: The overall objective of the work proposed in this application and accomplished during the past 34 years the grant has been active is to define and understand the mechanisms regulating the growth of gastrointestinal tract mucosa. This objective is unaltered in the current proposal which focuses on elucidating the mechanism by which polyamines are required for apoptosis of intestinal epithelial cells. During the past period of support we proved that polyamine depletion activates several signal pathways which prevented normal apoptosis. We now seek to identify the target for polyamine involvement that sets these pathways in motion. This is a major shift in the thinking about the action of polyamines, for it means that in the absence of polyamines, cell functions are activated instead of prevented from happening. Based on our published and preliminary data, we hypothesize that in the absence of polyamines Src-kinase is constitutively activated, and that it tyrosine phosphorylates PP2A (protein phosphatase 2A), inactivating it. The inactivation of PP2A, a serine threonine phosphatase, allows anti-apoptotic proteins, such as ERK, Akt, and Bcl-2, to remain active since their activity requires serine phosphorylation. On the other hand, proapoptotic proteins such as Bad and IkappaB-alpha, are inactivated by serine phosphorylation, and these proteins, thus remain inactive. To date experiments have been conducted using the IEC (intestinal epithelial cell)-6 cell line. In the first specific aim, we will extend our studies to whole animals, and determine whether the pattern of protection from apoptosis that we observed in cells is the same in polyamine depleted mice exposed to gamma-irradiation. We will then determine the role of PP2A in the protection from apoptosis observed in polyamine depleted cells and the role of Src-kinase in that resistance and whether it is responsible for inactivating PP2A. The final specific aim will examine STAT-3 and its part in protecting polyamine depleted cells from apoptosis. STAT-3 is activated by polyamine depletion and by Src-kinase. Therefore, if STAT-3 is involved in the resistance to apoptosis, it will further support our hypothesis. The finding that normal polyamine levels are required for apoptosis is new and of great potential clinical significance, since it suggests a way to protect normal cells during cancer therapy. Understanding the mechanisms involved in that protection will increase our knowledge of cancer, mucosal healing, inflammation and any other condition in which GI mucosal growth is altered or involved.
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