POLYAMINES AND EPITHELIAL TUMORIGENESIS

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Ornithine decarboxylase (ODC) is a key regulatory enzyme in the biosynthesis of polyamines which are essential for normal cell growth and differentiation. The applicants have demonstrated that elevated levels of ODC and polyamines cooperate with a mutated Ha-ras gene to promote epithelial tumor formation and invasion. The applicant's hypothesis is that elevated levels of ODC and polyamines "activate" keratinocytes and dermal stromal cells to produce a phenotype similar to that in wounded skin, which is characterized by increased proliferation and migration, neoangiogenesis, altered differentiation, and increased expression of proteases. A corollary is that conditions (or oncogenes) that sustain this polyamine-induced activation of cells will result in the development of a tumor. The overall goal of this project is to identify downstream effectors that play a key role in mediating ODC-promoted tumorigenesis in skin. The objective is complicated when tumors arise as a consequence of a mutated ras (as is generally found in initiated skin) since multiple effector pathways contribute to Ras-mediated transformation of cells. Therefore, in order to achieve these goals, the applicant plans to dissect out the downstream effectors of polyamines as well as the downstream effectors of an activated ras that cooperate with ODC and polyamines to drive epithelial tumorigenesis. To this end, Dr, Gilmour proposes the following specific aims: 1. To determine if elevated levels of ODC and polyamines activate keratinocytes and dermal stromal cells in a manner similar to that in wounded skin, ODC will be expressed in both normal quiescent adult epidermis (using an inducible ODC transgenic mouse model), and in proliferative activated epidermis (abrading K6/ODC transgenic mouse skin). By controlling expression of ODC in either sedentary or activated keratinocytes, the applicants will evaluate this noninitiated (i.e. no mutated ras gene) skin for the induction of: A. a wound healing phenotype, and B. the formation of benign skin tumors dependent upon sustained ODC-induced activation of cells. 2. Using the inducible ODC transgenic mouse model, mRNA from ODC overexpressing skin will be compared with that from normal skin using representational difference analysis (RDA) and microarray approaches. Genes that are upregulated or downregulated by ODC will be evaluated for their role in mediating the tumorigenic and invasive effects of ODC when expressed with a mutated H-ras. 3. Dr. Gilmour will determine which downstream effectors of an activated Ha-Ras can cooperate with elevated levels of ODC and polyamines to convert epidermal cells to a malignant invasive phenotype. In particular, she plans to investigate possible cooperation between ODC and various partial function effector mutants of an activate Ha-Ras that specifically induce either the raf-1/ERK, RalGDS, or phosphoinostitie 3-OH kinase pathway.