The extracellular matrix (ECM) has such a significant influence on normal growth and differentiation that it is likely to play a role in diseases involving abnormal growth and development, such as cancer. The long-term objective of this research is to define alterations in cell-extracellular matrix interactions which play a role in the development this disease. The ECM of cultured chicken embryo fibroblasts, infected with temperature-sensitive (ts) mutants of Rous Sarcoma virus, is modified during the early stages of transformation by the transient synthesis and deposition of a small protein (Mr approximately 21,000). The amphiphilic properties of this protein suggest that it could bind to, and perhaps link together, a number of different matrix components. The 21K protein does, in fact, bind to hyaluronic acid. Furthermore, it appears to be related to another 21K protein detected in the media of transforming cells.
The specific aims of this project are to: isolate, purify and characterize the 21K protein using chromatographic, electrophoretic and immunological techniques; to investigate its relationship to the media 21K protein; and, to determine its role in the transformation process. Monoclonal antibodies to the 21K protein will be used to aid in its characterization and to describe its distribution in the matrix by immunofluorescence microscopy. A peptide corresponding to the NH2- terminal amino acid sequence of the 21K protein will be synthesized for use as antigen in the acid sequence of the 21K protein will be synthesized for use as antigen in the production of polyclonal antibodies. These will facilitate bulk purification of the protein by affinity chromatography. Physical and chemical characterization and the potential interaction of the 21K protein with other matrix components will be established by electrophoresis, autoradiography and binding studies. The potential role of the 21K protein in expression of the transformed phenotype will be investigated by its ability to induce established transformation parameters in normal cells and ts virus-infected cells at the permissive temperature. In particular the contribution of the protein to altered adhesive properties and loss of growth control, exhibited by transforming cells, will be investigated.
