TRANSFORMATION AND EPITHELIAL CELL POLARITY

上皮细胞以不对称的方式组织起来，起到界面的作用 在多细胞有机体的隔室之间。单元格的极点特征 表面，如质膜结构域和连接复合体 通过非对称排列在细胞内并行 细胞骨架和细胞器，如高尔基复合体。AN的联系人 有底物的上皮细胞可能启动极化过程。 该项目的长期目标是确定两极分化是否 源于非对称小区接触事件的分层过程。在……里面 在建议的研究中，极化上皮细胞系MDCK将 通过引入野生型或温度进行致癌转化的 通过逆转录病毒载体获得敏感的ras癌基因。的影响 对细胞极性的转换将通过检查 特定细胞表面和细胞骨架转化子的定位 使用生化和免疫细胞化学分析标记蛋白质。这个 转化子与基膜成分的相互作用 细胞将合成IV型胶原、层粘连蛋白和纤维连接蛋白 通过细胞结合分析和放射性标记/免疫沉淀测定 分别进行实验。极地组织和细胞附着都将 也可以在条件转换中进行检查，因为它们从 变换的(非极性)到未变换的(极性)状态。最后，MDCK 体细胞杂交的产生将扰乱细胞的极性 非极性细胞系(特定目标5)。混合动力车的两极将是 使用相同的一套极性和细胞附着分析进行评估 变形金刚。这项工作将建立一个实验系统，使用 极性诱导的条件变式可以是 进行了详细的研究。此外，还获得了关于 上皮细胞的致癌转化和细胞杂交形成 极性将促进我们对人类致癌机制的理解。

Epithelial cells are organized asymmetrically to function as an interface between compartments in multicellulr oganisms. Polar features at the cell surface such as plasma membrane domains and junctional complexes are paralleled intracellularly by the asymmetric arrangement of the cytoskeleton and organelles such as the Golgi complex. Contact of an epithelial cell with the substratum may initiate the polarization process. The long-term objective of this project is to determine if polarization is a hierarchical process stemming from the asymmetric cell contact event. In the proposed studies, a polarized epithelial cell line MDCK will be oncogenically transformed by introduction of the wild-type or temperature sensitive ras oncogene via a retrovirus vector. The effects of transformation on cell polarity will be assessed by examining the localization in the transformants of specific cell surface and cytoskeletal marker proteins using biochemical and immunocytochemical assays. The interaction of the transformants with basal lamina components and the synthesis of collagen type IV, laminin, and fibronectin by the cells will be determined by cell binding assays and radiolabeling/immunoprecipitation experiments respectively. Both polar organization and cell attachment will also be examined in the conditional transformants as they are shifted from the transformed (nonpolar) to untransformed (polar) state. Finally, MDCK cell polarity will be perturbed by creation of somatic cell hybrids with nonpolar cell lines (Specific Aim 5). The polarity of the hybrids will be assessed using the same set of polarity and cell attachment assays used for the transformants. This work will establish an experimental system using the conditinal transformants in which the induction of polarity can be studied in detail. In addition, knowledge gained about the effects of oncogenic transformation and cell hybrid formation on epithelial cell polarity will advance our understanding of carcinogenesis in humans.