INTEGRIN DISTRIBUTION AND FUNCTION IN TUMOR CELLS

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Integrins are a complex family of cell surface receptors involved in both cell-matrix and cell-cell adhesion. Most of the known integrins recognize extracellular matrix ligands, including fibronectin, laminin, collagens, vitronectin, and tenascin. Some integrins mediate cell-cell interactions by binding to other membrane proteins, like I-CAM. Strong initial evidence suggests that along with other with other types of cell adhesion receptors, many members of the integrin family contribute to tumor metastasis. Integrins were discovered only a few years ago,and since then, about 20 members of this major gene family have been described. The expression of most of these is developmentally regulated, and in some cases restricted to a specific cell type. It is very likely that many additional numbers of the family are yet to be discovered. Improved understanding of the structure and function of tumor cell integrins might lead to the design of novel diagnostic and/or therapeutic techniques in the fight against cancer. The long-term objective of the proposed research is to analyze the role of integrins in tumor metastasis. As a first step, the distribution of integrins in tumor cells of different metastatic potential will be studied. A novel technique, homology-based PCR amplification, will be used to identify and clone tumor cell integrins. This technique makes it possible to clone all the integrins expressed in a given cell type, including previously unidentified members of the family. Short stretches of invariant amino acid sequence shared by all known members of the family are used as primers to initiate PCR amplification of both known and novel integrin mRNA sequences. The resulting mixture of fragments corresponding to different integrins is cloned into a plasmid vector, and individual clones are characterized by DNA sequencing. Cloned cDNA fragments will be used as probes to analyze the expression of integrins in cultured tumor cells of varying metastatic potential, and in sections of tumor tissues. Integrin cDNA fragments will also be expressed in bacteria as fusion proteins, which will be injected into rabbits to raise anti-integrin antibodies of predetermined specificity. These antibodies will be important tools to analyze subunit association, processing and cell surface expression of the respective antigens, and to study integrin distribution in normal and tumor tissues. The functional properties of tumor cell integrins will be analyzed using antibodies or antisense RNA techniques in a variety of cell adhesion and invasion assays. These studies will provide the basis for a more detailed functional analysis by expression of cDNAs in heterologous cells.