Imaging of DNA damage repair using 53BP1 protein and establishment of therapeutic strategy for cancer treatment.

肿瘤抑制因子P53结合蛋白1(53BP1)被迅速招募到DNA双链断裂(DSB)部位，参与细胞对DNA损伤的反应。我们利用53BP1在DSB位点上聚集的特性，利用53BP1-GFP融合蛋白在细胞中稳定表达的特殊形式，研制了检测DNA损伤的生物传感器。此外，我们还建立了一种通过细胞核内定位模式来区分细胞周期阶段差异的体系。表达这两种荧光融合蛋白的U2OS细胞株(U2RDP-LE53-21)可以观察到发生在不同细胞周期的双链断裂，这些细胞仍然存活。在这里，我们展示了如何在正常的培养条件下观察到53BP1焦点，以及如何由化学试剂诱导DSB 53BP1焦点，如新癌抑素(NCS)或DNA拓扑异构酶I抑制剂喜树碱(CPT)，这些都是众所周知的DSB诱导剂。与正常培养条件下的细胞相比，这些化学试剂增加了53BP1病灶的数量，延长了细胞的周期分布。U2OS细胞株可用于筛选诱导癌细胞产生双链断裂的化疗药物和放射增敏剂。

Tumor suppressor p53 Binding Protein 1(53BP1) is recruited rapidly to sites of DNA double-strand breaks (DSBs) and participates in the cellular response to DNA damage. We exploited property of 53BP1 that accumulate in DSB sites and developed biosensor detecting DNA damage using the specical form of 53BP1-GFP fusion protein that could express stably in the cell. Futhermore, we built a system that can identify the difference of cell cycle stage by intranuclear localization patterns of PCNA-DsRed. U2OS cell lines (U2RDP-LE53-21) expressing both these fluorescent fusion proteins make it possible to observe DSBs occurred in different cell cycle stage, these cells remain living. Here, we show how 53BP1 foci are observed in normal culturing conditions, and also how DSBs 53BP1 foci are induced by chemical agents such as Neocarcinostatine (NCS) or DNA Topoisomerase I inhibitor Camptothesin (CPT) that are well-known DSBs inducers. The number of 53BP1 foci was increased by these chemical agents and cell cycle distribution was prolonged compare to cells in normal culture condition. U2OS cell lines may be available for the screening chemotherapy drugs and radiosensitizers that induce DSBs in cancer cells.