The overall objective of the work proposed in this application and accomplished during the past 25 years the grant has been active is to define and understand the mechanisms regulating the growth of GI mucosa. Studies supported by this grant during the past 8 years indicate that the rate of mucosal cell proliferation depends on the supply of polyamines to dividing cells. The work outlined in the current proposal focuses on elucidating the mechanisms of action of the polyamines in supporting mucosal growth. Preliminary data indicate that polyamines are essential for the signal cascade initiated by growth factors. This will be investigated by l) examining tyrosine phosphorylation of proteins in response to EGF in the presence and absence of polyamines, 2) identifying key regulatory proteins dependent on polyamines for phosphorylation, 3) determining whether changes are due to kinase inhibition or phosphatase stimulation, 4) using mutant cell lines to localize effects along the Ras/MAP kinase signal cascade, and 5) determining the role of polyamines on trafficking of the EGF receptor. These studies will provide the first insight into the mechanism of action of polyamines in growth that has not been directed at the nucleus. The interaction between growth factors and luminal stimulants of polyamine synthesis will be examined by l) describing the interaction between EGF and asparagine on ODC (ornithine decarboxylase) activity , 2) determining the role of cAMP in that regulation, 3) determining the involvement of the Ras/MAP kinase cascade in the activation of ODC, and 4) determining whether EGF and asparagine alter ODC gene transcription or mRNA stability. These experiments will provide information to understand the mechanism of activation of ODC and for the first time the interaction between luminal stimulants and mitogens. Finally the role of polyamines and the importance of mitogen stimulated ODC activity in the growth response will be assessed by examining protooncogene (c-fos, c-jun, c-myc) mRNA levels to determine whether they are dependent on polyamine levels and/or ODC activity, 2) determine whether polyamines alter the stability or transcription of their genes, and 3) determine where polyamines act in the signal cascade to affect their levels. These experiments also will differentiate between the effects of polyamines synthesized immediately after giving a mitogen and those dependent on the endogenous supply of polyamines. This distinction has not been made to date. These studies have clinical application in GI cancer, wound healing and any condition where GI mucosal growth is altered or involved.
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