Multiplexed Quantification of DNA Damage Response

描述（由申请人提供）： 香烟烟雾，阳光和诊断计算机断层扫描（CT）是许多可能导致DNA损伤的环境暴露中的一部分。相关的致癌物包括烟草特有的亚硝胺、紫外线和电离辐射。每一次接触和DNA破坏剂都会增加癌症的发病率，并引发一个问题：“为什么有些人在接触后会患上癌症，而另一些人则不会？”“分子水平上的个体间差异被认为是暴露诱导疾病的主要决定因素，低于平均水平的DNA修复能力与癌症发病率增加有关1 -4。细胞DNA修复能力的测量可能是指导生活方式或临床程序决策的有用工具，但测量DNA修复能力的测定在技术上具有挑战性，通量低且昂贵。DNA修复能力最终由DNA损伤反应（DDR）决定，DDR是与损伤诱导的信号转导、细胞周期和DNA修复途径相关的百余种蛋白质网络2，5-6。该系统中所有组分的完整性、蛋白质水平和蛋白质修饰状态最终决定了细胞DNA修复能力。因此，定义和量化细胞DNA修复能力是一项复杂的任务，需要与DDR相关的数百种蛋白质的水平、修饰状态和完整性的详细信息。我们建议进一步开发一种多重蛋白质定量（MPQ）分析，以测量60个DDR蛋白质靶点的水平和翻译后修饰状态。为了实施我们的测定，我们将使用可从MesoScale Discoveries（MSD）获得的现有高通量平台。该技术基于抗体捕获和特定蛋白质的电化学发光检测。MSD技术的独特功能包括宽动态范围（6个对数）、测量毫微微克数量的能力、扩展到1，564个靶点的能力、每个板5分钟的读数以及可轻松转移到诊断实验室的ELISA类技术。我们将验证用于MPQ测定的60种DDR靶点特异性抗体，靶点特异性针对响应电离辐射损伤的DNA修复途径。将使用DDR受损细胞进一步验证该MPQ测定。我们还将使用DDR特异性MPQ测定结合表型终点对8种相似但遗传异质的细胞系的DNA修复能力进行评分。将计算分析所得数据以鉴定DNA修复能力的基于蛋白质的生物标志物特征。作为临床可行性和测定灵敏度的证明，我们还将对我们的测定进行临床试验，以分析CT诱导的DDR蛋白水平变化，并记录对辐射的体内反应。最终，我们将通过我们提出的研究来验证我们的假设，即MSD技术平台可以适用于测量实验室和临床系统中的DNA修复能力。 公共卫生相关性： DNA损伤反应蛋白质的多重定量细胞DNA损伤反应中的缺陷与环境诱导的癌症的发生有关。因此，定量细胞DNA修复能力的诊断测定将为临床和人群研究提供重要工具，因为测量DNA修复能力的稳健测定可用于鉴定易感人群。我们建议进一步开发一种多路复用的蛋白质定量工具，该工具将测量许多DNA修复蛋白的水平和修饰状态，我们将测试这种工具作为细胞DNA修复能力的衡量标准。使用来自患者的白细胞样本之前和之后的计算机断层扫描，我们也将证明我们的分析的临床可行性，并确定在体内的DNA修复途径激活这种受控暴露于电离辐射。该项目的成功完成将有助于为人口和临床研究提供诊断工具，并为癌症生物学和化疗开发提供资源。

DESCRIPTION (provided by applicant): Project Summary/Abstract Cigarette smoke, sunlight, and diagnostic computerized tomography (CT) are some of the many environmental exposures that can cause DNA damage. Associated carcinogens include tobacco specific nitrosoamines, UV- and ionizing-radiation. Each exposure and DNA damaging agent can increase cancer incidence and fuel the question "Why do some people get cancer after exposure while others do not?" Interindividual variability at the molecular level is believed to be a major determinant of exposure induced disease, with lower-than-average DNA repair capacity associated with increased cancer onset1-4. Measures of cellular DNA repair capacity could be useful tools to guide lifestyle or clinical procedure decisions, but assays that measure DNA repair capacity are technically challenging, low throughput, and expensive. DNA repair capacity is ultimately dictated by the DNA damage response (DDR), a hundred-plus protein network associated with damage-induced signal transduction, cell cycle, and DNA repair pathways2,5-6. The integrity, protein levels, and protein modification status of all components in this system ultimately dictate cellular DNA repair capacity. Defining and quantifying cellular DNA repair capacity is thus a complex task that requires detailed information on the levels, modification status, and integrity of hundreds of proteins associated with the DDR. We propose to further develop a multiplexed protein quantification (MPQ) assay to measure the levels and post translational modification status of 60 DDR protein targets en masse. In order to implement our assay we will use an existing high throughput platform available from MesoScale Discoveries (MSD). This technology is based on antibody capture and electrochemiluminescence detection of a specific protein. Unique features of the MSD technology include a wide dynamic range (six logs), the ability to measure femtogram quantities, the ability to expand to 1,564 targets, 5-minute readout for each plate, and ELISA-like technology that is easily transferable to diagnostic labs. We will validate antibodies specific to 60 DDR targets for use in a MPQ assay, with targets specific to DNA repair pathways that respond to ionizing radiation damage. This MPQ assay will be further validated using DDR compromised cells. We will also use the DDR specific MPQ assay in conjunction with phenotypic endpoints to score the DNA repair capacity of 8 similar yet genetically heterogeneous cell lines. The resulting data will be computationally analyzed to identify protein- based biomarker signatures of DNA repair capacity. As proof of clinical feasibility and assay sensitivity we will also perform a clinical test of our assay to analyze CT induced changes in DDR protein levels and to document the in vivo response to radiation. Ultimately we will us proposed studies to test our hypothesis that the MSD technology platform can be adapted to measure DNA repair capacity in laboratory and clinical systems. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Multiplexed Quantification of DNA Damage Response Proteins Deficiencies in the cellular DNA damage response have been implicated in the onset of environmentally induced cancers. Diagnostic assays to quantitate cellular DNA repair capacity would thus provide an important tool for clinical and population studies, as a robust assay to measure DNA repair capacity could be used to identify susceptible populations. We propose to further develop a multiplexed protein quantification tool that will measure the levels and modification status of many DNA repair proteins, and we will test this tool as a measure for cellular DNA repair capacity. Using leukocyte samples derived from patients before and after computerized tomography we will also demonstrate the clinical feasibility of our assay and identify the in vivo DNA repair pathways activated by this controlled exposure to ionizing radiation. Successful completion of this project would help provide a diagnostic tool for population and clinical studies, and provide a resource for cancer biology and chemotherapeutic development.