CD44/VARIANT-CYTOSKELETON IN ADHESION AND BREAST CANCERS

CD44s(CD44的标准型)是一种跨膜糖蛋白，其 外部结构域通过结合表现出外部基质黏附特性 透明质酸(HA)和胶原。CD44s的胞浆结构域 通过直接与锚定蛋白结合而与细胞骨架相互作用。在这 提议，我们计划检验CD44之间相互作用的假设 而细胞骨架蛋白，Ankyrin，是调节 CD44s细胞表面表达及其黏附功能最近，一位 肿瘤细胞和组织的数量，包括乳腺癌，已经被 表达CD44变异体(CD44v)亚型。因此，我们也 建议分析几种CD44变异体(CD44v)的表达 专用于转移性人类乳腺癌。 在提案的第一部分，我们建议使用各种 生化、免疫学和分子生物学技术以阐明 CD44s与细胞骨架之间的结构联系。我们将聘用 构建CD44s缺失和定点突变的体外诱变技术 用于确定CD44s功能区的定向突变体 锚定蛋白结合所需。这些突变的多肽将被表达 在CD44s阴性的T淋巴瘤细胞及其结构上的变化 与它们的表面表达和粘附性相关。在 提案的第二部分，我们计划确定CD44变异体(CD44v) 转移性乳腺癌组织中S异构体的RT-PCR检测 聚合酶链式反应、DNA序列分析和原位杂交。我们还将检查 CD44v与细胞骨架的相互作用、黏附功能及其后续研究 CD44v基因转染人乳腺上皮细胞的转移行为 CDNA.最后，我们计划制造多克隆和单克隆抗体 对抗含有不同CD44变异外显子(独一无二)的融合蛋白 到转移性乳腺癌)以便建立有用的转移性 乳腺癌的标志物。我们相信这些提议的结果 实验不仅将使我们更好地理解 细胞骨架调控CD44s和CD44v的黏附功能 将提供有关CD44表位变化的有用的新信息 (抗原决定簇)可能与乳房相关的表达 肿瘤转移，预后差。抗体的可得性 抗CD44v(转移性乳腺癌特有的)应该被证明是 非常有用的试剂，用于监测进展和/或预测 转移性乳腺癌的复发。

CD44s (standard form of CD44) is a transmembrane glycoprotein whose external domain displays external matrix adhesion properties by binding both hyaluronic acid (HA) and collagen. The cytoplasmic domain of CD44s interacts with the cytoskeleton by binding directly to ankyrin. In this proposal, we plan to test the hypothesis that the interaction between CD44s and the cytoskeletal protein, ankyrin, is required for the modulation of CD44s cell surface expression and its adhesion function. Recently, a number of tumor cells and tissues,k including breast carcinomas, have been shown to express CD44 variant (CD44v) isoforms. Therefore, we also propose to analyze athe expression of several CD44 variant (CD44v) isoforms specific for metastatic human breast cancers. In the first part of the proposal, we propose to use a variety of biochemical, immunological and molecular biological techniques to elucidate the structural association between CD44s and cytoskeleton. We will employ in vitro mutagenesis techniques to construct CD44s deletion and site- directed mutants in order to determine the CD44s functional domains required for ankyrin binding. These mutant polypeptides will be expressed in CD44s-negative T-lymphoma cells and their structural changes will be correlated with their surface expression and adhesion properties. In the second part of athe proposal, we plan to identify the CD44 variant (CD44v) isoform(s) associated with metastatic breast carcinoma tissues using RT- PCR, DNA sequence analyses and in situ hybridization. We will also examine the CD44v-cytoskeleton interactions, adhesion functions and the subsequent metastatic behavior of human breast epithelial cells transfected with CD44v cDNA. Finally, we plan to generate polyclonal and monoclonal antibodies against "fusion" proteins containing different CD44 variant exons (unique to metastatic breast carcinomas) in order to establish a useful metastatic marker for breast cancer. We believe the results of these proposed experiments will not only provide a better understanding of the role of the cytoskeleton in regulating adhesion functions of CD44s an CD44v, but also will provide useful, new information regarding the changes in CD44 epitope (antigenic-determinant) expression what may be associated with breast cancer metastasis and poor prognosis. The availability of antibodies against CD44v (unique for metastatic breast carcinomas) should prove to be very useful reagents for monitoring the progression and/or predicting the recurrence of metastatic breast carcinomas.