, H. Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells. Biochem Biophys Res Commun 307, 281-289 (2003).
50. Mesnildrey, S., Agou, F. & Veron, M. The in vitro DNA binding properties of NDP kinase are related to its oligomeric state. FEBS Lett 418, 53-57 (1997).
51. Hildebrandt, M., Lacombe, M.L., Mesnildrey, S. & Veron, M. A human NDP-kinase B specifically binds single-stranded poly-pyrimidine sequences. Nucleic Acids Res 23, 3858-3864 (1995).
52. Min, K., et al. Crystallization and preliminary X-ray crystallographic analysis of human nucleoside diphosphate kinase A. Acta Crystallogr D Biol Crystallogr 56, 503-504 (2000).
53. Bosnar, M.H., et al. Subcellular localization of A and B Nm23/NDPK subunits. Exp Cell Res 298, 275-284 (2004).
54. Lee, E., et al. Multiple functions of Nm23-H1 are regulated by oxido-reduction system. PLoS One 4, e7949 (2009).
55. Hartsough, M.T. & Steeg, P.S. Nm23/nucleoside diphosphate kinase in human cancers. J Bioenerg Biomembr 32, 301-308 (2000).
56. Understanding the molecular basis of the interaction between NDPK-A and AMPK alpha1. Mol Cell Biol 28, 5827 (2008).
57. Suzuki, E., et al. nm23-H1 reduces in vitro cell migration and the liver metastatic potential of colon cancer cells by regulating myosin light chain phosphorylation. Int J Cancer 108, 207-211 (2004).
58. Aktary, Z., et al. Plakoglobin interacts with and increases the protein levels of metastasis suppressor Nm23-H2 and regulates the expression of Nm23-H1. Oncogene 29, 2118-2129 (2010).
59. Baba, H., et al. Two isotypes of murine nm23/nucleoside diphosphate kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a murine melanoma line. Cancer Res 55, 1977-1981 (1995).
60. Tokunaga, Y., et al. Reduced expression of nm23-H1, but not of nm23-H2, is concordant with the frequency of lymph-node metastasis of human breast cancer. Int J Cancer 55, 66-71 (1993).
61. Leone, A., Flatow, U., VanHoutte, K. & Steeg, P.S. Transfection of human nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: effects on tumor metastatic potential, colonization and enzymatic activity. Oncogene 8, 2325-2333 (1993).
62. Backer, M.V., et al. Overexpression of NM23-1 enhances responsiveness of IMR-32 human neuroblastoma cells to differentiation stimuli. Anticancer Res 20, 1743-1749 (2000).
63. Lee, J.H., Marshall, J.C., Steeg, P.S. & Horak, C.E. Altered gene and protein expression by Nm23-H1 in metastasis suppression. Mol Cell Biochem 329, 141-148 (2009).
64. Fournier, H.N., Albiges-Rizo, C. & Block, M.R. New insights into Nm23 control of cell adhesion and migration. J Bioenerg Biomembr 35, 81-87 (2003).
65. An, R., et al. Over-expression of nm23-H1 in HeLa cells provides cells with higher resistance to oxidative stress possibly due to raising intracellular p53 and GPX1. Acta Pharmacol Sin 29, 1451-1458 (2008).
66. Fan, Z., Beresford, P.J., Oh, D.Y., Zhang, D. & Lieberman, J. Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 112, 659-672 (2003).
67. Lombardi, D. Commentary: nm23, a metastasis suppressor gene with a tumor suppressor gene aptitude? J Bioenerg Biomembr 38, 177-180 (2006).
68. Miyazaki, H., et al. Overexpression of nm23-H2/NDP kinase B in a human oral squamous cell carcinoma cell line results in reduced metastasis, differentiated phenotype in the metastatic site, and growth factor-independent proliferative activity in culture. Clin Cancer Res 5, 4301-4307 (1999).
69. Howlett, A.R., Petersen, O.W., Steeg, P.S. & Bissell, M.J. A novel function for the nm23-H1 gene: overexpression in human breast carcinoma cells leads to the formation of basement membrane and growth arrest. J Natl Cancer Inst 86, 1838-1844 (1994).
70. Roymans, D., Willems, R., Van Blockstaele, D.R. & Slegers, H. Nucleoside diphosphate kinase (NDPK/NM23) and the waltz with multiple partners: possible consequences in tumor metastasis. Clin Exp Metastasis 19, 465-476 (2002).
71. Ni, X., et al. Isolation and characterization of a novel human NM23-H1B gene, a different transcript of NM23-H1. J Hum Genet 48, 96-100 (2003).
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