成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	林慧如 Lin, Hui-Ju
論文名稱：	探討在急性淋巴白血病中Aurora kinases抑制劑VE-465的作用機制 Studying the molecular mechanism of Aurora kinase inhibitor, VE-465, in acute lymphoblastic leukemia (ALL)
指導教授：	洪良宜 Hung, Liang-Yi 張文昌 Chang, Wen-Chang
學位類別：	碩士 Master
系所名稱：	醫學院 - 藥理學研究所 Department of Pharmacology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	82
中文關鍵詞：	VE-465
外文關鍵詞：	VE-465
相關次數：	點閱：103 下載：0
分享至:	分享至facebook 分享至twitter

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

　許多文獻報導Aurora kinases 會在人類癌症中過度表現，包括肝癌、乳癌、直腸癌、胰臟癌、卵巢癌、胃癌和血癌。 Aurora kinases 的成員在細胞週期中扮演一個重要的調控角色，當Aurora kinases 有異常表現時，會造成基因的不穩定和細胞質分裂失敗，進而導致癌症。近幾年來，Aurora kinases已經成為具有展望性的小分子抑制劑的標的，被用在癌症的標靶治療上。在我的論文當中，將急性淋巴白血病的細胞處理VE-465 – 一種Aurora kinases 抑制劑，用以評估VE-465 在急性淋巴細胞中的效力及其分子作用機制。實驗成果發現，細胞在處理 VE-465 後，皆能有效地抑制Aurora kinases的活性，但是在九株急性淋巴白血病的細胞中，卻觀察到他們對藥物有不同的感受性。其中，RS4;11是對VE-465 有感受性的細胞，在處理VE-465之後，會使得細胞周期停留在G2/M，最後導致細胞死亡；而RPMI8402為對VE-465具抗藥性的細胞，細胞不會有死亡的現象，但是可觀察到多倍體細胞的產生。分析這些ALL細胞株Aurora kinases 及其活化因子，發現並無顯著性的差異。有趣的是，在藥物處理後，RS4;11細胞中CDKN1A的表現有顯著性地上升；相對地， RPMI8402 則維持在極低的表現。最後，利用在RS4;11中knockdown，以及在RPMI8402中overexpression CDKN1A 的實驗，我們證實了CDKN1A 的確參與在細胞對於VE-465不同的感受性中。

　Overexpression of Aurora kinases has been reported in a variety of human cancers, including hepatoma, breast, colon, pancreatic, ovarian, stomach and leukemia. Members of Aurora kinases play important roles in cell-cycle control. Aberrant expression of Aurora kinases result in genomic instability, cytokinesis failure, and finally lead to tumorigenesis. Recently, Aurora kinases have been greaty expected to be a target for therapeutic purpose in cancer therapy. In this study, we used VE-465, a pan-Aurora kinases inhibitor, to treat human acute lymphoblastic leukemia (ALL) cancer cell lines and then to assess its efficacy and investigate the underline mechanism. Cells treated with VE-465 can inhibit the activation of Aurora kinases effectively. Among nine ALL cell lines, some of them are resistant to VE-465 treatment and others are sensitive. In the VE-465-sentive cell lines, the addition of VE-465 can result in an increased G2/M and sub-G1 populations. Analyzing the expression levels of Aurora kinases and their activators, it exists no difference between the two kinds of cells. Importantly, the mRNA and protein expression levels of CDKN1A are up-regulated upon VE-465 treatment in the drug-sensitive cell – RS4;11, but keep in a very low levels and no significance change under VE-465 treatment in the drug-resistance cell – RPMI8402. Here, we uncover the role of CDKN1A involved in the susceptibility for VE-465 treatment by the manner of overexpression and knockdown CDKN1A.

