請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77286完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林亮音(Liang-In Lin) | |
| dc.contributor.author | Fang-Yu Lo | en |
| dc.contributor.author | 羅方妤 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:54:14Z | - |
| dc.date.available | 2021-07-10T21:54:14Z | - |
| dc.date.copyright | 2019-08-28 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-09 | |
| dc.identifier.citation | 1. Dohner, H., et al., Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood, 2010. 115(3): p. 453-74.
2. Bennett, J.M., et al., Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol, 1976. 33(4): p. 451-8. 3. Saultz, J. and R. Garzon, Acute Myeloid Leukemia: A Concise Review. Journal of Clinical Medicine, 2016. 5(3): p. 33. 4. Arber, D.A., et al., The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood, 2016. 127(20): p. 2391-405. 5. Dohner, H., et al., Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood, 2017. 129(4): p. 424-447. 6. Dohner, H., D.J. Weisdorf, and C.D. Bloomfield, Acute Myeloid Leukemia. N Engl J Med, 2015. 373(12): p. 1136-52. 7. Mayer, R.J., et al., Intensive postremission chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia Group B. N Engl J Med, 1994. 331(14): p. 896-903. 8. Bagrintseva, K., et al., Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of acquired drug resistance to PTK inhibitors in FLT3-ITD–transformed hematopoietic cells. Blood, 2004. 103(6): p. 2266-2275. 9. Meshinchi, S. and F.R. Appelbaum, Structural and functional Alterations of FLT3 in Acute Myeloid Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 2009. 15(13): p. 4263-4269. 10. Stirewalt, D.L. and J.P. Radich, The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer, 2003. 3(9): p. 650-665. 11. Wander, S.A., M.J. Levis, and A.T. Fathi, The evolving role of FLT3 inhibitors in acute myeloid leukemia: quizartinib and beyond. Therapeutic Advances in Hematology, 2014. 5(3): p. 65-77. 12. Swords, R., C. Freeman, and F. Giles, Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia. Leukemia, 2012. 26(10): p. 2176-85. 13. Whitman, S.P., et al., Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study. Cancer Res, 2001. 61(19): p. 7233-9. 14. Donnell MR, T.M., Abboud CN, Altman JK, Appelbaum FR, Arber DA, et al. , Acute myeloid leukemia, version 3.2017, NCCN Clinical Practice Guidelines in Oncology. . J Natl Compr Canc Netw, 2017. 15: p. 926–57. 15. Cortes JE, K.H., Kadia TM, Borthakur G, Konopleva and G.-M.G. M, Crenolanib besylate, a type I panFLT3 inhibitor, to demonstrate clinical activity in multiply relapsed FLT3-ITD and D835 AML. J Clin Oncol, 2016. 34: p. 7008. 16. Kronke, J., et al., Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia. Blood, 2013. 122(1): p. 100-8. 17. Daver, N., et al., Targeting FLT3 mutations in AML: review of current knowledge and evidence. Leukemia, 2019. 33(2): p. 299-312. 18. Stone, R.M., et al., Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood, 2005. 105(1): p. 54-60. 19. Smith, B.D., et al., Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. Blood, 2004. 