MECHANISMS OF CELL ADHESION AND COLON CARCINOMA

项目一的长期目标是了解 整合素受体介导大肠癌细胞粘附、迁移 和生长，并评估这些受体如何影响肿瘤 行为在最后一次授予期间，α 6 β 4和α 2 β 1 整联蛋白被鉴定为结肠癌层粘连蛋白受体。在 此外，还发现了一种新的细胞-层粘连蛋白相互作用模式， 以快速形成但短暂的粘附接触为特征， 仅由α 6 β 4整联蛋白控制。这种“动态”的特性 粘附”将促进肿瘤细胞迁移，并表明， α 6 β 4介导的迁移不同于β 1整合素介导的迁移。 迁移最近，我们发现β 4胞质结构域 调节细胞周期蛋白激酶抑制剂p21的生长和表达， 表达野生型p53的细胞。α 6 β 4介导的机制， 调节动态粘附，迁移和生长将在 详细β 4胞质结构域中调节 这些过程将通过β 4- 缺陷型结肠癌细胞系转染的突变形式 β 4 cDNA。延时视频显微镜将用于比较 结肠癌细胞的迁移机制， α 6 β 4和α 2 β 1层粘连蛋白受体以及仅表达α 6 β 4和α 2 β 1层粘连蛋白受体的细胞 β 1整合素层粘连蛋白受体。同样， α 6 β 4和α 2 β 1的定位和功能 整联蛋白是由于它们与不同的细胞骨架蛋白 将使用光学和电子显微镜检查蛋白质。 这些 研究将包括使用β 4胞质结构域突变体来定义 空间定位和细胞骨架所需的特定序列 协会.的配体结合和信号传导功能， 与迁移和动态粘附相关的α 6 β 4整合素 可能涉及特定蛋白质与β 4 胞质结构域这些蛋白质将通过几种方法鉴定 包括捕捉它们与β 4特异性免疫系统的联系， 配合物 α 6 β 4整合素如何表达的重要问题 影响肿瘤行为将通过使用野生型和显性 调节结肠中α 6 β 4表达的负β 4构建体 癌细胞系，并通过评估这种调节对 它们的致瘤性、侵袭潜力和转移潜力。 最后，我们将评估α 6 β 4整合素及其受体的表达。 结构变异体以及E-钙粘蛋白和α-连环蛋白在肿瘤中 标本与病理学和分子生物学核心合作。

The long-term objectives of Project I are to understand the role of integrin receptors in mediating colorectal carcinoma adhesion, migration and growth on laminin, and to assess how such receptors influence tumor behavior. During the last granting period, the alpha6beta4 and alpha2beta1 integrins were identified as colon carcinoma laminin receptors. In addition, a novel mode of cell-laminin interaction was discovered that is characterized by rapidly formed but transient adhesive contacts mediated solely by the alpha6beta4 integrin. The properties of this "dynamic adhesion" would facilitate tumor cell migration and suggest that alpha6beta4-mediated migration is distinct from beta1-integrin-mediated migration. More recently, we have found that the beta4 cytoplasmic domain regulates the growth and expression of the cyclin kinase inhibitor p21 in cells that express wild type p53. The alpha6beta4-mediated mechanisms that regulate dynamic adhesion, migration and growth will be examined in detail. Specific sequences in the beta4 cytoplasmic domain that regulate these processes will be identified by functional analysis of beta4- deficient colon carcinoma cell lines transfected with mutant forms of the beta4 cDNA. Time-lapse video microscopy will be used to compare the mechanics of migration of colon carcinoma cells that express both alpha6beta4 and alpha2beta1 laminin receptors and cells that express only beta1 integrin laminin receptors. Also, the possibility that differences in the localization and function of the alpha6beta4 and alpha2beta1 integrins results from their association with distinct cytoskeletal proteins will be examined using light and electron microscopy. These studies will include the use of beta4 cytoplasmic domain mutants to define specific sequences required for spatial localization and cytoskeletal associations. The ligand binding and signaling functions of the alpha6beta4 integrin associated with migration and dynamic adhesion most likely involve the interaction of specific proteins with the beta4 cytoplasmic domain. Such proteins will be identified by several approaches that involve capturing their association with beta4-specific immune complexes. The important issue of how alpha6beta4 integrin expression affects tumor behavior will be addressed by using wild-type and dominant negative beta4 constructs to modulate alpha6beta4 expression in colon carcinoma cell lines and by assessing the effect of this modulation on their tumorigenicity, invasive potential, and metastatic potential. Finally, we will assess the expression of the alpha6beta4 integrin and its structural variants, as well as E-cadherin and alpha-catenin, in tumor specimens in collaboration with the Pathology and Molecular Biology Cores.