The ATM inhibitor KU55933 sensitizes radioresistant bladder cancer cells with DAB2IP gene defect
The ATM inhibitor KU55933 sensitizes radioresistant bladder cancer cells with DAB2IP gene defect
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DOI：
10.3109/09553002.2015.1001531
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发表时间：
2015-04-01
影响因子：
2.6
通讯作者：
Kong, Zhaolu
中科院分区：
文献类型：
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作者：
Zhang, Tingting;Shen, Yijun;Kong, Zhaolu
目的：我们的初步研究结果表明，差异表达的卵巢癌-2/残疾同源2（DOC-2/DAB 2）相互作用蛋白（DAB 2 IP），一个假定的肿瘤抑制基因，在膀胱癌（BCa）与侵略性表型下调。在这项研究中，我们调查了DAB 2 IP敲低如何影响BCa细胞对电离辐射（IR）的反应，并讨论了可能的途径，以提高细胞radiosensitivity.Methods和Materials：小干扰RNA（siRNA）系统的实施，以抑制内源性DAB 2 IP在两个人BCa细胞系，T24和5637的表达。通过集落形成测定（CFA）测量细胞对单独IR或组合处理的敏感性。用Western blot法检测共济失调-毛细血管扩张突变（ATM）、DNA依赖性蛋白激酶催化亚基（DNA-PKcs）及相关DNA损伤修复（DDR）蛋白的磷酸化水平。采用免疫荧光和流式细胞术分别检测DNA双链断裂（DSB）修复和细胞周期分布。siDAB 2 IP）与对照细胞（即，siCON）表达内源水平的DAB 2 IP。siDAB 2 IP细胞中ATM表达和激活增加、S期细胞分布增加以及DSB修复动力学加快可解释其机制。2-吗啉-4-基-6-噻喃-1-基-吡喃-4-酮（KU 55933）可显著增强siDAB 2 IP细胞对IR的敏感性，这是由于抑制IR后ATM及其下游靶点的磷酸化和减慢DSB修复动力学。KU 55933在照射后抑制ATM磷酸化，可用于具有DAB 2 IP基因缺陷的BCa患者的放射治疗。
Purpose: Our preliminary results showed that differentially expressed in ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP), a putative tumor suppressor gene, is down-regulated in bladder cancer (BCa) with aggressive phenotypes. In this study, we investigated how DAB2IP knockdown influenced BCa cell response to ionizing radiation (IR) and discussed possible ways to enhance cell radiosensitivity.Methods and materials: The small interfering RNA (siRNA) system was implemented to inhibit endogenous DAB2IP expression in two human BCa cell lines, T24 and 5637. Cell sensitivity to IR alone or combined treatment was measured by a colony formation assay (CFA). Western blot was used to determine the phosphorylation levels of ataxia-telangiectasia mutated (ATM), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and related DNA damage repair (DDR) proteins. Immunofluorescence as well as a flow cytometry assay were employed to detect DNA double-strand break (DSB) repair and cell cycle distribution, respectively.Results: DAB2IP-knockdown of BCa cells (i.e., siDAB2IP) exhibit increased clonogenic survival in response to IR compared with control cells (i.e., siCON) expressing an endogenous level of DAB2IP. The mechanism in siDAB2IP cells could be explained by elevated ATM expression and activation, increased S phase cell distribution as well as faster DSB repair kinetics. 2-morpholin-4 -yl-6-thianthren-1-yl-pyran-4-one (KU55933) significantly sensitized siDAB2IP cells to IR due to inhibition of the phosphorylation of ATM and its downstream targets following IR and slower DSB repair kinetics.Conclusions: Loss of DAB2IP expression in BCa cells signifies their radioresistance. KU55933, which suppresses ATM phosphorylation upon irradiation, could be applied in the radiotherapy of BCa patients with a DAB2IP gene defect.