High Throughput Technology for Assessing Global DSB Repair Capacity

描述（由申请人提供）： 项目摘要/摘要提出的是一个强大的高通量廉价的方法来测量个人的全球DNA修复动力学在人类的发展和现场测试。潜在的应用是与癌症治疗策略相关的流行病学研究，以及促进各种疾病的预防策略的发展-无论是在独立的流行病学研究中，还是补充基于分子的关联研究。所提出的方法建立在当前全自动高通量生物剂量测定系统RABIT（快速自动生物剂量测定工具）的基础上，该系统使用基于手指针刺的血液样品和原位多孔板平台测量淋巴细胞中3-H2 AX病灶的产量。RABIT系统目前仅检测一种DNA修复蛋白（3-H2 AX），并且每个样品仅检测一次;该系统将适于通过以下方式表征DNA修复动力学：A）添加自动化辐照器，以将DNA双链断裂引入手指针刺样品中，B）扩展基于机器人的分析系统以允许在辐照挑战之后自动测量时间函数，C）扩展系统以允许同时测量多于一种DNA修复蛋白。该系统将能够以高通量（e1，000个样本/天）表征整体DSB修复动力学，通过3-H2 AX、ATM[ser 1981]、53 BP 1和Mdc 1修复蛋白的全自动免疫组织化学测量进行分析-选择这些蛋白作为主要DSB修复途径中一系列节点的代表。我们的目标如下：1.设计并将创新的紧凑型自动毛细管辐照器集成到RABIT中，以将DNA DSB引入手指针刺血样中。因为我们仅照射短毛细管，并且在一个接一个的顺序基础上，照射器可以非常紧凑，所以可以使用低活性90 Sr/90 Y放射性种子。这些放射性粒子的使用最大限度地减少了屏蔽要求，因为它们的放射性很低，而且这些粒子会发射短程β粒子。2.调整RABIT，使其在自动辐照后多次（0.5、2、4、8、24小时）自动测定每份手指针刺样本的3-H2 AX、ATM[ser 1981]、53 BP 1和Mdc 1。3.已经优化了用于我们的全自动原位系统的3-H2 AX测定的生物学方案，对其他三种修复蛋白也将进行同样的操作。4.建立一种定量方法，用于比较不同生物标志物/不同样本的DNA修复动力学测量值，并能够识别离群值。5.通过首次测量健康人群中的整体DSB修复动力学，展示该系统的实用性和可扩展性。将从150名健康志愿者中采集手指针刺毛细血管血样本，并使用改进的RABIT系统进行自动分析。将分析其动力学的四种修复蛋白代表主要DSB修复途径中的一系列节点。 公共卫生相关性： 项目叙述提出的是一个强大的高通量廉价的方法来测量个人的全球DNA修复动力学在人类的发展和现场测试。潜在的应用是与癌症治疗策略有关的流行病学研究，也有助于制定各种疾病的预防策略。

DESCRIPTION (provided by applicant): Project Summary/Abstract Proposed is development and field testing of a robust high-throughput inexpensive approach for measuring individual global DNA repair kinetics in humans. Potential applications are for epidemiological studies relating to cancer therapy strategies, and also to facilitate development of preventive strategies for a variety of diseases - either in standalone epidemiological studies, or to complement molecularly-based association studies. The proposed approach builds on a current fully-automated high-throughput biodosimetry system, the RABIT (Rapid Automated BIodosimetry Tool), which measures yields of 3-H2AX foci in lymphocytes, using fingerstick-based blood samples and an in-situ multi-well plate platform. The RABIT system currently assays only one DNA repair protein (3-H2AX), and at only one time for each sample; the system will be adapted to characterize DNA repair kinetics by A) adding an automated irradiator, to introduce DNA double strand breaks into the fingerstick sample, B) extending the robotically-based analysis system to allow automated measurements of a function of time after the irradiation challenge, C) extending the system to allow simultaneous measurements of more than one DNA repair protein. The system will be able to characterize, with high throughput (e1,000 samples / day) global DSB repair kinetics, as assayed by fully automated immunohistochemical measurements of 3-H2AX, ATM[ser1981], 53BP1, and Mdc1 repair proteins - chosen as representative of a range of nodal points in the main DSB repair pathways. Our goals are as follows: 1. Design and incorporate into the RABIT an innovative compact automated capillary irradiator, to introduce DNA DSBs into the fingerstick blood sample. Because we irradiate only short capillary tubes, and on a one- by-one sequential basis, the irradiator can be very compact, so low-activity 90Sr/90Y radioactive seeds can be used. Use of these radioactive seeds minimizes the shielding requirements, both because of the low activity and because these seeds emit short-ranged beta particles. 2. Adapt the RABIT so that it automatically assays each fingerstick sample for 3-H2AX, ATM[ser1981], 53BP1, and Mdc1, and at multiple times (0.5, 2, 4, 8, 24 hrs) after the automated irradiation. 3. Having already optimized the biological protocol for the 3-H2AX assay for use in our fully automated in-situ system, the same will be done for the other three repair proteins. 4. Establish a quantitative approach for comparing the measured DNA repair kinetics of different biomarkers / different samples, and to be able to identify outliers. 5. Demonstrate the practicality and scalability of the system by measuring, for the first time, global DSB repair kinetics in a healthy human population. Fingerstick capillary blood samples will be collected from 150 healthy volunteers, and automatically analyzed using the modified RABIT system. The four repair proteins whose kinetics will be analyzed represent a range of nodal points in the main DSB repair pathways. PUBLIC HEALTH RELEVANCE: Project Narrative Proposed is development and field testing of a robust high-throughput inexpensive approach for measuring individual global DNA repair kinetics in humans. Potential applications are for epidemiological studies relating to cancer therapy strategies, and also to facilitate the development of preventive strategies for a variety of diseases.