103(10): p. 3669-76. 20. Pratz, K.W., et al., A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response. Blood, 2009. 113(17): p. 3938-46. 21. Fiedler, W., et al., A phase I/II study of sunitinib and intensive chemotherapy in patients over 60 years of age with acute myeloid leukaemia and activating FLT3 mutations. Br J Haematol, 2015. 169(5): p. 694-700. 22. Stone RM, M.S., Sanford BL, Laumann K, Geyer SM, and B. CD, The addition of midostaurin to standard chemotherapy decreases cumulative incidence of relapse (CIR) in the international prospective randomized, placebo-controlled, double-blind trial (CALGB 10603 / RATIFY [Alliance]) for newly diagnosed acute myeloid leukemia (AML) patients with FLT3 mutations. . Blood, 2017. 130: p. 2580. 23. Levis, M., Midostaurin approved for FLT3-mutated AML. Blood, 2017. 24. Dhillon, S., Gilteritinib: First Global Approval. Drugs, 2019. 25. Yakes, F.M., et al., Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth. Mol Cancer Ther, 2011. 10(12): p. 2298-308. 26. Fathi, A.T., et al., A Phase I Study of the Multi-Targeted Tyrosine Kinase Inhibitor Cabozantinib in Patients with Acute Myeloid Leukemia. Blood, 2016. 128(22): p. 5218-5218. 27. Deeks, E.D., Cabozantinib: A Review in Advanced Hepatocellular Carcinoma. Target Oncol, 2019. 14(1): p. 107-113. 28. Schoffski, P., et al., Phase II randomised discontinuation trial of cabozantinib in patients with advanced solid tumours. Eur J Cancer, 2017. 86: p. 296-304. 29. Tolaney, S.M., et al., Phase II and Biomarker Study of Cabozantinib in Metastatic Triple-Negative Breast Cancer Patients. Oncologist, 2017. 22(1): p. 25-32. 30. Fathi, A.T., et al., Cabozantinib is well tolerated in acute myeloid leukemia and effectively inhibits the resistance-conferring FLT3/tyrosine kinase domain/F691 mutation. Cancer, 2018. 124(2): p. 306-314. 31. Lu, J.W., et al., Cabozantinib is selectively cytotoxic in acute myeloid leukemia cells with FLT3-internal tandem duplication (FLT3-ITD). Cancer Lett, 2016. 376(2): p. 218-25. 32. Levine, A.J., p53, the cellular gatekeeper for growth and division. Cell, 1997. 88(3): p. 323-31. 33. Peller, S. and V. Rotter, TP53 in hematological cancer: low incidence of mutations with significant clinical relevance. Hum Mutat, 2003. 21(3): p. 277-84. 34. Lane, D.P., Cancer. p53, guardian of the genome. Nature, 1992. 358(6381): p. 15-6. 35. Bueso-Ramos, C.E., et al., The human MDM-2 oncogene is overexpressed in leukemias. Blood, 1993. 82(9): p. 2617-23. 36. Bykov, V.J., et al., Mutant p53-dependent growth suppression distinguishes PRIMA-1 from known anticancer drugs: a statistical analysis of information in the National Cancer Institute database. Carcinogenesis, 2002. 23(12): p. 2011-8. 37. Bykov, V.J., et al., Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat Med, 2002. 8(3): p. 282-8. 38. Lambert, J.M., et al., PRIMA-1 reactivates mutant p53 by covalent binding to the core domain. Cancer Cell, 2009. 15(5): p. 376-88. 39. Lehmann, S., et al., Targeting p53 in vivo: a first-in-human study with p53-targeting compound APR-246 in refractory hematologic malignancies and prostate cancer. J Clin Oncol, 2012. 