Probes for Cytometry

描述（由申请人提供）： 该项目的长期目标是继续开发探针和方法，用于通过流式细胞术或图像细胞术分析单个细胞，适用于肿瘤诊断和预后以及凋亡，细胞增殖和抗肿瘤药物筛选的研究。具体目标代表了最近启动的四个项目的延续：（1）DNA损伤：组蛋白H2 AX磷酸化揭示DNA双链断裂（DSB）的位点。我们将：（a）设计区分抗肿瘤药物诱导的（DI）原发性DSB与肿瘤相关的继发性DSB的方法;（B）评估作为药物剂量和治疗持续时间的函数的H2 AX磷酸化和去磷酸化的动力学;（c）将H2 AX磷酸化的程度与细胞凋亡的早期事件相关联;（d）研究DNA复制和/或转录的速率如何影响DI DSB的发生率;（e）探索H2 AX的磷酸化是否可以提供DNA复制细胞的新的、方便的标志物;（f）揭示H2 AX磷酸化是否可以用作DNA修复的标志物;以及（g）调整激光扫描细胞仪（LSC）以量化每个细胞核的DSB的数量。(2)细胞凋亡过程中丝氨酸蛋白酶的激活。使用荧光染料标记的丝氨酸蛋白酶抑制剂（FLISP）作为活性丝氨酸蛋白酶的亲和配体，我们将：（a）进一步表征这些配体的特异性;（B）在内源性和外源性凋亡模型中将丝氨酸蛋白酶的激活与启动子和效应子半胱天冬酶的激活相关联;和（c）测试荧光染料标记的丝氨酸蛋白酶膦酸酯抑制剂作为蛋白酶激活的替代标记物。(3)细胞凋亡过程中转氨酶（TGase 2）的激活。将使用一种新的TGase 2活化细胞计数试验：（a）通过细胞计数法测定在有与无TGase 2活化的情况下发生细胞凋亡的细胞的形态学和其他物理/细胞化学属性的差异;和（B）定义在有与无TGase 2活化的情况下发生细胞凋亡的条件/原因。(4)开发微应答器（MTP）作为测量细胞活力的平台，目标是创建高含量的抗癌药物筛选测定。为实现这一目标：（a）确定附着于MTP的细胞生长的最佳条件，并将这些表面上的细胞生长和存活力（通过DNA和RNA含量的差异测量）与标准固相支持物上的细胞生长进行比较;和（B）测试测量抗肿瘤药物对肿瘤细胞系的细胞抑制/细胞毒性作用的能力，并与标准MTT细胞毒性测定进行比较。

DESCRIPTION (provided by applicant): The long-term objective of this project is to continue development of probes and methodologies for analysis of individual cells by flow or image cytometry applicable to tumor diagnosis and prognosis and for studies of apoptosis, cell proliferation and antitumor drug screening. The specific aims represent the continuation of four recently initiated projects: (1) DNA damage: Histone H2AX phosphorylation reveals the site of DNA double strand breaks (DSBs). We will: (a) design methods to distinguish antitumor drug induced (DI), primary DSBs from apoptosis-associated, secondary DSBs; (b) assess the kinetics of H2AX phosphorylation and dephosphorylation as a function of drug dose and treatment duration; (c) correlate the extent of H2AX phosphorylation with early events of apoptosis; (d) study how the rate of DNA replication and/or transcription affects the incidence of DI DSB; (e) explore whether phosphorylation of H2AX can provide a new, convenient marker of DNA-replicating cells; (f) reveal whether H2AX phosphorylation can serve as a marker of DNA repair, and (g) adapt the laser scanning cytometer (LSC) to quantify the number of DSBs per nucleus. (2) Activation of serine (Ser) proteases during apoptosis. Using fluorochrome-labeled inhibitors of Ser proteases (FLISP) as affinity ligands to active Ser proteases we will: (a) further characterize the specificity of these ligands; (b) correlate activation of Ser proteases with activation of initiator and effector caspases in intrinsic and extrinsic models of apoptosis; and (c) test fluorochrome-tagged phosphonate inhibitors of Ser proteases as alternative markers of protease activation. (3) Activation of transglutaminase (TGase 2) during apoptosis. A new cytometric assay of TGase 2 activation will be used to: (a) characterize the differences in morphology and in other physical/cytochemical attributes measured by cytometry of cells that undergo apoptosis with vs without TGase 2 activation; and (b) define conditions/causes under which apoptosis occurs with vs without TGase 2 activation. (4) Develop microtransponders (MTPs) as a platform for measuring cell viability with the goal of creating a high-content anticancer drug screening assay. Towards this goal: (a) the optimal conditions for growing cells attached to MTPs will be determined and cell growth and viability on these surfaces (by differential measurement of DNA and RNA content) will be compared with cell growth on standard solid-phase support; and (b) the ability to measure the cytostatic/cytotoxic effects of antitumor drugs on tumor cell lines will be tested and compared with the standard MTT cytotoxicity assay.