Multiparameter Assay for Mechanistic Understanding of Carcinogens

多参数分析用于了解致癌物的机理

• 批准号: 8199654
• 负责人: Peter Krutzik
• 金额: $ 19.58万
• 依托单位: PRIMITY, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8199654
• 关键词: Animal Model; Antibiotic Resistance; Antigens; Automobile Driving; Biological Assay; Biosensor; Cancer Etiology; Carcinogenicity Tests; Carcinogens; Cell Count; Cell Cycle; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chemicals; Clinical; Clone Cells; Cloning; Complementary DNA; Complex; DNA; DNA Damage; Detection; Development; Electroporation; Elements; End Point Assay; Ensure; Exposure to; Flow Cytometry; Genes; Genomics; Genotoxic Stress; Grant; Health; Human; Individual; Industry; Measurement; Measures; Metabolism; Methods; Monitor; Mutagens; Noise; Oncogenic; Pattern; Plasmids; Population; Preparation; Procedures; Process; Proteins; Regulation; Reliance; Reporter; Reporter Genes; Running; Sampling; Screening procedure; Signal Pathway; Signal Transduction; Site; Small Business Innovation Research Grant; Solutions; System; TP53 gene; Testing; Transfection; base; chemical carcinogen; cost; high throughput screening; hygromycin A; improved; promoter; response; transcriptional coactivator p75; vector

DESCRIPTION (provided by applicant): The determination of carcinogenic activity in experimental compounds is a long and expensive process that negatively impacts many core industries and can threaten human health. Faster, more reliable methods that can be used to accurately determine human oncogenic activity are needed to rapidly identify environmental problems and reduce the reliance on animal models. The pleiotropic effects of carcinogens have made them difficult to detect using standard high-throughput assays that measure a single cellular change. A solution to this problem is to monitor several aspects of cell function simultaneously. Although these methods have the appropriate sensitivity, their implementation has been difficult due to long and complicated sample preparation procedures which decrease the throughput and dramatically increase the assay variability. The solution we propose in this grant is the multiplexed measurement of cellular responses in one sample using flow cytometry combined with cellular barcoding. In this system, multiple biosensors are encoded in a single sample to provide nine distinct measures of cellular responses to carcinogens. In addition, cell viability and cell cycle status are obtained for each sample, enabling multi-dimensional analysis of compound activity. This system benefits from the increased sensitivity of multiplexed measurement while retaining the precision of a homogeneous high-throughput assay. PUBLIC HEALTH RELEVANCE: Chemical carcinogens induce complex changes in cellular DNA and metabolism, making it difficult to identify carcinogens using single end-point assays. The platform described here uses high-content, multiplexed measurements of known oncogenic signaling pathways to improve the detection of carcinogens in clinical and environmental samples and provide information about their mechanism of action.

描述（由申请人提供）：确定实验化合物中的致癌活性是一个漫长而昂贵的过程，对许多核心行业产生负面影响，并可能威胁人类健康。需要更快、更可靠的方法来准确地确定人类致癌活性，以快速识别环境问题并减少对动物模型的依赖。致癌物的多效性使得它们难以使用测量单个细胞变化的标准高通量测定来检测。这个问题的解决方案是同时监测细胞功能的几个方面。虽然这些方法具有适当的灵敏度，但由于长时间和复杂的样品制备程序，它们的实施一直很困难，这降低了通量并显著增加了测定的可变性。我们在该授权中提出的解决方案是使用流式细胞术结合细胞条形码在一个样品中多重测量细胞反应。在这个系统中，多个生物传感器被编码在一个单一的样本中，以提供细胞对致癌物反应的九种不同的测量。此外，获得每个样品的细胞活力和细胞周期状态，从而能够对化合物活性进行多维分析。该系统受益于多重测量的灵敏度增加，同时保持均匀高通量测定的精度。 公共卫生关系：化学致癌物诱导细胞DNA和代谢的复杂变化，使得难以使用单一终点测定来识别致癌物。本文描述的平台使用已知致癌信号通路的高含量、多路复用测量来改善临床和环境样本中致癌物的检测，并提供有关其作用机制的信息。