中文摘要	I
ABSTRACT	II
ACKNOWLEDGEMENT	III
BIBLIOGRAPHY	IV
LIST OF FIGURES	VIII
LIST OF APPENDICES	X
ABBREVIATIONS	XI
I.	INTRODUCTION	1
1-1. Cell cycle progression	1
1-2. Entry into each phase of the cell cycle is controlled	1
1-3. Cancer is a disease of uncontrolled cell proliferation	2
1-4. Aurora kinase family	2
1-5. Aurora kinases aberrations in human cancer	4
1-6. The potential of Aurora kinase inhibitors as a targeted cancer therapeutics	5
1-7. Philadelphia chromosome	6
1-8. Inhibitors of BCR/ABL	6
1-9. VX-680 (MK-0457)	7
1-10. Objectives of the present study	8
II.	MATERIALS AND METHODS	9
2-1. Cell culture	9
2-2. Drug treatment	11
2-3. Oligonucleotide transfection	11
2-4. Transduction with lentivirus vectors	13
2-5. Cell proliferation assay	14
2-6. RNA extraction and Reverse transcriptase polymerase chain reaction	15
2-7. Western blotting	21
2-8. In vitro measurement of apoptosis	25
2-9. Cell cycle assay	26
2-10. Functional analyses	27
2-11. Statistical analysis	27
III.	RESULTS	28
3-1. Expression levels of Aurora kinases in human acute lymphoblastic leukemia (ALL) cell lines	28
3-2. The cytotoxicity of VE-465 in nine human acute lymphoblastic leukemia (ALL) cancer cell lines	28
3-3. Lower poliferative cells have high sensitivity to VE-465	29
3-4. Activation status of Aurora kinases and expression levels of their activator	30
3-5. VE-465 can efficiently inhibit the Aurora kinases’ activity in ALL cell lines	31
3-5. VE-465 suppresses of the activities of Aurora kinases in RS4;11 VE-465-sensitive cell line and RPMI8402 VE-465-resistant cell line	31
3-6. Effect of VE-465 on growth arrest	32
3-7. VE-465 induces G2/M arrest in RS4;11 and polypoidy in RPMI 8402	32
3-8. VE-465 induces a cell cycle distribution change	33
3-9. Role of Aurora kinase inhibition-mediated apoptosis in the response to VE-465	34
3-10. Identification of genes that confer cell sensitivity to VE-465	35
3-11. RS4;11 expresses a higher level of CDKN1A (p21) than RPMI8402	35
3-12. Down-regulation of p21 by a shRNA blunts the ability of VE-465 to induce apoptosis of RS4;11 cells	36
3-13. Overexpression of CDKN1A can modulate the drug susceptibility of cells	37
3-14. Role of p53 in the response to Aurora kinase inhibition by VE-465	37
3-15. VE-465 has activity in a solid tumor	38
3-16. The increased expression of CDN1A might contribute to VE-465 sensitivity also observed in colon cancer cells	39
IV.	DISCUSSION	40
4-1. VE-465 is a candidate in MLL-AF4 acute lymphocytic leukemia	40
4-2. VE-465 induces apoptosis in Aurora A-low cells	40
4-3. The potential anticancer effects of VE-465	42
4-4. The molecular mechanism of Aurora kinase inhibitor, VE–465, in acute lymphoblastic leukemia (ALL)	43
V.	CONCLUSIONS	45
VI.	PROSPECT	46
VII.	REFERENCE	47
Fig. 1 The expression levels of Aurora kinases in the tested cell lines	54
Fig. 2 The effects of VE-465 on human acute lymphoblastic leukemia (ALL) cancer cell lines.	55
Fig. 3 Antiproliferative activity of VE-465 versus VX-680.	56
Fig. 4 The poliferative rate in RS4;11 and RPMI8402 cells.	57
Fig. 5 The expression status of phosphorylated Aurora A and B and the activators of Aurora kinases.	58
Fig. 6 Efficacy of VE-465 on Aurora kinases.	59
Fig. 7 Evaluation of the inhibition of Aurora kinase	60
Fig. 8 VE-465 induces the phosphorylation of Histone H3.	61
Fig. 9 VE-465 affects Cell-cycle distribution.	62
Fig. 10 VE-465 affects Cell-cycle distribution.	63
Fig. 11 VE-465 induces caspase 3 activation.	64
Fig. 12 Functional analysis from IPA in dataset-1	65
Fig. 13 Functional analysis from IPA in dataset-2	66
Fig. 14 Integrated miRNA and mRNA expression by bioinformatics analyses.	67
Fig. 15 Analysis of TP53 downstream genes.	68
Fig. 16 Effect of CDKN1A silencing.	69
Fig. 17 Effect on ectopic p21 expression.	70
Fig. 18 Analysis of TP53 expression.	71
Fig. 19 The different VE465 sensitivity of human colorectal cancer cell lines – SW480 and SW620 is due to the different expression levels of CDKN1A.	72
Fig. 20 Overexpression of CDKN1A.	73
Appendix I: The development of Aurora kinase inhibitors	74
Appendix II: The information of the nine cell lines, by which cell viability assay, was carried out.	75
Appendix III: Status of TP53 in RS4;11 and RPMI8402 cells and comparision of RS4;11 and RPMI8402 cells	76
Appendix IV: TP53 regulated signaling pathway	77
Appendix V: (A) The effect of VE-465 and VX-680 as well as (B) the expression level of p21 in MLL-AF4 leukemia cells	78
Appendix VI: Inhibition of Aurora A in RS4;11 cells	79
Appendix VII: Inhibition of Aurora B in RPMI8402 cells	80
Appendix VIII: The potential anticancer effects of VE-465	81
自述	82


