Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells
Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells
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DOI：
10.1016/j.exphem.2008.10.013
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发表时间：
2009-02-01
影响因子：
2.6
通讯作者：
Holyoake, Tessa L.
中科院分区：
文献类型：
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作者：
Myssina, Svetlana;Helgason, G. Vignir;Holyoake, Tessa L.
Objective.本研究调查了两种方法，短发夹RNA（shRNA）和有效的ABL抑制剂，达沙替尼，单独和一起，以实现费城阳性（Ph+）细胞中BCR-ABL活性的完全抑制。通过慢病毒转导或电穿孔将BCR-ABL b3 a2特异性shRNA递送至K562细胞，使用或不使用达沙替尼。通过定量逆转录聚合酶链反应、流式细胞术和蛋白质印迹法测定mRNA和蛋白质敲低。通过细胞内流式细胞术评估pCrkL的BCR-ABL活性。采用台盼蓝拒染法、Annexin-V和活性caspase-3染色检测细胞死亡和凋亡。转染或电穿孔shRNA后48小时，BCR-ABL mRNA和蛋白质分别减少75%和> 90%，并持续5天。慢病毒递送）和电穿孔同样有效。pCrkL的抑制与细胞死亡有关。到转导或电穿孔后5天，活细胞占输入的50%，与扩增6倍的对照相比减少12倍。当shRNA（通过绿色荧光蛋白滴定为低和高）与达沙替尼（浓度范围，0 - 10 nM）组合时，低shRNA与低达沙替尼（0.6和1 nM）相加，导致pCrkL的抑制、激活的caspase-3的诱导、Annexin-V的表达以及活细胞的显著减少。这些结果证实，通过用shRNA降低BCR-ABL水平，可以用较低浓度的达沙替尼实现癌蛋白活性的完全抑制，从而为在高靶点表达的情况下结合这些方法提供了理论基础，例如在晚期疾病和干细胞室中发现的。(C)2009 ISEH -血液学和斯坦细胞学会。爱思唯尔公司出版
Objective. This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph+) cells.Materials and Methods. shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, How cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining.Results. Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral deliver), and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0 - 10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells.Conclusion. These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment. (C) 2009 ISEH - Society for Hematology and Stein Cells. Published by Elsevier Inc.