High-content screening for modifiers of the DNA damage response

描述（由申请人提供）：项目总结：对DNA损伤的协调和综合反应对于细胞在遗传毒性应激后存活和增殖至关重要。细胞包含一个复杂的分子机制，在各种各样的外在或内在损伤（如暴露于紫外线和电离辐射、环境毒素、正常代谢产物以及基因转录和DNA复制期间形成的不稳定DNA结构）后，停止细胞周期并修复受损的DNA。虽然DNA损伤在正常组织中具有明显的诱变和肿瘤诱导潜力，但由于其细胞毒性作用，它矛盾地形成了现代癌症治疗的支柱。通过DNA损伤治疗如放射治疗的选择性肿瘤杀伤依赖于正常组织和肿瘤组织之间对遗传毒性应激的不同反应。不幸的是，大量的肿瘤类型对放射治疗有抵抗力。鉴定能够调节细胞对电离辐射的反应的小分子将在破译DNA损伤反应的分子细节方面具有巨大的效用，为探索环境诱导的遗传损伤的基本细胞生物学提供化学工具，并鉴定用于开发临床有用的辐射增敏剂或辐射防护剂的先导化合物。在该提案中，我们改进、调整和优化了一种基于自动显微镜的测定，以同时询问人类癌细胞中DNA损伤反应的多个组分，用于NIH分子库生产中心网络作为NIH分子库和成像路线图计划的一部分对化合物进行高通量筛选。该检测使用新型数字图像分析技术来创建单个细胞的多个定量测量的独特数据集，从而优化后续高通量小分子筛选的成功机会。叙述在这项提案中，我们完善，调整和优化一个自动化的基于显微镜的测定，同时询问人类癌细胞中DNA损伤反应的多个组分，用于NIH分子库生产中心网络作为NIH分子库和成像路线图计划的一部分的化合物的高通量筛选。这一项目的成果应有助于开发化学工具，以研究细胞对环境辐射和破坏基因组的毒素的反应，并有助于确定可用作辐射防护剂和辐射增敏剂的化合物，以加强目前各种形式的癌症治疗。

DESCRIPTION (provided by applicant): Project Summary: A coordinated and integrated response to DNA damage is critical for cells to survive and proliferate after genotoxic stress. Cells contain an elaborate molecular machinery that halts the cell cycle and repairs damaged DNA after a wide variety of extrinsic or intrinsic insults such as exposure to ultraviolet and ionizing radiation, environmental toxins, products of normal metabolism, and unstable DNA structures formed during gene transcription and DNA replication. Although DNA damage has clear potential for mutagenesis and tumor induction in normal tissues, it paradoxically forms a mainstay of modern cancer treatment due to its cytotoxic effect. Selective tumor killing by DNA damaging treatments such as radiation therapy relies on a differential response to genotoxic stress between normal and neoplastic tissue. Unfortunately, a significant number of tumor types are resistant to radiation treatment. The identification of small molecules capable of modulating the response of cells to ionizing radiation would be of tremendous utility in deciphering the molecular details of the DNA damage response, providing chemical tools for exploring the basic cell biology of environmentally-induced genetic damage, and identifying lead compounds for the development of clinically useful radiation sensitizers or radioprotective agents. In this proposal we refine, adapt and optimize an automated microscopy-based assay to simultaneously interrogate multiple components of the DNA damage response in human cancer cells for use in high- throughput screening of compounds by NIH Molecular Libraries Production Centers Network as part of the NIH Molecular Libraries and Imaging Roadmap Initiative. This assay uses novel digital image analysis techniques to create a unique dataset of multiple quantitative measurements on individual cells that optimizes the chances of success in subsequent high-throughput small molecule screening. Narrative In this proposal we refine, adapt and optimize an automated microscopy-based assay to simultaneously interrogate multiple components of the DNA damage response in human cancer cells for use in high-throughput screening of compounds by NIH Molecular Libraries Production Centers Network as part of the NIH Molecular Libraries and Imaging Roadmap Initiative. The results of this project should facilitate the development of chemical tools for studying the response of cells to environmental radiation and toxins that damage the genome, and help identify compounds that could be used as radio- protective and radio-sensitizing agents to enhance current forms of cancer treatment.