                                    

1. Smith JA, Martin L (1973) Do cells cycle? Proc Natl Acad Sci U S A 70: 1263-1267.

2. Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432: 316-323.

3. Massague J (2004) G1 cell-cycle control and cancer. Nature 432: 298-306.

4. Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8: 379-393.

5. Andrews PD, Knatko E, Moore WJ, Swedlow JR (2003) Mitotic mechanics: the auroras come into view. Curr Opin Cell Biol 15: 672-683.

6. Carmena M, Earnshaw WC (2003) The cellular geography of aurora kinases. Nat Rev Mol Cell Biol 4: 842-854.

7. Nigg EA (2001) Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2: 21-32.

8. Farruggio DC, Townsley FM, Ruderman JV (1999) Cdc20 associates with the kinase aurora2/Aik. Proc Natl Acad Sci U S A 96: 7306-7311.

9. Nguyen HG, Chinnappan D, Urano T, Ravid K (2005) Mechanism of Aurora-B degradation and its dependency on intact KEN and A-boxes: identification of an aneuploidy-promoting property. Mol Cell Biol 25: 4977-4992.

10. Taguchi S, Honda K, Sugiura K, Yamaguchi A, Furukawa K, et al. (2002) Degradation of human Aurora-A protein kinase is mediated by hCdh1. FEBS Lett 519: 59-65.

11. Crane R, Kloepfer A, Ruderman JV (2004) Requirements for the destruction of human Aurora-A. J Cell Sci 117: 5975-5983.

12. Marumoto T, Zhang D, Saya H (2005) Aurora-A - a guardian of poles. Nat Rev Cancer 5: 42-50.

13. Crane R, Gadea B, Littlepage L, Wu H, Ruderman JV (2004) Aurora A, meiosis and mitosis. Biol Cell 96: 215-229.

14. Andrews PD (2005) Aurora kinases: shining lights on the therapeutic horizon? Oncogene 24: 5005-5015.

15. Ruchaud S, Carmena M, Earnshaw WC (2007) Chromosomal passengers: conducting cell division. Nat Rev Mol Cell Biol 8: 798-812.

16. Adams RR, Carmena M, Earnshaw WC (2001) Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol 11: 49-54.

17. Vader G, Maia AF, Lens SM (2008) The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond. Cell Div 3: 10.

18. Li X, Sakashita G, Matsuzaki H, Sugimoto K, Kimura K, et al. (2004) Direct association with inner centromere protein (INCENP) activates the novel chromosomal passenger protein, Aurora-C. J Biol Chem 279: 47201-47211.

19. Sampath SC, Ohi R, Leismann O, Salic A, Pozniakovski A, et al. (2004) The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell 118: 187-202.

