The overall objective of the work proposed in this application and accomplished during the past 29 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 determining the mechanisms of action of polyamines on the growth of intestinal epithelial cells. The PI will test the principal hypothesis that polyamines are actively involved in regulating signal transduction pathways that determine whether cells progress through the cell cycle, undergo cell cycle arrest, or undergo apoptosis. This novel hypothesis is a major shift in thinking about polyamine action. In the past, numerous studies demonstrated that in polyamine depleted cells various activities of the cell were depressed. In other words, """"""""things"""""""" failed to occur. The PI's studies of the past 3 years, however, indicate that the absence of polyamines stimulates or activates pathways resulting in changes of cell function. Their data indicate that polyamine depleted IEC-6 cells are actively shifted into GI cell cycle arrest and that apoptosis is inhibited. These data will be expanded and their hypothesis tested by examining the mechanisms behind these events by determining the role of polyamines in the regulation of the cell cycle. The PI has proposed a total of eleven specific aims organized into 3 specific areas. The PI will examine the role of the Ras/MAPK cascade in cell cycle arrest induced by polyamine depletion, determine whether polyamines affect the levels and activities of cell cycle regulatory proteins, and use p53 mutated cell lines to elucidate the role of p53 in the cell cycle arrest caused by depletion of polyamines. Next, the PI will investigate the role of polyamines in apoptosis by determining whether polyamines protect cells from apoptosis regardless of the stimulus and by determining whether polyamine depletion alters the upstream regulators of caspase activity. The PI will then elucidate the roles and significance of NF-kappaB, STAT-3, p53 and the bcl-2 family of proteins in the anti-apoptotic response to polyamine depletion. These experiments will define the mechanisms resulting in cell cycle arrest in response to polyamine depletion and indicate their points of interaction with pathways that prevent apoptosis. The finding that normal polyamine levels are required for apoptosis is new and of great potential clinical significance, for it suggests a way to protect normal cells during cancer therapy. The basic knowledge gained from these studies will increase our understanding of mucosal healing and of any condition in which GI mucosal growth is altered or involved.
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