Fluorescent peptidyl aldehyde (C-terminal aldehyde) transition-state analog inhibitors of proteases will be synthesized to visualize and quantify phenotypic differences in protease expressions with specific oncogene transformations and other tumor cell phenotypic characteristics such as the potential for tumorigenesis and metastasis. Selective probes will give a single cell assay characterizing cathepsin B-like, plasminogen activator-like, and elastase-like activities associated with a cell. Other probes will visualize general trypsin or chymotrypsin-like activities. Protease expressions will be visualized by fluorescent microscopy and quantified by spectrofluorimetry. In addition, separation of cells based on their phenotypic protease expressions will be carried out in a fluorescence activated cell sorter and over cell affinity chromatography resins. Specific oncogene expressions to be characterized include: (i) Src infected field vole cells and revertant src infected vole cells in which the activity of the pp60-src protein is decreased; (ii) Chick embryo fibroblasts transformed by the temperature sensitive Schmidt-Ruppin virus src oncogene, in which system the changes in protease expression over the time course of transformation will be studied; (iii) Combination c-HA-ras, pSVv-myc, and plasmids encoding for the polyoma virus middle and large T antigens transfected into rat embryo fibroblasts and immortalized Rat-1 cells. In these systems differences in protease phenotypic expressions will be correlated with oncogene expressions in oncogene defined pre-malignant and transformed states. In related work, tissue cross sections of neoplasms will be treated with the transition-state analog fluorescent probes. The research will look for correlations in protease expressions with tumor cell invasiveness and metastatic potential. The technique will have a usefulness in the pathological determination of transformed cells and in the staging of neoplasms. This research will obtain direct visual and quantitative data on the relationship of protease phenotypic expressions to different oncogene expressed states. An ability to accurately determine the actual and potential phenotypic character of transformed cells will be useful for the diagnosis and treatment of cancer.