20. Sasai K, Katayama H, Stenoien DL, Fujii S, Honda R, et al. (2004) Aurora-C kinase is a novel chromosomal passenger protein that can complement Aurora-B kinase function in mitotic cells. Cell Motil Cytoskeleton 59: 249-263.

21. Tang CJ, Lin CY, Tang TK (2006) Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis. Dev Biol 290: 398-410.

22. Warner SL, Bearss DJ, Han H, Von Hoff DD (2003) Targeting Aurora-2 kinase in cancer. Mol Cancer Ther 2: 589-595.

23. Keen N, Taylor S (2004) Aurora-kinase inhibitors as anticancer agents. Nat Rev Cancer 4: 927-936.

24. Tatsuka M, Katayama H, Ota T, Tanaka T, Odashima S, et al. (1998) Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1-like midbody-associated protein mitotic kinase in human cancer cells. Cancer Res 58: 4811-4816.

25. Katayama H, Ota T, Jisaki F, Ueda Y, Tanaka T, et al. (1999) Mitotic kinase expression and colorectal cancer progression. J Natl Cancer Inst 91: 1160-1162.

26. Carvajal RD, Tse A, Schwartz GK (2006) Aurora kinases: new targets for cancer therapy. Clin Cancer Res 12: 6869-6875.

27. Fu J, Bian M, Jiang Q, Zhang C (2007) Roles of Aurora kinases in mitosis and tumorigenesis. Mol Cancer Res 5: 1-10.

28. Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PN, Jr., et al. (2008) Aurora kinases as anticancer drug targets. Clin Cancer Res 14: 1639-1648.

29. Liu Q, Ruderman JV (2006) Aurora A, mitotic entry, and spindle bipolarity. Proc Natl Acad Sci U S A 103: 5811-5816.

30. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, et al. (2003) Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 161: 267-280.

31. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, et al. (2003) The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 161: 281-294.

32. Yang H, Burke T, Dempsey J, Diaz B, Collins E, et al. (2005) Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase. FEBS Lett 579: 3385-3391.

33. Sawyers CL, Druker B (1999) Tyrosine kinase inhibitors in chronic myeloid leukemia. Cancer J Sci Am 5: 63-69.

34. Chai SK, Nichols GL, Rothman P (1997) Constitutive activation of JAKs and STATs in BCR-Abl-expressing cell lines and peripheral blood cells derived from leukemic patients. J Immunol 159: 4720-4728.

35. Ilaria RL, Jr., Van Etten RA (1996) P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members. J Biol Chem 271: 31704-31710.

36. Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ, Wen SC, et al. (1995) Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells. Blood 86: 726-736.

37. Tauchi T, Okabe S, Miyazawa K, Ohyashiki K (1998) The tetramerization domain-independent Ras activation by BCR-ABL oncoprotein in hematopoietic cells. Int J Oncol 12: 1269-1276.

38. Deininger M, Buchdunger E, Druker BJ (2005) The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood 105: 2640-2653.

39. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, et al. (2001) Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 344: 1038-1042.

40. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, et al. (2001) Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293: 876-880.

41. Hofmann WK, Jones LC, Lemp NA, de Vos S, Gschaidmeier H, et al. (2002) Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood 99: 1860-1862.

42. Nardi V, Azam M, Daley GQ (2004) Mechanisms and implications of imatinib resistance mutations in BCR-ABL. Curr Opin Hematol 11: 35-43.

43. Shah NP, Tran C, Lee FY, Chen P, Norris D, et al. (2004) Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305: 399-401.

44. Weisberg E, Manley PW, Breitenstein W, Bruggen J, Cowan-Jacob SW, et al. (2005) Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 7: 129-141.

45. O'Hare T, Corbin AS, Druker BJ (2006) Targeted CML therapy: controlling drug resistance, seeking cure. Curr Opin Genet Dev 16: 92-99.

46. Soverini S, Colarossi S, Gnani A, Rosti G, Castagnetti F, et al. (2006) Contribution of ABL kinase domain mutations to imatinib resistance in different subsets of Philadelphia-positive patients: by the GIMEMA Working Party on Chronic Myeloid Leukemia. Clin Cancer Res 12: 7374-7379.

47. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, et al. (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 10: 262-267.

48. Carter TA, Wodicka LM, Shah NP, Velasco AM, Fabian MA, et al. (2005) Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc Natl Acad Sci U S A 102: 11011-11016.

49. Giles FJ, Cortes J, Jones D, Bergstrom D, Kantarjian H, et al. (2007) MK-0457, a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the T315I BCR-ABL mutation. Blood 109: 500-502.

50. Pollard JR, Mortimore M (2009) Discovery and development of aurora kinase inhibitors as anticancer agents. J Med Chem 52: 2629-2651.

51. Young MA, Shah NP, Chao LH, Seeliger M, Milanov ZV, et al. (2006) Structure of the kinase domain of an imatinib-resistant Abl mutant in complex with the Aurora kinase inhibitor VX-680. Cancer Res 66: 1007-1014.

52. Mountzios G, Terpos E, Dimopoulos MA (2008) Aurora kinases as targets for cancer therapy. Cancer Treat Rev 34: 175-182.

53. Lin ZZ, Hsu HC, Hsu CH, Yeh PY, Huang CY, et al. (2009) The Aurora kinase inhibitor VE-465 has anticancer effects in pre-clinical studies of human hepatocellular carcinoma. J Hepatol 50: 518-527.

54. Akahane D, Tauchi T, Okabe S, Nunoda K, Ohyashiki K (2008) Activity of a novel Aurora kinase inhibitor against the T315I mutant form of BCR-ABL: in vitro and in vivo studies. Cancer Sci 99: 1251-1257.

55. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, et al. (2002) Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol 158: 617-623.

56. Matthews N, Visintin C, Hartzoulakis B, Jarvis A, Selwood DL (2006) Aurora A and B kinases as targets for cancer: will they be selective for tumors? Expert Rev Anticancer Ther 6: 109-120.

57. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW (1991) Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51: 6304-6311.

58. Fuster JJ, Sanz-Gonzalez SM, Moll UM, Andres V (2007) Classic and novel roles of p53: prospects for anticancer therapy. Trends Mol Med 13: 192-199.

59. Leibovitz A, Stinson JC, McCombs WB, 3rd, McCoy CE, Mazur KC, et al. (1976) Classification of human colorectal adenocarcinoma cell lines. Cancer Res 36: 4562-4569.

60. Pui CH, Chessells JM, Camitta B, Baruchel A, Biondi A, et al. (2003) Clinical heterogeneity in childhood acute lymphoblastic leukemia with 11q23 rearrangements. Leukemia 17: 700-706.

61. Shi Y, Reiman T, Li W, Maxwell CA, Sen S, et al. (2007) Targeting aurora kinases as therapy in multiple myeloma. Blood 109: 3915-3921.

62. Okada H, Mak TW (2004) Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer 4: 592-603.

63. Margolis RL, Lohez OD, Andreassen PR (2003) G1 tetraploidy checkpoint and the suppression of tumorigenesis. J Cell Biochem 88: 673-683.

64. Vogel C, Kienitz A, Hofmann I, Muller R, Bastians H (2004) Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy. Oncogene 23: 6845-6853.

65. Kallio MJ, McCleland ML, Stukenberg PT, Gorbsky GJ (2002) Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis. Curr Biol 12: 900-905.

66. Liu Q, Kaneko S, Yang L, Feldman RI, Nicosia SV, et al. (2004) Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215. J Biol Chem 279: 52175-52182.

67. Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, et al. (2004) Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet 36: 55-62.

68. Kaestner P, Stolz A, Bastians H (2009) Determinants for the efficiency of anticancer drugs targeting either Aurora-A or Aurora-B kinases in human colon carcinoma cells. Mol Cancer Ther 8: 2046-2056.

69. Kojima K, Konopleva M, Tsao T, Nakakuma H, Andreeff M (2008) Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis along with reduced endoreduplication in acute myelogenous leukemia. Blood 112: 2886-2895.

校內：2016-08-01公開
校外：不公開電子論文尚未授權公開，紙本請查館藏目錄

簡易檢索 / 詳目顯示

相關論文