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.
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