30(29): p. 3633-9. 40. Issaeva, N., et al., Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med, 2004. 10(12): p. 1321-8. 41. Zhao, C.Y., et al., Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA. Cell Cycle, 2010. 9(9): p. 1847-55. 42. Burmakin, M., et al., Dual targeting of wild-type and mutant p53 by small molecule RITA results in the inhibition of N-Myc and key survival oncogenes and kills neuroblastoma cells in vivo and in vitro. Clin Cancer Res, 2013. 19(18): p. 5092-103. 43. Grinkevich, V.V., et al., Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis. Cancer Cell, 2009. 15(5): p. 441-53. 44. Hu, B., et al., MDMX overexpression prevents p53 activation by the MDM2 inhibitor Nutlin. J Biol Chem, 2006. 281(44): p. 33030-5. 45. Vassilev, L.T., et al., In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science, 2004. 303(5659): p. 844-8. 46. Andreeff, M., et al., Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia. Clin Cancer Res, 2016. 22(4): p. 868-76. 47. Ding, Q., et al., Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development. J Med Chem, 2013. 56(14): p. 5979-83. 48. Reis, B., et al., Acute myeloid leukemia patients' clinical response to idasanutlin (RG7388) is associated with pre-treatment MDM2 protein expression in leukemic blasts. Haematologica, 2016. 101(5): p. e185-8. 49. Yee, K., et al., Phase 1/1b Study of RG7388, a Potent MDM2 Antagonist, in Acute Myelogenous Leukemia (AML) Patients (Pts). Blood, 2014. 124(21): p. 116-116. 50. Chatterjee, N. and G.C. Walker, Mechanisms of DNA damage, repair, and mutagenesis. Environ Mol Mutagen, 2017. 58(5): p. 235-263. 51. Scully, R., et al., DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat Rev Mol Cell Biol, 2019. 52. Lambert, S. and B.S. Lopez, Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms. Embo j, 2000. 19(12): p. 3090-9. 53. Henning, W. and H.W. Sturzbecher, Homologous recombination and cell cycle checkpoints: Rad51 in tumour progression and therapy resistance. Toxicology, 2003. 193(1-2): p. 91-109. 54. Xia, S.J., M.A. Shammas, and R.J. Shmookler Reis, Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase. Mol Cell Biol, 1997. 17(12): p. 7151-8. 55. Vispe, S., et al., Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation. Nucleic Acids Res, 1998. 26(12): p. 2859-64. 56. Ohnishi, T., et al., In vitro and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene. Biochem Biophys Res Commun, 1998. 245(2): p. 319-24. 57. Slupianek, A., et al., BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance. Mol Cell, 2001. 8(4): p. 795-806. 58. Seedhouse, C.H., et al., DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412. Leukemia, 2006. 20(12): p. 2130-6. 59. Maifrede, S., et al., Tyrosine kinase inhibitor-induced defects in DNA repair sensitize FLT3(ITD)-positive leukemia cells to PARP1 inhibitors. Blood, 2018. 132(1): p. 67-77. 60. Esposito, M.T. and C.W. So, DNA damage accumulation and repair defects in acute myeloid leukemia: implications for pathogenesis, disease progression, and chemotherapy resistance. Chromosoma, 2014. 