Tumors exhibit inappropriate regulation of growth-regulatory proteins that are thought to give rise to a selected growth advantage. Overexpression of the growth-related enzyme ornithine decarboxylase (ODC) causes cellular transformation and is central to TPA-induced papilloma formation in mouse skin. Cytoskeletal disruptors abrogate ODC induction and papilloma formation, which is especially interesting as we have recently observed an ODC pool associated with the epithelial cytoskeleton. ODC localization is altered by cytoskeletal disruptors and the cytoskeleton is disrupted when ODC levels are altered. Thus we know that there is functional cross-talk between ODC and the cytoskeleton. This proposal uses epithelial keratinocytes to explore regulation of ODC localization and its effects on cytoskeletal organization and cell growth. Molecular means are used to perturb or modify the expression levels and/or activity of ODC in cells to analyze the resultant cellular consequences.
The specific aims are: (1) to determine the effects of expression of recombinant ODC's, i.e., phosphorylation-defective, inactive, and active, on ODC distribution by subcellular fractionation and immunohistochemistry, and the response of such cells to treatments known to regulate cytoskeletal integrity or ODC levels, including TPA, cytochalasin, alpha-difluoromethylornithine (alpha- DFMO), and putrescine; (2) to assess the function of regulated ODC localization on cytoskeletal organization and on cellular characteristics, by expression of the recombinant ODC's in cells depleted of endogenous ODC by antisense oligonucleotide treatment. Cellular parameters to be examined include growth (DNA synthetic rates), proliferation (expression of the keratin subtypes), and cell-substrate adhesion (cell surface integrin beta1 expression); and (3) to test the ODC/cytoskeleton functional linkage by determining if overexpression of ODC mutants cooperate with pp60v-src or ras to cause anchorage- independent growth; this information will provide insight into how interaction of ODC with the cytoskeleton may influence the cell's ability to adhere, communicate, and grow, as related to tumor growth. We believe that these studies represent an in-depth analysis of a potentially highly important signal transduction system involved in regulating cell growth and subcellular architecture, and should yield useful information on the targeting of specific cellular pathways for cancer treatment.
