Altered Apoptotic Responses in Chemically-Resistant Mammalian Cell Variants

耐化学性哺乳动物细胞变体中细胞凋亡反应的改变

• 批准号: 9974741
• 负责人: James Wyche
• 金额: $ 18万
• 依托单位: Brown University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974741&HistoricalAwards=false
• 关键词: Altered; Apoptotic; Responses; Chemically; Resistant

Tissue and organ homeostasis in all multicellular organisms is maintained by a rigid balance between cellular proliferation and cell death. One of the most important mechanisms of cell death is a genetically regulated process of cellular suicide known as apoptosis. Several forms of this process have been shown to involve (a) the apoptotic suppressor, Bcl-2, (b) a factor, Bid, which when activated induces cytochrome c efflux from mitochondria, and (c) several specific proteases, including caspase-3 and caspase-8. The overall purpose of this project is to elucidate the molecular and biochemical roles of these gene products in the model system of apoptosis induced by staurosporine in human HL-60 cultured cells. Experiments will be undertaken to answer three specific questions. (1) What is the staurosporine-activated caspase that processes Bid? The caspase will be purified biochemically. If novel, it will be sequenced, and its gene will be cloned. (2) How does Bcl-2 regulate staurosporine-induced caspase-8 activation? Several possibilities will be considered including the formation of signaling complexes with other components. (2) Does Bcl-2 form heterodimers with Bid in the cytosol and affect the susceptibility of Bid to caspase action? The possible formation of such dimers and their substrate activities will be assayed directly. It is expected that the results will yield fundamental new information about apoptosis applicable to a wide variety of multicellular organisms.

所有多细胞生物的组织和器官稳态都是通过细胞增殖和细胞死亡之间的严格平衡来维持的。细胞死亡最重要的机制之一是基因调控的细胞自杀过程，即细胞凋亡。这一过程的几种形式已被证明涉及(a)凋亡抑制因子Bcl-2， (b)一个因子Bid，当它被激活时，会诱导线粒体的细胞色素c外排，以及(c)几种特定的蛋白酶，包括caspase-3和caspase-8。本项目的总体目的是阐明这些基因产物在staurosporine诱导人HL-60培养细胞凋亡模型系统中的分子和生化作用。将进行实验来回答三个具体问题。(1)处理Bid的staurosporine激活的caspase是什么？半胱天冬酶将被生化纯化。如果是新的，将对其进行测序，并克隆其基因。(2) Bcl-2如何调控staurosporine诱导的caspase-8激活？将考虑几种可能性，包括与其他组分形成信号复合物。(2) Bcl-2是否在细胞质中与Bid形成异源二聚体并影响Bid对caspase的易感性？这种二聚体的可能形成及其底物活性将直接测定。预计这些结果将产生适用于多种多细胞生物的关于细胞凋亡的基本新信息。