123(6): p. 545-61. 61. Chakraborty, C., et al., Zebrafish: a complete animal model for in vivo drug discovery and development. Curr Drug Metab, 2009. 10(2): p. 116-24. 62. Lu, J.W., et al., Zebrafish as a Model for the Study of Human Myeloid Malignancies. Biomed Res Int, 2015. 2015: p. 641475. 63. Kojima, K., J. Ishizawa, and M. Andreeff, Pharmacological activation of wild-type p53 in the therapy of leukemia. Exp Hematol, 2016. 44(9): p. 791-8. 64. Cavenee, W.K., et al., Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature, 1983. 305(5937): p. 779-84. 65. Perdrix, A., et al., PRIMA-1 and PRIMA-1(Met) (APR-246): From Mutant/Wild Type p53 Reactivation to Unexpected Mechanisms Underlying Their Potent Anti-Tumor Effect in Combinatorial Therapies. Cancers (Basel), 2017. 9(12). 66. Zhang, Q., et al., APR-246 reactivates mutant p53 by targeting cysteines 124 and 277. Cell Death Dis, 2018. 9(5): p. 439. 67. Parrales, A. and T. Iwakuma, Targeting Oncogenic Mutant p53 for Cancer Therapy. Front Oncol, 2015. 5: p. 288. 68. Kazemi, A., M. Safa, and A. Shahbazi, RITA enhances chemosensivity of pre-B ALL cells to doxorubicin by inducing p53-dependent apoptosis. Hematology, 2011. 16(4): p. 225-31. 69. Nabinger, S.C., et al., Mutant p53 enhances leukemia-initiating cell self-renewal to promote leukemia development. Leukemia, 2019. 33(6): p. 1535-1539. 70. Zhang, D., et al., CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells. Front Neurol, 2018. 9: p. 255. 71. De Kouchkovsky, I. and M. Abdul-Hay, 'Acute myeloid leukemia: a comprehensive review and 2016 update'. Blood Cancer J, 2016. 6(7): p. e441. 72. Talati, C. and K. Sweet, Recently approved therapies in acute myeloid leukemia: A complex treatment landscape. Leuk Res, 2018. 73: p. 58-66. 73. Prokocimer, M., A. Molchadsky, and V. Rotter, Dysfunctional diversity of p53 proteins in adult acute myeloid leukemia: projections on diagnostic workup and therapy. Blood, 2017. 130(6): p. 699-712. 74. Quintas-Cardama, A., et al., p53 pathway dysfunction is highly prevalent in acute myeloid leukemia independent of TP53 mutational status. Leukemia, 2017. 31(6): p. 1296-1305. 75. Secchiero, P., et al., The MDM-2 antagonist nutlin-3 promotes the maturation of acute myeloid leukemic blasts. Neoplasia, 2007. 9(10): p. 853-61. 76. Secchiero, P., et al., Synergistic cytotoxic activity of recombinant TRAIL plus the non-genotoxic activator of the p53 pathway nutlin-3 in acute myeloid leukemia cells. Curr Drug Metab, 2007. 8(4): p. 395-403. 77. Perdrix, A., et al., PRIMA-1 and PRIMA-1(Met) (APR-246): From Mutant/Wild Type p53 Reactivation to Unexpected Mechanisms Underlying Their Potent Anti-Tumor Effect in Combinatorial Therapies. Cancers, 2017. 9(12): p. 172. 78. Mohell, N., et al., APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells. Cell Death Dis, 2015. 6: p. e1794. 79. Dittmer, D., et al., Gain of function mutations in p53. Nat Genet, 1993. 4(1): p. 42-6. 80. Lang, G.A., et al., Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell, 2004. 119(6): p. 861-72. 