Translational Control of Radiation-Induced Apoptosis

描述（申请人提供）：放射治疗是最重要的癌症治疗方法之一。与放射治疗一起使用的无毒放射增敏剂将提高放射治疗的有效性，同时减少总辐射剂量，以防止不必要的副作用。DNA双链断裂（DSBs）是电离辐射引起的主要DNA损伤，也是辐射敏感性的主要原因。众所周知，缺乏DSB修复在哺乳动物细胞中是致命的。DNA依赖性蛋白激酶（DNA- pk）是DNA非同源末端连接（NHEJ）途径的关键组成部分，是哺乳动物细胞DSB修复的主要途径。我们已经证明，抑制DNA-PK激酶活性和/或消除辐射诱导的DNA-PKcs （DNA-PK的催化亚基）的自磷酸化会导致严重的辐射敏感性。由于DNA-PKcs的自磷酸化是NHEJ介导的DSBs修复的必要步骤，抑制自磷酸化而不影响DNA-PK蛋白水平或其酶活性的分子可以选择性地使细胞放射敏感而不影响其他细胞过程；而抑制DNA-PK激酶活性的分子通常也会抑制其他PI-3K样蛋白激酶（ATM和ATR），并引起显著的细胞毒性。在本提案中，我们计划开发基于裂解物和基于细胞的高通量筛选（HTS）方法，以鉴定化学物质或天然化合物，这些化学物质或天然化合物可以阻断DNA-PKcs的自磷酸化或抑制DNA-PKcs的激酶活性。关于方法，

DESCRIPTION (provided by applicant): Radiation treatment is one of the most important cancer-therapeutic approaches. Non-toxic radiosensitizers, used in conjunction with radiation therapy, would enhance the effectiveness of the radiotherapy and, at the same time, reduce the total radiation dose to prevent unwanted side effects. DNA double-strand breaks (DSBs) are the major DNA lesion induced by ionizing radiation (IR) and the dominant cause of radiation sensitivity. It is well established that lack of DSB repair can be lethal in mammalian cells. DNA-dependent protein kinase (DNA-PK) is the key component of the DNA nonhomologous end-joining (NHEJ) pathway that is the predominant pathway for DSB repair in mammalian cells. We have shown that inhibition of DNA-PK kinase activity and/or abrogation of radiation-induced autophosphorylation of DNA-PKcs (the catalytic subunit of DNA-PK) results in severe radiation sensitivity. As the autophosphorylation of DNA-PKcs is a requisite step for NHEJ mediated DSBs repair, molecules that inhibit autophosphorylation without affecting the protein levels of DNA-PK or its enzymatic activity could selectively radiosensitize cells without affecting other cellular processes; whereas molecules that inhibit the kinase activity of DNA-PK also commonly inhibit other PI-3K like protein kinases (ATM and ATR) and cause significant cellular toxicity. In this proposal, we plan to develop both lysate-based and cell-based high throughput screening (HTS) assay to identify chemicals or natural compounds that would either block the autophosphorylation of DNA-PKcs or inhibit the kinase activity of DNA-PKcs. With regard to the approaches,