A substantial body of correlative data has indicated that the serine protease, plasminiogen activator (PA) is highly-regulated in normal cells, expressed at elevated levels in malignant cells and catalytically involved in specific cell behavior patterns. In order to understand the cellular and genetic mechanisms involved in PA regulation and provide direct experimental evidence for its catalytic role in normal and malignant cell behavior, cell function will be linked directly to structural aspects of PA and PA- associated proteins and to PA gene expression. Using the well-defined primary culture system of normal chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV), we propose to examine the cellular regulation of PA in terms of its catalytic activity, its structure, its genomic regulation and the involvement of PA-related protein. The procurement of molecular, immunological and nucleotide probes will form the initial experimental approaches. A newly-acquired monoclonal antibody, specific for avian PA, will be used to purify microgram to milligram quantities of PA. In addition to allowing for PA structural studies, the purified PA also will be employed to produce a monospecific polyclonal antibody to PA. The latter probe will be used to directly monitor PA production (PA zymogen and active PA) in normal and transformed cells and PA expression in vectors carrying RSVCEF cDNA for the eventual isolation of a PA cDNA. Once acquired these three probes (PA, anti-PA IgG and PA cDNA) will be used to examine the cellular regulation and catalytic manifestation of PA. A coordinate search for a specific PA receptor on the surface of normal and transformed cells also will be carried out as this molecule could have a profound influence on PA expression at close cell-cell and cell-matrix contacts. The cellular balance of zymogen PA, active PA, and PA receptors will be quantitated and related directly to transformed cell behavior through the use of specific antibodies, the binding of purified PA to PA-deficient cells, and the selective employment of the conditional, temperature-sensitive mutant of RSV. The genomic expression of PA will be examined under a variety of culture conditions using the cDNA probe and finally PA genomic DNA will be isolated and its structure related to any cell physiological events and factors that affect PA expression.
