CD44/VARIANT-CYTOSKELETON IN ADHESION AND BREAST CANCERS

CD 44 s（CD 44的标准形式）是一种跨膜糖蛋白， 外部域通过结合显示外部基质粘附特性 透明质酸（HA）和胶原蛋白。 CD 44 s的胞浆区 通过直接结合锚蛋白与细胞骨架相互作用。 在这 我们计划验证CD 44之间的相互作用的假设， 和细胞骨架蛋白，锚蛋白，是调节 CD 44 s细胞表面表达及其粘附功能 近日一 许多肿瘤细胞和组织，包括乳腺癌， 显示表达CD 44变体（CD 44 v）同种型。 所以我们也 建议分析几种CD 44变体（CD 44 v）亚型的表达 对转移性人类乳腺癌特异。 在提案的第一部分，我们建议使用各种 生物化学、免疫学和分子生物学技术来阐明 CD 44和细胞骨架之间的结构关联。 我们会委聘 体外诱变技术构建CD 44 s缺失和位点- 定向突变体，以确定CD 44 s功能结构域 锚蛋白结合所必需的。 这些突变多肽将被表达 CD 44 s阴性T淋巴瘤细胞及其结构变化将 与它们的表面表达和粘附性质相关。 在 第二部分，我们计划识别CD 44变体（CD 44 v） 与转移性乳腺癌组织相关的同种型 PCR、DNA序列分析和原位杂交。 我们亦会研究 CD 44 v-细胞骨架相互作用、粘附功能以及随后的 转染CD 44 v的人乳腺上皮细胞的转移行为 cDNA。 最后，我们计划产生多克隆和单克隆抗体 针对含有不同CD 44变体外显子的“融合”蛋白（独特的 转移性乳腺癌）以建立有用的转移性 乳腺癌的标志物 我们认为这些建议的结果 实验不仅可以更好地了解 细胞骨架在调节CD 44 s和CD 44 v粘附功能中的作用， 将提供关于CD 44表位变化的有用的新信息， （抗原决定簇）表达可能与乳腺癌相关 癌转移和预后差。 抗体的可用性 抗CD 44 v（转移性乳腺癌特有）应被证明是 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.