Altered Apoptotic Responses in Chemically-Resistant Mammalian Cell Variants

在所有多细胞生物中，组织和器官的动态平衡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一个受基因控制的细胞自杀过程，称为细胞凋亡。这一过程的几种形式已被证明涉及(A)凋亡抑制因子bl-2，(B)当被激活时诱导细胞色素c从线粒体外流的Bid因子，以及(C)几种特定的蛋白水解酶，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星状孢子素诱导的人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，以回答三个具体问题。(1)星孢菌素激活的处理BID的半胱氨酸氨基转移酶是什么？半胱氨酸天冬氨酸氨基转移酶将进行生化提纯。如果是新的，它将被测序，其基因将被克隆。(2)Bcl2是如何调节星形孢子素诱导的caspase-8活化的？将考虑几种可能性，包括与其他成分形成信号复合体。(2)bc l-2是否与胞浆中的Bid形成异二聚体，从而影响Bid对caspase作用的敏感性？这些二聚体的可能形成和它们的底物活性将被直接检测。预计这些结果将产生适用于多种多细胞生物体的关于细胞凋亡的基本新信息。

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.