A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.
A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.
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DOI：
10.1038/srep18231
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发表时间：
2015-12-15
影响因子：
4.6
通讯作者：
Masutani M
中科院分区：
文献类型：
--
作者：
Fujimori H;Sato A;Kikuhara S;Wang J;Hirai T;Sasaki Y;Murakami Y;Okayasu R;Masutani M
利用shrna文库/功能聚类分析对HeLa细胞中的放射致敏靶点进行了全面的全基因组筛选，并确定DNMT3B为候选靶点。DNMT3B RNAi提高了HeLa、A549和HCT116细胞对γ辐照和碳离子束辐照的敏感性。DNMT3B RNAi降低了γ辐照诱导的DNA损伤反应的激活，包括HP1β-、γH2AX-和rad51 -灶的形成。DNMT3B RNAi破坏了损伤依赖性的H2AX积累，并显示γ辐照后γH2AX诱导水平降低。DNMT3B在未辐照条件下与HP1β相互作用，而辐照消除了DNMT3B/HP1β复合物，但诱导了DNMT3B与H2AX的相互作用。与放射致敏一致，γ-辐照后DNMT3B敲低细胞中TP63、BAX、PUMA和NOXA的表达均受到诱导。这些结果表明，DNMT3B RNAi通过损伤依赖性HP1β灶的形成和有效的γ - H2AX诱导机制（包括H2AX积累）的损害诱导了放射性致敏。在异种肿瘤移植模型中也观察到DNMT3B RNAi增强的放射敏感性。综上所述，目前的研究表明，伴随聚类分析的全面筛查能够确定放射致敏目标。DNMT3B的下调是使用该方法确定的靶标之一，通过干扰多种DNA损伤反应使癌细胞放射致敏。
A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses.