The goal of this work is to understand the mechanism by which ornithine decarboxylase (ODC) activity is induced during carcinogenesis by oncogenic ras, andto define the role of ODC in this process. Results in the Progress Report show for the first time that induction of ODC activity in Ras-transformed cells requires the coordinate activation of the Raf/MEK/ERK and PI 3-kinase pathways. ODC RNA and protein are increased, with the primary regulation being post-transcriptional. We will use constitutively active forms of Ras, Raf and Akt to activate the effector pathways of interest in RIE-1 epithelial cells. These cells are appropriate since the Raf/ME/ERK and PI 3-kinase pathways cooperate in their transformation. We will use this model to study translational regulation of ODC in response to these signaling cascades, and to assess the role of sustained ODC activation in transformation, cell cycle control, and survival. We also show that inhibiting ODC with the irreversible inactivator alpha-difluoromethylornithine (DFMO) delays the onset of spontaneous tumors in mice overexpressing an active MEK mutant in the skin (K14-MEK mice), and causes regression of established tumors. To test the hypothesis that suppression of ODC blocks the promotion of target cells initiated by the Raf/MEK/ERK pathway, we will cross K14-MEK mice with two transgenic lines overexpressing antizyme (AZ), which binds to ODC and targets it for degradation. We have shown using K5-AZ and K6-AZ mice that AZ suppresses tumor growth in the two-stage model of skin carcinogenesis. The K6 promoter requires hyperproliferation for maximal expression. Since MEK overexpression causes hyperplasia, MEK/K6-AZ mice represent a model to test if ODC inhibition is effective in preventing tumor formation after establishment of a carcinogenic environment. The K5 promoter is constitutive, and MEK/K5-AZ mice have a constant expression of AZ from birth. These mice represent a chemoprevention model, in which ODC is inhibited at the time of the initiating stimulus. We will examine tumors from MEK/AZ mice to determine effects on cell cycle proteins, apoptosis, and proliferation. To specifically address the role of ODC in cell cycle progression during tumorigenesis, we will use transgenic mice that overexpress AZ and cyclin D1 in the skin. Keratinocytes from D1/AZ mice will also be used to analyze direct interactions of AZ with cyclin D1, to test whether other properties of AZ are important for deregulation of cell proliferation in tumor development.
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