ODC, THE CYTOSKELETON, AND CELL GROWTH

肿瘤对生长调节蛋白的调节不当 它们被认为会带来特定的增长优势。 生长相关酶鸟氨酸脱羧酶的过表达 (ODC)导致细胞转化，是TPA诱导的核心 小鼠皮肤形成乳头状瘤。细胞骨架干扰物废除ODC 诱导和乳头状瘤的形成，这是特别有趣的，因为我们 最近观察到一个与上皮细胞相关的ODC池 细胞骨架。细胞骨架干扰物改变ODC的定位 当ODC水平改变时，细胞骨架就会被破坏。因此， 我们知道在ODC和 细胞骨架。这项建议使用上皮角质形成细胞来探索 ODC定位调控及其对细胞骨架的影响 组织和细胞生长。分子手段被用来干扰或 修改ODC在细胞中的表达水平和/或活性以进行分析 由此产生的细胞后果。具体目标是：(1) 确定重组ODC的表达效果，即， 磷酸化-缺陷、非活性和活性，对ODC分布的影响 亚细胞分裂和免疫组织化学，以及对 这样的细胞被用来调节细胞骨架完整性或ODC的治疗 水平，包括TPA、细胞松弛素、α-二氟甲基鸟氨酸 α-DFMO，和腐胺；(2)评估受调控的功能 ODC在细胞骨架组织和细胞上的定位 通过在细胞中表达重组ODC的特性 反义寡核苷酸治疗耗尽内源性ODC。 待检测的细胞参数包括生长(DNA合成速率)、 增殖(角蛋白亚型的表达)和细胞-底物 粘附性(细胞表面整合素Beta1表达)；以及(3)测试 通过确定ODC/细胞骨架是否过表达来实现功能连接 Odc突变体与pp60v-src或ras协同作用导致锚定- 独立增长；这些信息将提供有关如何 ODC与细胞骨架的相互作用可能会影响细胞的 与肿瘤生长有关的附着、沟通和生长的能力。 我们认为，这些研究代表了对 潜在的高度重要的信号转导系统参与 调节细胞生长和亚细胞结构，并应产生 靶向特定细胞通路的有用信息 癌症治疗。

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.