The extracellular matrix (ECM) has such a significant influence on normal growth and differentiation that 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 of 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 synthesis and deposition of a small protein (Mr about 21,000). Sufficient amounts of the 21K protein have been purified to determine its function in the matrix and potential role in transformation. The protein promotes the detachment of transforming cells from the ECM and stimulates proliferation of quiescent cells. Amino acid sequence data and other properties indicate that the protein is related, but not identical, to tissue inhibitor of metalloproteinases (TIMP/erythroid potentiating activity (EPA). We have detected five metalloproteinase inhibitor activities (Mr about 20,000-26,000) in cultured chicken embryo fibroblasts; these are designated ChIMPs (Chicken Inhibitor of MetalloProteinases). ChIMP-3 is the 21K protein. This proposal is designed to analyze the structure of ChIMP-3 using biochemical, molecular and physical techniques and to test the hypothesis that the protein promotes cell detachment from the ECM during oncogenic transformation. Electrophoresis, chromatography and mass spectrometry will be used to define post-translational modifications. Polyclonal antibodies to ChIMP-3 will be used to aid in its characterization and to describe its distribution in the matrix by immunofluorescence microscopy. The potential interaction of ChIMP-3 with other matrix components will be established by electrophoresis, autoradiography and binding studies. The role of ChIMP-3 in expression of the transformed phenotype will be investigated by inhibition studies with antisense oligodeoxynucleotides. Site-directed mutagenesis of ChIMP-3 will be used to determine if the biological properties of this protein are mediated through its metalloproteinase inhibitory activity.
