Probes for Cytometry

描述（由申请人提供）：项目摘要/摘要本项目的广泛的长期目标是继续开发新的生物探针和方法，通过流式细胞术和图像细胞术分析单个细胞。这些分析将在DNA损伤、DNA修复、致癌/诱变、细胞凋亡的研究中以及在鉴定抗肿瘤药物的作用机制中找到实用性。第一种方法分析DNA结合的荧光配体的荧光的最大像素强度的变化，染色质松弛的报告者，细胞的DNA损伤反应（DDR）的初始事件。待开发的其他探针利用磷酸化特异性抗体和多参数细胞术来测量关键DDR的磷酸化/活化：组蛋白H2 AX、ATM、Chk 2和p53。将评估探针测量响应于不同类型的DNA损伤的DDR的能力，所述DNA损伤包括由药物、诱变剂和辐射引起的DNA损伤，并且与细胞周期阶段和细胞凋亡诱导相关。将探索图像细胞术定量单个核病灶频率的能力，每个核病灶代表DDR期间发生的DNA双链断裂（DSB）。我们还将开发一种综合的、多路复用的方法，该方法将DDR（3 H2 AX或ATM、Chk 2和p53磷酸化的诱导）的检测与通过单细胞DNA电泳（彗星试验）评估的实际DNA损伤的测量相结合。激光扫描细胞仪的“图像合并”能力将用于整合DDR分析与相同单个细胞中的DNA损伤测定。另一种待开发的多重测定将结合联合收割机的DDR与DNA修复的分析，测量为非程序性DNA合成（UDS）。将探索和开发5-溴-2 '-脱氧尿苷（BrdU）和5-乙炔基-2'-脱氧尿苷（EdU）的掺入，后者随后进行环加成反应（“点击”化学），以便可以在同一细胞中评估UDS和DDR。使用上述方法，将评估细胞计数中常用的超活体探针（Hoechst 33342、DRAQ 5、DyeCycle Violet和SYTO 17）诱导的潜在DNA损伤和DDR。将特别注意探索在这些探针用于分离干细胞作为“侧群”的条件下是否发生DNA损伤。在细胞计数分离的干细胞中检测DNA损伤，尤其是DSB，将预测分离程序的致癌/致突变潜力。评价肿瘤抑制因子p53在由不同遗传毒性剂（包括靶向DNA的抗肿瘤药物）引起的DDR中的作用是本申请的另一个项目。我们建议开发的测定法将在许多不同的领域中找到广泛的应用，包括药理学，诱变，致癌，毒理学，实验和临床肿瘤学以及细胞和分子生物学。 公共卫生相关性： 该项目的目的是开发新的方法来评估细胞遗传物质（DNA）的损伤，以及受损DNA的修复，因为它发生在暴露于辐射，环境诱变剂和癌症治疗期间。与年龄相关的渐进性DNA损伤也是衰老的主要原因，并使个体易患癌症。所提出的方法将广泛应用于筛选诱变剂、预防癌症、开发新的抗癌药物，以及在临床肿瘤学中快速检测抗癌疗法的有效性，从而允许选择最有效的癌症治疗。

DESCRIPTION (provided by applicant): Project Summary/Abstract The broad, long-term objective of this project is to continue the development of new bioprobes and methodologies for analysis of individual cells by flow and image cytometry. These assays will find utility in studies of DNA damage, DNA repair, carcinogenesis/mutagenesis, apoptosis, and in identifying the mechanism of action of antitumor drugs. The first approach analyzes changes in the maximal pixel intensity of fluorescence of DNA-bound fluorescent ligands, a reporter of chromatin relaxation, the initial event in a cell's DNA damage response (DDR). Other probes to be developed utilize phospho-specific antibodies and multiparameter cytometry to measure phosphorylation/activation of key DDRs: histone H2AX, ATM, Chk2 and p53. The probes ability to measure DDR in response to different types of DNA damage will be assessed including that caused by drugs, mutagens and radiation, and correlated with cell cycle phase and induction of apoptosis. The capability of image cytometry to quantify the frequency of individual nuclear foci each representing a DNA double-strand break (DSB) occurring during DDR will be explored. We will also develop an integrated, multiplexed methodology that combines the detection of DDR (induction of 3H2AX, or ATM, Chk2 and p53 phosphorylation) with the measurement of actual DNA damage, assessed by single-cell DNA electrophoresis (comet assay). The "image-merge" capability of the laser scanning cytometry will be used to integrate the analysis of DDR with the assay of DNA damage in the same individual cells. Another multiplexed assay to be developed will combine the analysis of DDR with DNA repair, measured as unscheduled DNA synthesis (UDS). The incorporation of 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'deoxyuridine (EdU), the latter followed by the cyclo-addition reaction ("click" chemistry) will be explored and developed so that UDS and DDR can be assessed in the same cell. Using the approaches described above, the potential DNA damage and DDR induced by supravital probes commonly used in cytometry (Hoechst 33342, DRAQ5, DyeCycle Violet and SYTO 17) will be assessed. Particular attention will be given to explore whether DNA damage occurs under conditions in which these probes are used to isolate stem cells as "side populations". The detection of DNA damage, especially DSBs, in cytometrically isolated stem cells will be predictive of the carcinogenic/mutagenic potential of the isolation procedure. Evaluation of the role of the tumor suppressor p53 in DDR caused by different genotoxic agents, including antitumor drugs targeting DNA, is another project in this application. The assays we propose to develop will find wide application in many diverse fields including pharmacology, mutagenesis, carcinogenesis, toxicology, experimental and clinical oncology as well as in cell and molecular biology. PUBLIC HEALTH RELEVANCE: Relevance The aim of this project is to develop new methods to evaluate damage to the genetic material (DNA) of cells, and the repair of damaged DNA, as it occurs upon exposure to radiation, to environmental mutagens and also during treatment of cancer. Progressive age-related DNA damage is also the main cause of aging and predisposes individuals to cancer. The proposed methods will find wide application in screening for mutagens, prevention of cancer, development of new anticancer drugs, and in clinical oncology to rapidly detect the effectiveness of anticancer therapies thereby allowing selection of the most effective cancer treatment.