Probes for Cytometry

描述(由申请者提供)：项目摘要/摘要该项目广泛的长期目标是继续开发新的生物探针和方法，用于通过流式细胞术和图像细胞术分析单个细胞。这些检测方法将在DNA损伤、DNA修复、致癌/突变、细胞凋亡的研究以及确定抗肿瘤药物的作用机制方面发挥作用。第一种方法分析DNA结合的荧光配体的最大荧光像素强度的变化，染色质松弛的报告，细胞DNA损伤反应(DDR)的初始事件。其他待开发的探针利用磷酸特异性抗体和多参数细胞术来测量关键DDR：组蛋白H2 AX、ATM、Chk2和P53的磷酸化/激活。将评估探针在不同类型DNA损伤时测量DDR的能力，包括药物、诱变剂和辐射造成的损伤，并与细胞周期时相和诱导细胞凋亡相关。本课程将探讨影像细胞术量化个别核灶频率的能力，每个核灶代表在DDR期间发生的DNA双链断裂(DSB)。我们还将开发一种集成的多重方法，将DDR(诱导3H2 AX或ATM、Chk2和P53磷酸化)的检测与实际DNA损伤的测量相结合，通过单细胞DNA电泳(彗星试验)进行评估。激光扫描细胞术的“图像合并”能力将被用来将DDR分析与同一单个细胞中的DNA损伤分析相结合。另一种将开发的多重分析将结合DDR的分析和DNA修复，测量为非程序DNA合成(UDS)。将探索和发展5-溴-2‘-脱氧尿苷(BrdU)和5-乙炔基-2’-脱氧尿苷(EDU)的结合，后者随后进行环加成反应(“点击”化学)，以便在同一细胞中评估UDS和DDR。使用上述方法，将评估细胞分析中常用的超活性探针(Hoechst 33342、DRAQ5、DyeCycle Violet和SYTO 17)引起的潜在dna损伤和DDR。将特别注意探索在使用这些探针将干细胞作为“侧群”分离的条件下是否会发生DNA损伤。细胞分离干细胞中DNA损伤的检测，特别是DSB的检测，将预测分离过程的致癌/突变潜力。评估肿瘤抑制基因P53在不同遗传毒性药物引起的DDR中的作用，包括针对DNA的抗肿瘤药物，是这一应用中的另一个项目。我们建议开发的检测方法将在许多不同的领域得到广泛应用，包括药理学、诱变性、致癌性、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： 这个项目的目的是开发新的方法来评估细胞遗传物质(DNA)的损害，以及受损DNA的修复，因为它发生在辐射、环境诱变剂和癌症治疗过程中。与年龄相关的渐进性DNA损伤也是衰老的主要原因，并使个人容易患上癌症。所提出的方法将在诱变剂筛选、癌症预防、新的抗癌药物开发以及临床肿瘤学中得到广泛应用，以快速检测抗癌治疗的有效性，从而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.