Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL(+) leukemic cells and induces apoptosis
Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL(+) leukemic cells and induces apoptosis
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DOI：
10.1006/bcmd.1997.0155
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发表时间：
1997-01-01
影响因子：
2.3
通讯作者：
Lydon, NB
中科院分区：
文献类型：
--
作者：
GambacortiPasserini, C;leCoutre, P;Lydon, NB
BCR/ABL融合蛋白转化髓系干细胞。慢性髓性白血病（CML）和急性淋巴细胞白血病（ALL）的一个亚群都与BCR/ABL蛋白的表达有关。这一知识尚未转化为任何特定的工具来控制ABL驱动的肿瘤细胞生长，CGP57148B是ABL蛋白激酶的atp竞争性抑制剂；在体外和体内均显示其抑制ABL的激酶活性，抑制v-abl和bcr/ ABL转染物的生长，以及在一些CML患者中存在生长因子的情况下骨髓（BM）衍生菌落的体外形成。本研究旨在探讨CGP57148B对新鲜细胞和培养细胞、白血病细胞和正常细胞、BCR/ABL阳性细胞和阴性细胞的自发增殖活性，并探讨其作用机制。6个来源于BCR/ABL+白血病的细胞系（K562、BV173、KCL22、KU812、MC3、LAMA84）， 13个BCR/ABL阴性细胞系，包括肿瘤细胞系（KG1、SU-DHL-1、U937、Daudi、NB4、NB4.306）和来源于正常细胞（PHA母细胞、LAK、成纤维细胞、LCL、肾上皮细胞、内皮细胞、CD34（+）细胞），以及14个新鲜白血病样本，使用氚化胸腺嘧啶摄取法进行了测试。western blot检测BCR/ABL蛋白在体内的磷酸化水平，annexin V/丙啶结合试验检测细胞凋亡水平。采用多抗体免疫荧光法检测诱导分化情况。所有6株BCR/ABL（+）细胞系均表现出剂量依赖性的自发增殖率抑制，且不伴有分化。治疗在几分钟内引起BCR/ABL蛋白的去磷酸化，随后16-24小时内循环细胞减少并诱导凋亡。在低于或等于3 μ M的浓度下，除成纤维细胞和CD34细胞外，在任何BCR/ABL阴性的正常或肿瘤细胞系中均未观察到明显的DNA合成抑制，当使用从两个Ph+ ALL和12个连续CML患者获得的新鲜样品时，也观察到增殖抑制。在这些样品中也观察到细胞凋亡的诱导。CGP57148B的活性可以在离体分离或培养细胞中使用简单且可重复的试验进行监测，而无需外源添加生长因子。该分子可能通过抑制BCR/ABL的激酶活性和随后的细胞凋亡来发挥其作用，而不诱导细胞分化。一些正常细胞也受到影响。
The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has nor yet been translated into any specific tool to control ABL driven neoplastic cells growth, CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic(KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies.All six BCR/ABL(+) lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis, No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations less than or equal to 3 mu M, with the exception of fibroblasts and CD34 cells, Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too.The activity of CGP57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected.