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.
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