81. Guerra, V.A., C. DiNardo, and M. Konopleva, Venetoclax-based therapies for acute myeloid leukemia. Best Pract Res Clin Haematol, 2019. 32(2): p. 145-153. 82. Wiegering, A., et al., Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin. Neoplasia, 2017. 19(4): p. 301-309. 83. Rosell, R., et al., Adaptive resistance to targeted therapies in cancer. Transl Lung Cancer Res, 2013. 2(3): p. 152-9. 84. Seipel, K., et al., MDM2- and FLT3-inhibitors in the treatment of FLT3-ITD acute myeloid leukemia, specificity and efficacy of NVP-HDM201 and midostaurin. Haematologica, 2018. 103(11): p. 1862-1872. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77286 | - |
| dc.description.abstract | 急性骨髓性白血病(AML)是由於骨髓系造血前驅細胞異常增生且無法分化所導致的血液惡性腫瘤。FLT3是一種酪胺酸激酶受體(receptor tyrosine kinase, RTK)，大約有20%-30%的AML病人帶有FLT3基因突變，其中又以FLT3內部串聯重複(FLT3-ITD)最常見，並且具有FLT3-ITD突變的病人通常預後不佳。過去實驗室的研究發現，FLT3標靶藥物Cabozantinib (CBZ)能夠選擇性的抑制FLT3-ITD突變的AML細胞株MV4-11和Molm13之增生，在動物實驗中也發現CBZ能有效抑制其腫瘤的生長。目前臨床上使用FLT3標靶藥物最大的困境就是產生抗藥性問題。對於產生抗藥性的原因至今仍不清楚，因此發展一個新的治療策略是必要的。
為探討CBZ的抗藥性，實驗室先前分別建立了對於CBZ具有抗藥性的細胞株MV4-11-XR和Molm13-XR，我們利用RNA次世代定序(RNA-seq)分析母株細胞及抗藥性細胞株間mRNA表達量的差異。我們發現MV4-11-XR和Molm13-XR共同具有87個差異性下調的基因，經Metascape做基因富集路徑分析發現這些差異性下調的基因和p53訊號傳遞路徑最有相關。同時，以即時監控聚合酶鏈鎖反應(Real-time quantitative PCR)驗證這兩株對CBZ具有抗藥性的細胞株之p53訊號傳遞路徑下游相關基因表達量相較於母株細胞皆明顯下調。 p53訊號傳遞路徑功能失活在多種癌症中都有被發現，但p53功能喪失和癌症抗藥性之間的關係，並未被研究地很透徹。因此，我們以西方墨點法進一步探討MV4-11-XR和Molm13-XR細胞之p53訊號傳遞路徑下調的原因，結果顯示MV4-11-XR細胞之p53功能喪失的可能原因是野生型的TP53發生基因座缺失所致。而Molm13-XR細胞p53功能喪失的原因則可能和p53負調節蛋白質MDM2堆積有關。 依據上述實驗結果，我們針對其p53功能喪失的原因分別予以不同藥物激活p53活性以期能克服抗藥性細胞株對CBZ的抗藥性。過去文獻指出PRIMA-1及RITA可以透過和p53結合，使突變型的p53重新獲得野生型p53的功能，並得以正常活化下游相關基因。於是，我們將PRIMA-1及RITA分別合併CBZ處理MV4-11-XR細胞，結果顯示RITA合併CBZ可以達到協同作用，使CBZ用藥濃度從1µM降低至400nM即可達到半致死效果。 RG7388是第二代的MDM2抑制劑，我們以RG7388合併CBZ處理Molm13-XR細胞，結果顯示RG7388合併CBZ可以達到協同作用，使CBZ用藥濃度從400nM降低至100nM即可達到半致死效果。 透過小分子藥物重新活化p53合併FLT3抑制劑的用藥策略，在兩株抗藥性細胞中，都可顯著提升細胞內p53訊息路徑中之下游相關基因的表達及蛋白質總量，誘導細胞週期停滯且抑制細胞自我更新及增生能力；而在Molm13-XR細胞中更發現合併藥物使用可抑制DNA修補機制相關蛋白RAD51的總量，使細胞一直處於DNA損傷狀態，誘導細胞凋亡。此外，針對Molm13-XR細胞之合併藥物策略在斑馬魚動物實驗中也有顯著抑制效果。 整體來說，對CBZ具有抗藥性的MV4-11-XR和Molm13-XR細胞株都因不同原因而發生p53路徑功能喪失；而透過小分子藥物針對不同原因重新激活p53，可以有效增強抗藥性細胞株對CBZ的敏感性。上述實驗結果也揭示了重新活化p53並同時標靶FLT3，可能是未來臨床上有潛力的治療策略。 | zh_TW |
| dc.description.abstract | Acute myeloid leukemia (AML) is a hematological malignancy caused by abnormal proliferation of bone marrow-derived hematopoietic precursor cells and differentiation arrest. FLT3 is a receptor tyrosine kinase (RTK), with approximately 20%-30% of AML patients with FLT3 gene mutations, among which FLT3 internal tandem repeats (FLT3-ITD) are the most common one and patients with FLT3-ITD mutations usually showed a poor prognosis. In the past, our laboratory studies have found that the FLT3 target drug Cabozantinib (CBZ) can selectively inhibit the proliferation of FLT3-ITD mutant AML cell lines MV4-11 and Molm13. It has also been found that CBZ can effectively inhibit tumor growth in animal experiments. Currently, the resistance of leukemia cells to target therapy drugs becomes the main obstacle in the treatment of AML. The reasons for drug resistance are still unclear, so developing a new treatment strategy is necessary.
