Scappini Barbara, Gatto Simona, Onida Francesco, Ricci Clara, Divoky Vladimir, Wierda William G, Andreeff Michael, Dong Li, Hayes Kimberly, Verstovsek Srdan, Kantarjian Hagop M, Beran Miloslav
Department of Leukemia, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA.
Cancer. 2004 Apr 1;100(7):1459-71. doi: 10.1002/cncr.20131.
Although various mechanisms have been recognized as being associated with the development of resistance to imatinib mesylate in vitro and in clinical situations, their relative significance and contributions remain poorly understood, as is the sequence of events leading to the selection of the resistant phenotype. Experimental in vitro systems involving well defined cell lines and conditions can be used to some advantage to answer specific questions and to develop in vitro models of imatinib resistance that would reflect its potential heterogeneity.
Two cell lines, KBM5 and KBM7, which expressed p210 Bcr/Abl and which differed in their inherent sensitivity to imatinib, the number of copies of the BCR/ABL fusion gene, and the activation of apoptotic pathways, were grown in vitro in the presence of increasing concentrations of imatinib. The resistant cells were analyzed for cell cycle progression, apoptotic response after exposure to imatinib, expression of Bcr/Abl, tyrosine kinase activity, and the presence of mutations within the adenosine triphosphate (ATP) coding domain of BCR/ABL. At various levels of resistance, the cells were transferred into drug-free media, and the stability of the resistant phenotype was determined in the absence of the drug.
In KBM7 cells, the development of resistance was characterized by loss of apoptotic response to the drug, amplification of BCR/ABL, increased levels of expression of p210 Bcr/Abl, and decreased inhibition of Bcr/Abl tyrosine kinase (TK) activity by imatinib. No mutations within the ATP-binding domain of Bcr/Abl were identified, and resistance remained stable in the absence of the drug. In KBM5 cells, which previously were found to be characterized by the acquisition of a single C-T mutation at ABL nucleotide 944 (T315I) at high levels of resistance, this same mutation was detected at an intermediate level, but not at a low level, of resistance. The response of KBM5 cells to imatinib was characterized by a low level of apoptotic response, a marginal increase in BCR/ABL copy number, a modest increase in p210 expression, and a highly imatinib-resistant Bcr/Abl TK. Partial reversal of resistance was observed in highly resistant KBM5-STI571(R1.0) cells, which continued to display the C-T mutation. In KBM5 cells with an intermediate level of resistance, the T315I mutation was no longer detectable upon their reversal to the sensitive phenotype.
BCR/ABL amplification with subsequent overexpression of Bcr/Abl protein, loss of apoptotic response, or point mutation of the ATP-binding site of BCR/ABL was associated alternatively with the acquisition of the resistant phenotype, supporting the notion that multiple mechanisms are involved in the induction of resistance to imatinib. The initial number of BCR/ABL copies itself was not related directly to the degree of resistance. The reversibility of the resistance may be complete, partial, or irreversible, depending on the mechanism(s) involved and the degree of resistance. Both cell lines serve as models for further elucidation of various aspects of imatinib-resistance mechanisms.
尽管多种机制已被认为与甲磺酸伊马替尼在体外及临床环境中耐药性的产生有关，但其相对重要性和作用仍了解甚少，导致耐药表型选择的事件顺序亦是如此。涉及明确细胞系和条件的体外实验系统可用于回答特定问题，并建立能反映伊马替尼潜在异质性的耐药体外模型。
两种细胞系KBM5和KBM7，表达p210 Bcr/Abl，对伊马替尼的固有敏感性、BCR/ABL融合基因拷贝数及凋亡途径激活情况不同，在体外于伊马替尼浓度不断增加的情况下培养。对耐药细胞进行细胞周期进程、暴露于伊马替尼后的凋亡反应、Bcr/Abl表达、酪氨酸激酶活性以及BCR/ABL三磷酸腺苷（ATP）编码域内突变情况的分析。在不同耐药水平下，将细胞转移至无药培养基中，在无药情况下确定耐药表型的稳定性。
在KBM7细胞中，耐药性的产生表现为对药物的凋亡反应丧失、BCR/ABL扩增、p210 Bcr/Abl表达水平增加以及伊马替尼对Bcr/Abl酪氨酸激酶（TK）活性的抑制作用降低。未在Bcr/Abl的ATP结合域内鉴定到突变，且在无药情况下耐药性保持稳定。在KBM5细胞中，先前发现其在高耐药水平时以ABL核苷酸944处单个C-T突变（T315I）的获得为特征，在中等耐药水平时检测到相同突变，但在低耐药水平时未检测到。KBM5细胞对伊马替尼的反应表现为低水平的凋亡反应、BCR/ABL拷贝数略有增加、p210表达适度增加以及高度耐伊马替尼的Bcr/Abl TK。在高度耐药的KBM5-STI571(R1.0)细胞中观察到耐药性的部分逆转，这些细胞继续显示C-T突变。在中等耐药水平的KBM5细胞恢复至敏感表型时，T315I突变不再可检测到。
BCR/ABL扩增随后Bcr/Abl蛋白过表达、凋亡反应丧失或BCR/ABL的ATP结合位点点突变与耐药表型的获得交替相关，支持了多种机制参与伊马替尼耐药诱导的观点。BCR/ABL拷贝数的初始数量本身与耐药程度无直接关系。耐药性的可逆性可能是完全的、部分的或不可逆的，这取决于所涉及的机制和耐药程度。两种细胞系均作为进一步阐明伊马替尼耐药机制各方面的模型。
