A novel Chk inhibitor, XL-844, increases human cancer cell radiosensitivity through promotion of mitotic catastrophe
A novel Chk inhibitor, XL-844, increases human cancer cell radiosensitivity through promotion of mitotic catastrophe
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DOI：
10.1007/s10637-009-9361-2
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发表时间：
2011-06
影响因子：
3.4
通讯作者：
O. Riesterer;F. Matsumoto;Li Wang;J. Pickett;D. Molkentine;U. Giri;L. Milas;U. Raju
中科院分区：
文献类型：
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作者：
O. Riesterer;F. Matsumoto;Li Wang;J. Pickett;D. Molkentine;U. Giri;L. Milas;U. Raju
检查点激酶 (Chk) 在促进辐射暴露后 DNA 修复方面发挥着重要作用。我们测试了 Chk1 和 Chk2 的新型抑制剂 XL-844（由美国加利福尼亚州 Exelixis Inc. 提供）对培养中生长的人类癌细胞放射增敏的效力，并研究了其潜在机制。将 HT-29 细胞（人类结肠癌细胞系）暴露于 XL-844、辐射或两者，并评估克隆细胞存活率。通过蛋白质印迹评估对 Chk 蛋白磷酸化形式的治疗依赖性影响。对 HT-29 细胞的进一步机制研究包括通过流式细胞术进行细胞周期分析，以及通过免疫细胞化学 (ICC) 评估 DNA 修复动力学，以评估磷酸化形式的组蛋白 2AX 蛋白 (γ-H2AX) 染色的核外观。通过 ICC 染色的有丝分裂纺锤体标记 α 和 γ-微管蛋白的不规则模式来识别经历有丝分裂灾难的细胞。 XL-844 以剂量和方案依赖性方式增强放射敏感性，并且在 0.5 生存分数时增强因子为 1.42。从机制上讲，XL-844 消除了辐射诱导的 Chk2 磷酸化，诱导了全核 γ-H2AX，并延长了辐射诱导的 γ-H2AX 病灶的存在时间，并促进了有丝分裂灾难。总之，我们的数据表明，XL-844 对 Chk2 活性的抑制增强了癌细胞的放射敏感性，这与抑制 DNA 修复和诱导有丝分裂灾难有关。
Check point kinases (Chk) play a major role in facilitating DNA repair upon radiation exposure. We tested the potency of a novel inhibitor of Chk1 and Chk2, XL-844 (provided by Exelixis Inc., CA, USA), to radiosensitize human cancer cells grown in culture and investigated the underlying mechanisms. HT-29 cells (a human colon cancer line) were exposed to XL-844, radiation, or both, and assessed for clonogenic cell survival. Treatment-dependent effects on phosphorylated forms of Chk proteins were assessed by Western blots. Further mechanistic investigations in HT-29 cells included cell cycle analysis by flowcytometry and assessment of DNA repair kinetics by immuno-cytochemistry (ICC) for nuclear appearance of the phosphorylated form of histone 2AX protein (γ-H2AX) staining. Cells undergoing mitotic catastrophe were identified by irregular pattern of mitotic spindle markers α and γ-tubulin staining by ICC. XL-844 enhanced radiosensitivity in a dose and schedule-dependent manner and the enhancement factor was 1.42 at 0.5 survival fraction. Mechanistically XL-844 abrogated radiation-induced Chk2 phosphorylation, induced pan-nuclear γ-H2AX, and prolonged the presence of radiation-induced γ-H2AX foci, and promoted mitotic catastrophe. In conclusion, our data showed that inhibition of Chk2 activity by XL-844 enhanced cancer cell radiosensitivity that was associated with inhibition of DNA repair and induction of mitotic catastrophe.