臺灣博碩士論文加值系統

BCR-ABL融合蛋白質會造成持續性的色胺酸酵素活化，而被認為是造成慢性骨髓性白血病與費城染色體陽性之急性淋巴型白血病的一個主要致病原因。異常的BCR-ABL色胺酸激酶磷酸化會誘發細胞轉形與血球癌化。特定蛋白質抑制劑可以抑制此融合蛋白質所造成色胺酸活化，此類藥劑被認為在治療慢性白血病上是一種極為有效的治療方式。在許多色胺酸酵素抑制劑中，Imatinib是一種在治療慢性期的慢性白血病效果非常好的藥物。然而，在許多較嚴重與侵犯性病人最後都疾病復發並對Imatinib藥物產生抗藥性。此Imatinib抗藥性產生可能是由於ABL蛋白質基因突變而造成BCR-ABL融合蛋白質在訊息傳遞中再次被活化有關。為了要了解其中ABL基因突變與Imatinib抗藥性的關係，我們調查了19個用Imatinib治療後復發的病人檢體（18個慢性白血病人，與1個費城染色體陽性的急性淋巴白血病人）。我們利用聚合酵素連鎖反應產物與限制酵素作用分析，dHPLC方法以及DNA定序來分析ABL基因中的外顯子5-9區域。針對基因突變偵測，19個病人中有5個病人基因的第5到7外顯子出現基因突變情形。其中費城染色體陽性之急性淋巴型白血病病人身上同時存在有GAG→AAG (E255K)與ACT→ATT (T315I)突變。E255K位於ABL的第5外顯子區域，此基因突變是由於序列G到A 的改變，而T315I 位於ABL第7外顯子區域中有C到T改變。我們同時發現其他ABL基因突變包括有CTC→CCC (L213P)，TAC→CAC (Y253H)，ATG→ACG (M351T)，與一個3個核酸AAG的插入突變。在此研究中，我們發展以dHPLC偵測基因突變，與應用定量RT-PCR監測BCR-ABL基因轉錄產物。在白血病之Imatinib治療監控中，我們認為應該要同時進行包括殘餘腫瘤細胞數目監控與基因變異偵測，才能提供病人更好之治療方式。此外，基因突變之檢測，也能讓我們更瞭解蛋白質活化結構與調控，能幫助我們發展出對Imatinib藥物治療無效的新治療策略。

The BCR-ABL fusion protein is a constitutively activated tyrosine kinase that plays a central role in the pathogenesis of chronic myeloid leukemia (CML) and Philadelphia (Ph) chromosome-positive acute lymphoid leukemia (ALL). The aberrant tyrosine kinase activity of BCR-ABL induces cellular transformation and leukemogenesis. Targeting the tyrosine kinase activity of BCR-ABL is a very promising therapeutic strategy in treating these disorders. Imatinib is a tyrosine kinase inhibitor that has been shown a very effective therapy in patients with chronic phase CML. Although most patients with CML in the blast-crisis phase are initially responsive to Imatinib, majority of the patients relapse and develop resistance to this inhibitor. Studies have shown that many relapsed patients carry mutations in the ABL gene that increase the level of autophosphorylation of BCR-ABL kinase and promote the downstream signal transduction pathway.
In order to understand the molecular mechanisms by which ABL gene mutations induce Imatinib resistance, we investigated 19 patients (18 CML patients and 1 Ph (+) ALL patient) who either relapsed or showed no response after Imatinib treatment. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, denaturing high performance liquid chromatography (dHPLC), and direct DNA sequencing to analyze all possible mutations in exons 5 to 9 of the ABL gene. We also used quantitative PCR to monitor minimal residual disease of those patients. Various mutations were found in exons 5, 6, and 7 of the ABL gene in 5 of 19 patients with CML. The patient with Ph (+) ALL had a GAG→AAG (E255K) mutation in exon 5 and a ACT→ATT (T315I) mutation in exon 7, a finding consistent with the literatures reported previously. The E255K substitution had a G to A change, and the T315I substitution had a C to T change in the ABL gene. Other unique mutations found in this study include TAC→CAC (Y253H), ATG→ACG (M351T), CTC→CCC (L213P) and AAG tri-nucleotide insertion.
In summary, both quantitative monitoring of residual tumor cells and mutation detection are useful to predict outcome during Imatinib treatment. The different mutations are likely to interfere the Imatinib binding ability, and may alter intrinsic kinase activity by changing the protein structure conformation or phosphorylation status. The alteration of BCR-ABL kinase activity will count for the different transformation potency, which conferred the drug resistant and directly contribute to the relapse and disease progression. It is important to identify the gene mutations, which can provide a new insight into kinase function and better drug treatment strategies to overcome the drug resistance.

