Calcium Signaling and Cytochrome C Release in Apoptosis

细胞凋亡中的钙信号传导和细胞色素 C 释放

• 批准号: 6613758
• 负责人: Jianjie Ma
• 金额: $ 26.06万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613758
• 关键词: CHO cells; apoptosis; biological signal transduction; calcium; calcium channel; cell migration; cell proliferation; cytochrome c; cytoplasm; endoplasmic reticulum; epithelium; extracellular; fluorescence; green fluorescent proteins; homeostasis; intracellular; intracellular transport; mitochondria; mitochondrial membrane; neoplastic cell; ovary; prostate; prostate neoplasms; protein transport; tissue /cell culture; transcription factor; transforming growth factors

Apoptosis is a genetically controlled process which plays a major role in the development and maintenance of tissue homeostasis. Endoplasmic reticulum (ER) and mitochondria are two key cellular organelles known to actively participate in apoptosis. While perturbation in cellular Ca homeostasis (i.e. depletion of ER Ca and/or increase in cytosolic Ca) leads to apoptosis, translocation of cytochrome c from mitochondria to cytosol has also been shown to play a critical role in the apoptotic cell death pathway. A family of Bcl-2 related proteins (i.e. Bcl-xL and Bax) serves essential roles in apoptosis, but the functional effects of which on intracellular Ca signaling remain largely unknown. The experiments proposed here will test the hypothesis that ER and mitochondria communicate with each other to release cytochrome c into the cytosol with Ca serving as a messenger in the initiation of apoptosis, and the movements of intracellular Ca and Bax (or its partners) play important roles in determining the fate of the cells to undergo either apoptosis or necrosis. Aim 1 of this proposal will involve developing cultured cell lines(CHO - Chinese hamster ovary cells, and NRP- 154 - epithelial cells derived from prostate tumor tissue) that express both green fluorescent protein-tagged cytochrome c (cyt.c-GFP) and ryanodine receptor which functions as an intracellular Ca release channel. Through controlled release of Ca via activation of ryanodine receptor, and simultaneous measurement of Ca uptake into mitochondria and translocation of cyt.c-GFP from mitochondria, we will establish the spatio- temporal relationship between ER Ca movement and mitochondria cytochrome c translocation, and test the role of intracellular Ca release and extracellular Ca entry in the apoptotic and necrotic cell death pathway. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and/or ER membranes. Our preliminary data showed that overexpression of Bax can affect ER Ca homeostasis, and perturbation of intracellular Ca can feedback to the translocation process of Bax. Aim 2 of this proposal will probe the relationship between changes in Ca homeostasis and Bax movement in apoptosis, and investigate the cellular and molecular function of Bcl-xL and Bax in the Ca signaling process of apoptosis. The NRP-154 cells are sensitive to TGF-beta-mediated apoptosis, but are resistant to Bax overexpression-mediated apoptosis. This is in contrary to most other known cells where Bax has been described as an ubiquitous stimulator of cell death. Aim 3 of this proposal will search for putative genes that may constitute antagonistic co-factors for Bax-induced apoptosis in the NRP-154 cells, with the hope to further understand the role of Bcl-xL, Bax and Ca in TGF-beta-mediated apoptosis of prostate tissues. Our long term goal of this project is to understand the cellular and molecular mechanism of Ca signaling in apoptosis, and to provide alternative interventions in the therapy of prostate cancer.

凋亡是一个遗传控制的过程，在组织稳态的发展和维持中起着重要作用。 内质网（ER）和线粒体是两个关键的细胞细胞器，已知积极参与凋亡。 尽管细胞Ca稳态的扰动（即ER CA的耗竭和/或胞质CA中的增加）导致凋亡，但也已显示，从线粒体到细胞质的细胞色素C易位在凋亡细胞死亡途径中起着关键作用。 Bcl-2相关蛋白（即Bcl-XL和BAX）的家族在凋亡中起着至关重要的作用，但是其对细胞内CA信号传导的功能效应仍然很大程度上未知。 此处提出的实验将检验以下假设：ER和线粒体相互通信以将细胞色素C释放到胞质溶胶中，CA用作凋亡开始的使者，以及细胞内CA和BAX（或其伴侣）的运动在确定细胞的效果上，以确定较重要的细胞来确定较重要的细胞以确定无apopoptosis的效果。 该提案的目标1将涉及开发培养的细胞系（CHO-中国仓鼠卵巢细胞和NRP-154-衍生自前列腺肿瘤组织的上皮细胞），这些细胞表达出绿色荧光蛋白标记的细胞色素c（cyt.c-gfp）和ryanonodine受体的绿色荧光蛋白标记的细胞，和ryanodine受体，这些受体起作用。 通过通过激活Ryanodine受体的受控释放，以及将CA吸收到线粒体中的CA摄取和线粒体的Cyt.C-GFP的易位，我们将建立ER CA运动与线粒体Cytrome c procortoter ca Opertotor Ca propertoter opelliular ca propertoter and ectect opelliular ca procorto and apellect opelliular ca procorto and apellect opelliular ca the ect opelliular ca procorto the apelliular ca the ect opter theeclellular ca serecrolular ca的作用路径。 凋亡中的关键早期事件与从细胞质到线粒体和/或ER膜的Bax重新分布有关。 我们的初步数据表明，BAX的过表达会影响ER CA的稳态，并且细胞内CA的扰动可以反馈到BAX的易位过程。 该提案的目标2将探测凋亡中Ca稳态的变化与Bax运动的变化之间的关系，并研究BCl-XL和BAX在CA凋亡的CA信号传导过程中的细胞和分子功能。 NRP-154细胞对TGF-β介导的细胞凋亡敏感，但对BAX过表达介导的凋亡具有抗性。 这与大多数其他已知细胞相反，该细胞被描述为细胞死亡的无处不在刺激剂。该提案的目标3将搜索可能构成NRP-154细胞中Bax诱导的凋亡的拮抗辅助因子的假定基因，希望进一步了解BCL-XL，BAX和CA在TGF-BetA介导的前列腺组织中的作用。 我们该项目的长期目标是了解凋亡中CA信号传导的细胞和分子机制，并在前列腺癌治疗中提供替代干预措施。