ODC, THE CYTOSKELETON, AND CELL GROWTH

肿瘤表现出对生长调节蛋白的不适当调节 被认为能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，并且是TPA诱导的 小鼠皮肤乳头状瘤形成。 细胞骨架破坏剂消除ODC 诱导和乳头状瘤形成，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 ODC定位被细胞骨架破坏物改变 当ODC水平改变时，细胞骨架被破坏。 因此 我们知道，在ODC和 细胞骨架 这项建议使用上皮角质细胞来探索 ODC的定位调节及其对细胞骨架的影响 组织和细胞生长。 分子手段用于干扰或 修饰细胞中ODC的表达水平和/或活性以分析 所产生的细胞后果。 具体目标是：（1） 确定重组ODC表达的影响，即， 磷酸化缺陷、失活和活性对ODC分布的影响 亚细胞分级和免疫组化，以及 这些细胞接受已知可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 （2）评估调节的功能， ODC在细胞骨架组织和细胞膜上的定位 通过在细胞中表达重组ODC， 通过反义寡核苷酸处理耗尽内源性ODC。 待检查的细胞参数包括生长（DNA合成速率）， 增殖（角蛋白亚型的表达）和细胞基质 粘附（细胞表面整联蛋白β 1表达）;和（3）测试 ODC/细胞骨架功能性连接，通过确定是否过度表达 ODC突变体与pp 60 v-src或ras合作引起锚定- 独立增长;此信息将提供洞察如何 ODC与细胞骨架的相互作用可能影响细胞的功能， 粘附、交流和生长的能力，与肿瘤生长有关。 我们认为，这些研究代表了一个深入的分析， 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， 关于靶向特定细胞途径的有用信息， 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.