In order to study the drug resistance mechanism, we established CBZ -resistant cell lines from MV4-11 and Molm13 cells, named MV4-11 XR and Molm-13 XR, respectively.We used RNA next generation sequencing (RNA-seq) to analyze differences in mRNA expression between parental and drug-resistant cell lines. We found that 87 differentially down-regulated genes between MV4-11-XR and Molm13-XR. Then, we used Metascape for gene enrichment pathway analysis to find that these differentially down-regulated genes are most relevant to the p53 signaling pathway. We also used real-time quantitative PCR to verify that the expression of the downstream gene related to the p53 signal pathway was significantly down-regulated in the two cell lines resistant to CBZ. To explore the reasons for the loss of p53 pathway function in cell lines resistant to CBZ.We used western blot analysis, IGV analysis and microsatellite analysis to find that the loss of p53 function in MV4-11-XR cells may be due to mutations in the TP53 sequence, and further confirmed by microsatellite PCR analysis that the wild type TP53 is loss of heterozygosity in MV4-11-XR cells. On the other hand, the loss of p53 function in Molm13-XR cells may be related to the accumulation of p53 negative regulatory protein MDM2. Based on the above experimental results, we applied different drugs to activate p53 activity for the reason of its loss of p53 function in order to enhance the sensitivity of drug-resistant cell lines to CBZ. In the past, it was pointed out that PRIMA-1 and RITA can bind to p53, and the mutant p53 can regain the function of wild-type p53 and activate the downstream related genes. Therefore, we combined PRIMA-1 and RITA with CBZ to treat MV4-11-XR cells, respectively. The results showed that RITA combined with CBZ can achieve synergistic effect, and the CBZ concentration can be reduced from 1μM to 400nM to achieve semi-lethal effect. RG7388 is a second-generation MDM2 inhibitor. We combined RG7388 with CBZ to treat Molm13-XR cells. The results showed that RG7388 combined with CBZ can achieve synergistic effect, and the CBZ concentration can be reduced from 400nM to 100nM to achieve semi-lethal effect. The strategy of re-activating p53 combined with FLT3 inhibitors can significantly increase the expression of downstream related genes and total protein level in the p53 signaling pathway, induce cell cycle arrest and inhibit cell self-renewal and proliferation. In Molm13-XR cells, it was found that the combined use of drugs inhibited the total protein level of RAD51, DNA repair related protein, and the cells were always in DNA damage state then induced apoptosis. In addition, the combined drug strategy for Molm13-XR cells also showed significant inhibitory effects in zebrafish in vivo experiments. Overall, the causes of loss of p53 pathway function in MV4-11-XR and Molm13-XR cell lines are different. Reactivation of p53 by small molecule drugs for different reasons can effectively enhance the sensitivity of drug-resistant cell lines to CBZ. The above results also revealed that reactivation of p53 and simultaneous targeting of FLT3 may be a clinically potential therapeutic strategy in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:54:14Z (GMT). No. of bitstreams: 1 ntu-108-R06424020-1.pdf: 3994825 bytes, checksum: 1eae2264f66c4bd1a6e91673144c90e8 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
