The overall goal of this research is to investigate the genetic controls and molecular mechanisms regulating gene expression in apoptotic death associated with thymic involution. The experimental system used is the thymoma-derived murine cell line WEH17.2 which undergoes apoptosis as a response to glucocorticoids, cAMP, and agents which increase the intracellular Ca2+ concentration. We have a particular advantage with this system in that we have isolated a novel apoptosis-defective variant, WR256, which is resistant to death induced by glucocorticoids, cAMP, and calcium. The properties of this mutant strongly suggest that the independent cascades leading to death induced by these agents may share steps We have preliminary evidence to suggest that one of these shared events is the regulation of transcription, in part through the cAMP or Ca2+ activation of cAMP response element (CRE)-mediated gene expression.
The specific aims of this proposal are three: 1. Test the hypothesis that transcriptional activation by CREB, the cAMP response element binding protein, plays a central role in apoptotic death induced by cAMP, glucocorticoids and changes in intracellular Ca2+. 2. Test the hypothesis that Ca2= release from the endoplasmic reticulum is involved in apoptosis by comparing the effects of dexamethasone and thapsigargin (an inhibitor of the Ca2+/ATPase in the ER) on WEH17.2 and WR256. 3. Characterize the specific genes which are regulated by cAMP in WEH17.2, but which fail to be regulated in the apoptotic deathless mutant WR256. We will alter the effective concentration of CREB in the cell through a variety of approaches, measure the phosphorylation state of CREB as an indicator of transcriptional activity, an identify the kinase responsible for phosphorylating CREB. In addition, we will define the role of Ca2+ in the apoptotic response by determining the biochemical, genetic, and morphological responses of WEH17.2 cells to thapsigargin and dexamethasone, and comparing the results to those obtained with the """"""""deathless"""""""" WR256. Finally, we will isolate and analyze genes regulated by cAMP during apoptosis using the method of differential amplification.
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