DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

集成细胞生物测定法的开发和应用

• 批准号: 6217605
• 负责人: ROBERT H RICE
• 金额: $ 16.36万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6217605
• 关键词: DNA binding protein; aromatic hydrocarbon receptor; bioassay; biological signal transduction; cell differentiation; cell growth regulation; chemical carcinogenesis; cytotoxicity; diagnosis design /evaluation; environmental toxicology; enzyme activity; gel mobility shift assay; gene expression; guinea pigs; hazardous substances; human tissue; industrial waste; isozymes; keratinocyte; organ culture; protein kinase C; steroid hormone receptor; stress proteins; toxicant interaction; transcription factor; transfection

This project focuses on biologically-based detection of hazardous agents by methods exploiting their specific mechanisms of action. First, sensitive and rapid bioassays will be developed for several classes of environmentally important toxicants. In one approach, the ability of certain agents to stimulate gene expression by binding to and activating chemical-specific DNA transcription factors will be exploited in bioassays for certain classes of chemicals. For this purpose, cells will he stably transfected with an expression vector containing a luciferase reporter gene inducibly regulated by the high affinity interaction of one of several ligand-dependent receptors (including those for estrogens, dioxin and related polycyclic aromatic hydrocarbons) with transcriptional enhancer elements inserted upstream of the reporter gene. In a complementary approach, competitive in vitro binding microplate assays for the detection of these chemicals will be developed utilizing purified receptors which have been produced in large quantities by a baculovirus expression system. Initial efforts will be focused on xenobiotics which interact with the Ah and estrogen receptors and will be ex:tended to include other chemicals (such as metals and peroxisome proliferators) that also alter gene expression by chemical-dependent receptors. Second, the ability of hazardous agents to perturb biochemical processes important for cellular growth and differentiation will be examined using human epidermal keratinocytes as targets. The direct toxicity of samples will be measured as well as the ability of samples to sensitize cultures to toxicity from agents activated by metabolism such as polycyclic aromatic hydrocarbons. In addition, based on our original findings with arsenic, the phosphorylation state and levels of select kinase and phosphatase activities will be examined. Protein kinase C isozymes, critical for proper keratinocyte function, and known (keratinocyte transglutaminase) or suspected protein substrates will be of special interest. Moreover, protein tyrosine phosphate levels and effects on specific DNA transcription factors (AP2, steroid hormone receptors) and differentiation markers (involucrin) will be monitored as appropriate. This information will help in tracing signal transduction pathways with which toxicants interfere. Finally, heat shock protein isoform expression and subcellular localization will be examined as a measure of cell stress. Effects of chemical treatment will be compared to actual isoform perturbation in human skin tumors and arsenical keratoses. Validation of effects seen in cultured keratinocytes will be performed using human skin in organ culture. For each assay above, applicability to environmental samples provided by other Superfund components will be tested using purified and complex mixtures of model agents as well as extracts of combustion residues from plastics, metals and other constituents present at hazardous waste sites.

该项目的重点是基于生物学的有害物质检测 通过利用其特定作用机制的方法。第一、 将为几类生物开发灵敏和快速的生物测定法， 对环境有害的有毒物质。在一种方法中， 某些试剂通过结合并激活 化学特异性DNA转录因子将被利用， 某些化学品的生物测定。为此，细胞将 用含有荧光素酶的表达载体稳定转染 报告基因的高亲和性相互作用的诱导调节 几种配体依赖性受体（包括雌激素受体， 二恶英和相关的多环芳烃）与转录 插入报告基因上游的增强子元件。中 互补方法，竞争性体外结合微孔板试验 用于检测这些化学品将开发利用纯化 由杆状病毒大量产生的受体 表达系统最初的努力将集中在外源性物质上， 与Ah和雌激素受体相互作用，并将被延长， 包括其他化学品（如金属和过氧化物酶体增殖剂） 也会通过化学依赖性受体改变基因表达。第二、 有害物质扰乱生物化学过程的能力 重要的细胞生长和分化将检查使用 人表皮角质形成细胞作为靶细胞。样品的直接毒性 以及样品致敏培养物的能力 代谢激活剂的毒性，如多环 芳香烃.此外，根据我们最初的发现， 砷，磷酸化状态和选择激酶的水平， 检查磷酸酶活性。蛋白激酶C同工酶， 对于适当的角质形成细胞功能至关重要，并且已知（角质形成细胞 转氨酶）或疑似蛋白质底物将具有特殊的 兴趣此外，蛋白酪氨酸磷酸水平和对 特异性DNA转录因子（AP2，类固醇激素受体）， 适当时监测分化标志物（外皮蛋白）。 这些信息将有助于追踪信号转导途径， 哪些有毒物质会干扰。最后，热休克蛋白异构体表达 并将检查亚细胞定位作为细胞的衡量标准 应力将化学处理的效果与实际亚型进行比较 干扰人类皮肤肿瘤和砷角化病。验证 在培养的角质形成细胞中观察到的效果将使用人皮肤进行 器官培养。对于上述各项测定，环境适用性 由超级基金其他组成部分提供的样本， 模型剂的纯化和复杂混合物以及 塑料、金属和其他成分的燃烧残留物 危险废物处理场。