Multiparameter Cell Cycle Analysis

多参数细胞周期分析

基本信息

批准号：
7371063
负责人：
James W Jacobberger
金额：
$ 26.11万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-06-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7371063
关键词：
Abnormal Cell Affinity Anaphase Antibodies Apoptosis Apoptotic Binding Biological Assay Cell Cycle Cell Proliferation Cell Size Cells Cellular biology Chemistry Clinical Clinical Trials Color Cross Reactions Cyclin A Cyclin B Cyclins Cytokinesis Cytometry Diagnosis Diagnostic Encapsulated End Point Epitopes Film Fluorescence Frequencies Funding Genes Goals Grant Histocytochemistry Histone H3 Human Cell Line Label Lead Length Malignant Neoplasms Mammalian Cell Marketing Measurement Metaphase Methodological Studies Methodology Microscopic Microscopy Mitosis Mitotic Modeling Modems Molecular Probes Monoclonal Antibodies Mus Negative Axillary Lymph Node Newborn Infant Numbers Oryctolagus cuniculus Phase Phase Transition Phosphorylation Physiologic pulse Pik-off Ploidies Population Proliferating Prometaphase Prophase Pulse taking Range Rate Reading Reagent Reporting Role Route Sampling Series Serine Shapes Signal Transduction Somatic Cell Specificity Staging Staining method Stains Sterile coverings Streptavidin System Testing Therapeutic Time Validation Western Blotting Work Yeasts anaphase-promoting complex anticancer research aurora B kinase base cancer therapy cell fixation cyclin B1 design desire enzyme activity immunoreactivity instrumentation malignant breast neoplasm novel polyclonal antibody pre-clinical prognostic research study response size telophase

项目摘要

DESCRIPTION (provided by applicant): The long-range objective is to create a robust, precise, and accurate model of the mammalian cell cycle based on N dimensional clustering of cytometric measurements of the activity of rate limiting molecules for cell cycle traverse. At present, this is encapsulated by an intermediate Transition State Model of Mitosis that is based on measurements of DNA content, two morphological parameters (DNA pulse shape and cell size), the expression of cyclin A, cyclin B1, and phospho-serine 10 histone H3. In mitosis, quantification of cyclin A is a measurement of the activity of the Anaphase Promoting Complex (APC) in conjunction with Cdc20; Cyclin B1 reads out the activity of the APC in conjunction with Cdhl. Phospho-S10-histone H3 immunoreactivity reports the activity of Aurora B kinase. For human cells lines, the conjunction of these measurements results in the ability to quantify the frequency of multi-dimensional clusters with sharply defined boundaries that represent transition states that closely approximate morphological mitosis i.e., post- G2 -> prophase --> prometaphase -> metaphase --) anaphase/telophase --> cytokinesis --) new born GI. Because these clusters are frequencies of cells residing in each transition state, the size of the cluster is proportional to the time the analyte population existed in each state at sampling time. Within this analysis, but dependent on fewer parameters, the remainder of the cell cycle (G1, S, G2) can be quantified. The specific aims of this proposal are: (1) to enhance the current analysis by adding 2 parameters aimed at simultaneous quantification of GO and apoptotic cells, and add a third variable parameter; (2) develop a primary set of cytometry-grade cell cycle probes with a uniform and low Kd with the yeast display system. These include phospho-epitopes of substrates of Nek2, PIk-1, cyclin Bl/Cdkl, and Aurora B kinase. (3a) determine the relationship between classical mitosis and cytometrically-defined cell state clusters. (3b) test the hypothesis that the conjunction of phospho-cyclin B1 measurements and other mitotic enzyme activity measurements define unique, novel cell states. (3c) test the hypothesis that the Transition State model can be generalized through GI.

描述（由申请人提供）：长期目标是基于细胞周期穿越限速分子活性的细胞计数测量的 N 维聚类，创建稳健、精确且准确的哺乳动物细胞周期模型。目前，这是由有丝分裂的中间过渡态模型封装的，该模型基于 DNA 含量、两个形态参数（DNA 脉冲形状和细胞大小）、细胞周期蛋白 A、细胞周期蛋白 B1 和磷酸丝氨酸 10 组蛋白的表达的测量H3。在有丝分裂中，细胞周期蛋白 A 的定量是对后期促进复合物 (APC) 与 Cdc20 结合的活性的测量； Cyclin B1 与 Cdhl 一起读出 APC 的活性。 Phospho-S10-histone H3 免疫反应性报告了 Aurora B 激酶的活性。对于人类细胞系，这些测量的结合导致能够量化具有明确边界的多维簇的频率，这些边界代表非常接近形态有丝分裂的过渡状态，即 G2 后 -> 前期 -> 中期 ->中期 --) 后期/末期 --> 胞质分裂 --) 新生胃肠道。因为这些簇是驻留在每个过渡状态的细胞的频率，所以簇的大小与采样时分析物群体存在于每个状态的时间成正比。在该分析中，但依赖于较少的参数，可以量化细胞周期的剩余部分（G1、S、G2）。该提案的具体目标是：（1）通过添加两个旨在同时定量GO和凋亡细胞的参数来增强当前的分析，并添加第三个可变参数； (2) 使用酵母展示系统开发一套主要的细胞计数级细胞周期探针，其具有均匀且低 Kd 的特性。这些包括 Nek2、PIk-1、细胞周期蛋白 B1/Cdk1 和 Aurora B 激酶底物的磷酸表位。 (3a) 确定经典有丝分裂和细胞计数定义的细胞状态簇之间的关系。 (3b) 检验这样的假设：磷酸细胞周期蛋白 B1 测量值和其他有丝分裂酶活性测量值的结合定义了独特的、新颖的细胞状态。 (3c) 检验过渡态模型可以通过 GI 进行推广的假设。