The majority of human tumors originate in epithelial tissue. A characteristic of self-renewing epithelial tissue is its precisely controlled balance between growth and differentiation pathways. While the normal genetic mechanisms regulating these two pathways in epithelial cells are not known, it is clear that these controls are drastically altered in neoplastic tissue. In addition to known genetic changes such as oncogene activation, recent findings in both mouse and human tumor suggest that aberrant regulation of ornithine decarboxylase (ODC) may play an important role in tumor development. ODC is a key regulatory enzyme in the biosynthesis of polyamines which are essential for cell growth and differentiation. The hypothesis to be tested in this proposal is that a change in the regulation of polyamine biosynthesis, as regulated by ODC levels, is one of the determinants in whether initiated keratinocytes differentiate (leading to cell death) or proliferate, thus, upsetting the precise balance between these processes which is characteristic of normal epidermis. A corollary to this hypothesis is that altered ODC expression resulting in elevated levels of ODC activity is causally involved in mouse epidermal tumor development. In order to override the normally tight controls over polyamine synthesis, a replication-defective retroviral vector capable of overexpressing mouse ODC will be introduced into primary epidermal cell cultures and keratinocyte cell lines. These ODC overexpressor cells will be assessed for changes in their proliferative capacity and ability to undergo a program of terminal differentiation in vitro in response to differentiation signals such a high calcium levels or TPA addition. In addition to these in vitro experiments, the role of sustained high basal levels of ODC activity in epidermal tumorigenesis will be evaluated in vivo. An ODC expression vector will be introduced into normal primary keratinocytes or cultured papilloma cell lines, and these cells along with normal mouse dermal fibroblasts will be transplanted as skin grafts onto athymic nude mice. the reconstituted skin grafts will be analyzed for morphological alterations or tumor formation. These experiments will directly test the hypothesis that ODC overexpression and altered polyamine synthesis function with other genetic changes to lead to the development of a malignant phenotype in an epithelial tissue.
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