目錄 1 圖目錄 3 附圖 4 表目錄 5 縮寫表 6 第一章 前言 7 1.1 急性骨髓性白血病簡介 7 1.1.1 急性骨髓性白血病(Acute Myeloid Leukemia, AML) 7 1.1.2 AML之分類 7 1.1.3 AML之治療 8 1.2 FLT3之簡介 9 1.2.1 FLT3之治療 10 1.3 Cabozantinib (CBZ)抗藥性細胞株Molm13-XR與MV4-11-XR之簡介 11 1.4 p53之簡介 12 1.4.1 PRIMA-1 12 1.4.2 RITA 13 1.4.3 Idasanutlin 13 1.5 DNA修補機制和AML的關係 14 1.61斑馬魚 15 第二章 研究目的 16 第三章 材料與方法 17 3.1 材料 17 3.1.1 細胞株 17 3.1.2 儀器設備 17 3.1.3 藥品 18 3.1.4 抗體 20 3.1.5 試劑組 21 3.1.6 藥品與試劑配製 21 3.2 方法 23 3.2.1 細胞培養 23 3.2.2 斑馬魚飼養 23 3.2.3 細胞抑殺試驗 (MTS assay) 24 3.2.4 流式細胞儀(Flow cytometry)分析 24 I. 細胞凋亡(apoptosis)分析 24 II. 細胞週期分析 24 3.2.5 細胞生長曲線(Cell proliferation curve) 25 3.2.6 細胞群落分析(Colony formation assay) 25 3.2.7 細胞內蛋白質萃取 26 3.2.8 蛋白質濃度定量 26 3.2.9 西方墨點法 26 3.2.10 RNA萃取 27 3.2.11 反轉錄酶反應 (Reverse Transcription) 28 3.2.12 即時監控聚合酶連鎖反應 (Quantitative-PCR) 28 3.2.13 斑馬魚藥物毒性試驗 28 3.2.14 斑馬魚in vivo實驗 29 3.2.15 所使用到的軟體及網站 29 3.2.16 統計方法 29 第四章 實驗結果 30 4.1 對CBZ具抗藥性的細胞與其母株細胞間具差異性表達基因之基因富集分析 30 4.2 對CBZ具抗藥性的細胞MV4-11-XR 30 4.2.1 MV4-11-XR p53路徑功能下調原因的分析 30 4.2.2 MV4-11-XR TP53基因座缺失(Loss Of Heterozygosity, LOH) 31 4.2.3 PRIMA-1合併CBZ對MV4-11-XR細胞沒有協同作用 32 4.2.4 RITA合併CBZ對MV4-11-XR細胞有協同作用 33 4.2.5 RITA合併CBZ處理MV4-11-XR細胞後可顯著增加p53 訊息路徑中的下游相關基因表達量 33 4.2.6 RITA合併CBZ處理MV4-11-XR細胞24小時後對p53及其下游分子的調控情形 34 4.2.7 RITA合併CBZ處理MV4-11-XR細胞72小時後對細胞凋亡的影響 34 4.2.8 RITA合併CBZ處理MV4-11-XR細胞24小時後對細胞群聚形成的影響 34 4.2.9 RITA合併CBZ處理MV4-11-XR細胞後對細胞生長曲線的影響 35 4.2.10 RITA合併CBZ處理MV4-11-XR細胞24小時後，誘導細胞走向G2/M細胞週期停滯 35 4.2.11 探討RITA合併CBZ處理MV4-11-XR細胞24小時後對DNA 損傷及DNA修復機制相關蛋白質的影響。 35 4.3 對CBZ具抗藥性的細胞Molm13-XR 36 4.3.1 Molm13-XR p53路徑功能下調原因分析 36 4.3.2 RG7388合併CBZ對Molm13-XR細胞有協同作用 36 4.3.3 RG7388合併CBZ處理Molm13-XR細胞後可顯著增加p53 訊息路徑中的下游相關基因表達量 37 4.3.4 RG7388合併CBZ處理Molm13-XR細胞24小時後對p53及其下游分子的調控情形 37 4.3.5 RG7388合併CBZ處理Molm13-XR細胞72小時後對細胞凋亡的影響 38 4.3.6 RG7388合併CBZ處理Molm13-XR細胞24小時後對細胞群聚形成的影響 38 4.3.7 RG7388合併CBZ處理Molm13-XR細胞後對細胞生長曲線的影響 38 4.3.8 RG7388合併CBZ處理Molm13-XR細胞24小時後，誘導細胞走向G0/G1細胞週期停滯。 39 4.3.7 探討RG7388合併CBZ處理Molm13-XR細胞24小時後對DNA 損傷及DNA修復機制相關蛋白質的影響。 39 4.4 斑馬魚in vivo實驗 39 4.4.1斑馬魚藥物毒性測試 39 4.4.2 斑馬魚in vivo實驗 40 第五章 討論 41 第六章 參考文獻 45 圖 51 表 80 附圖 81 附表 87 | |
| dc.language.iso | zh-TW | |
| dc.subject | p53 | zh_TW |
| dc.subject | 急性骨髓性白血病 | zh_TW |
| dc.subject | FLT3-ITD | zh_TW |
| dc.subject | Cabozantinib抗藥性 | zh_TW |
| dc.subject | p53 | en |
| dc.subject | FLT3-ITD | en |
| dc.subject | Cabozantinib resistance | en |
| dc.subject | Acute myeloid leukemia | en |
| dc.title | 以小分子藥物調節p53功能以克服急性骨髓性白血病對Cabozantinib的抗藥性 | zh_TW |
| dc.title | Modulation of p53 function with small molecules to overcome Cabozantinib-resistance in AML | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 胡忠怡(Chung-Yi Hu),歐大諒(Da-Liang Ou),顧雅真(Ya-Chen Ko),侯信安(Hsin-An Hou) | |
| dc.subject.keyword | 急性骨髓性白血病,FLT3-ITD,Cabozantinib抗藥性,p53, | zh_TW |
| dc.subject.keyword | Acute myeloid leukemia,FLT3-ITD,Cabozantinib resistance,p53, | en |
| dc.relation.page | 89 | |
| dc.identifier.doi | 10.6342/NTU201902966 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-108-R06424020-1.pdf 未授權公開取用 | 3.9 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。