Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells: the role of ATM-Chk2-Cdc25A and ATM-p53-p21-pathways
Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells: the role of ATM-Chk2-Cdc25A and ATM-p53-p21-pathways
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DOI：
10.1007/s00432-007-0278-x
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发表时间：
2008-02-01
影响因子：
3.6
通讯作者：
Schmidmaier, Ralf
中科院分区：
文献类型：
--
作者：
Gaul, Leander;Mandl-Weber, Sonja;Schmidmaier, Ralf
目的多发性骨髓瘤是由浆细胞恶性转化引起的一种致死性血液病。苯达莫司汀已被证明是一个有效的替代美法仑在第3阶段的研究，但其分子模式的行动仍然知之甚少。方法四个骨髓瘤细胞株NCI-H929，OPM-2，RPMI-8226，和U266体外培养。Annexin V FITC和碘化丙啶染色后，用流式细胞仪检测细胞凋亡。碘化丙啶DNA染色法测定细胞周期分布。胞内水平的（磷酸化）蛋白质的测定通过western blot.Results我们表明，苯达莫司汀诱导细胞凋亡的IC 50为35-65 μ g/ml，并与半胱天冬酶3裂解。用10-30 μ g/ml孵育导致所有四细胞系的G2细胞周期停滞。主要的DNA损伤信号激酶ATM和Chk 2，而不是ATR和Chk 1，被激活。Chk 2底物Cdc 25 A磷酸酶被降解，Cdc 2被Tyr 15的抑制性磷酸化抑制，伴随着细胞周期蛋白B水平的增加。此外，p53的活化发生在丝氨酸15的磷酸化，丝氨酸15是ATM的磷酸化位点。p53通过上调p21促进Cdc 2抑制。通过选择性抑制剂SB 202190靶向p38 MAPK显著增加苯达莫司汀诱导的细胞凋亡。结论苯达莫司汀诱导ATM-Chk 2-Cdc 2介导的G2期阻滞和p53介导的细胞凋亡。抑制p38 MAPK可增强细胞凋亡并消除G2期阻滞，可被认为是与苯达莫司汀联合的一种新的治疗策略。
Purpose Multiple myeloma is a fatal hematological disease caused by malignant transformation of plasma cells. Bendamustine has been proven to be a potent alternative to melphalan in phase 3 studies, yet its molecular mode of action is still poorly understood.Methods The four-myeloma cell lines NCI-H929, OPM-2, RPMI-8226, and U266 were cultured in vitro. Apoptosis was measured by flow cytometry after annexin V FITC and propidium iodide staining. Cell cycle distribution of cells was determined by DNA staining with propidium iodide. Intracellular levels of (phosphorylated) proteins were determined by western blot.Results We show that bendamustine induces apoptosis with an IC50 of 35-65 mu g/ml and with cleavage of caspase 3. Incubation with 10-30 mu g/ml results in G2 cell cycle arrest in all four-cell lines. The primary DNA-damage signaling kinases ATM and Chk2, but not ATR and Chk1, are activated. The Chk2 substrate Cdc25A phosphatase is degraded and Cdc2 is inhibited by inhibitory phosphorylation of Tyr15 accompanied by increased cyclin B levels. Additionally, p53 activation occurs as phosphorylation of Ser15, the phosphorylation site for ATM. p53 promotes Cdc2 inhibition by upregulation of p21. Targeting of p38 MAPK by the selective inhibitor SB202190 significantly increases bendamustine induced apoptosis. Additionally, SB202190 completely abrogates G2 cell cycle arrest.Conclusion Bendamustine induces ATM-Chk2-Cdc2-mediated G2 arrest and p53 mediated apoptosis. Inhibition of p38 MAPK augments apoptosis and abrogates G2 arrest and can be considered as a new therapeutic strategy in combination with bendamustine.