CONTENT
致謝 I
ABSTRACT II
中文摘要 IV
CONTENT V
INTRODUCTION 1
I. The chronic myeloid leukemia 1
II. CML staging and diagnostic methods 2
III. Organization of BCR and ABL genes 7
IV. Molecular mechanisms for the BCR-ABL fusion gene in leukemia 8
V. Regulation of the c-ABL and BCR-ABL tyrosine kinase 9
VI. Rational drug design and pharmacological study of Imatinib 11
VII. Resistance mechanism to Imatinib 16
VIII. Mutation detection in the ABL gene 19
IX. Real-time quantitative PCR 21
X. Minimal residual disease (MRD) in leukemia 22
XI. Aims and strategies of study 24
MATERIALS AND METHODS 27
I. Materials 27
II. Methods 28
RESULTS 34
I. Patients’ history and summary of methods used and results obtained 34
II. Detection of point mutations in ABL gene and classification of types of BCR-ABL gene 34
III. Restriction enzyme analysis for the mutation of T315I 35
IV. Time course mutation analysis for the T315I by PCR- RFLP analysis 36
V. dHPLC analysis for exons 5 and 7 of the ABL gene 37
VI. Time course mutation analysis by dHPLC 38
VII. Mutation determination on samples purified by dHPLC 39
VIII. MRD monitoring of disease progression 40
DISCUSSION 42
REFERENCES 56
TABLES AND FIGURES 66
Table 1. Summary of primers and probes used in this study 66
Table 2. Patients' history and summary of methods used and results obtained 68
Table 3. Clinical course and mutation analysis of patients treated with Imatinib 69
Table 4. Clones generated and characterized in this study 70
Figure 1. Two different length PCR amplicons of ABL gene 71
Figure 2. Sequence alignment of the BCR-ABL fusion transcripts 72
Figure 3. PCR amplification of ABL exons 5 and 7 73
Figure 4. High resolution melting profile of BCR-ABL fusion transcripts analysis 75
Figure 5. The Dde I restriction analysis on the ABL exon 7 of TCU-ALL-1 patient 76
Figure 6. DNA sequence of the Ph(+) TCU-ALL-1 patient 77
Figure 7. Progressive appearance of the T315I (TCU-ALL-1) point mutation as demonstrated by PCR-RFLP 78
Figure 8. Melting profiles for exons 5, 7 and 8 of the ABL gene 81
Figure 9. TCU-ALL-1 exon 5 mutation detected by dHPLC analysis 83
Figure 10. dHPLC analysis for the exon 7 of the ABL gene 84
Figure 11. Time course analysis on the exon 5 of the ABL gene by dHPLC (TCU-ALL-1) 85
Figure 12. Time course analysis for the exon 7 of the ABL gene by dHPLC (TCU-ALL-1) 87
Figure 13. DNA sequence analysis on the exon 7 for the NTU-CML-12 patient 88
Figure 14. Mutation detection for the NTU-CML-12 patient 89
Figure 15. Mutation detection for the NTU-CML-13 patient 90
Figure 16. Mutation detection for the NTU-CML-14 patient 91
Figure 17. Mutation detection for the NTU-CML-7 patient 92
Figure 18. Mutation detection for the TCU-ALL-1 patient 93
Figure 19. Minimal Residual Disease Monitoring using the relative expression of BCR-ABL/GAPDH 96
Figure 20. Overall survival of patients with Imatinib treatment 97
APPENDICES 98
Appendix 1.Features and definitions of three phase CML 98
Appendix 2.The morphology of the blood smear from a CML patient 99
Appendix 3.The karyotype from a ALL patient (TCU-ALL-1) 100
Appendix 4.Types and breaking point of BCR-ABL fusion gene 101
Appendix 5.Gene structure of the BCR-ABL fusion 103
Appendix 6.Structure of Imatinib 104
Appendix 7.Mechanisms of the BCR-ABL actions and the Imatinib inhibition 105
Appendix 8.Point mutations in the ABL kinase domain 106
Appendix 9.ABL mRNA sequences and the encoded amino acid sequence 107
Appendix 10.Comparison of mutation detection methods 114
Appendix 11.Scheme of heteroduplex/homoduplex formation by dHPLC 115
Appendix 12.Comparison of methods used in the detection the minimal residual disease 116
Appendix 13.Scheme for the construction of the pICCML plasmid DNA 117
Appendix 14.The sequence of pICCML plasmid DNA 118
Appendix 15.The T315 position in the ABL protein kinase domain. 122
Appendix 16.The M351 position in the ABL protein kinase domain. 123
Appendix 17.The crystal structure of ABL protein domain complexes with STI-571 inhibitor 124
Appendix 18.Structure change of L213P substitution 125
Appendix 19.The AAG insertion in the ABL protein ATP binding site 126
Appendix 20.The Y253 position in the ABL protein kinase domain 127

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