One of the histopathological differences between normal epithelia and invasive tumors of epithelial orgin (breast, colon, lung, prostrate, uterus, pancreas) is that the normal epithelial cells deposit a thin, continuous basal lamina at the connective tissue interface whereas invasive epithelial tumors exhibit irregularities, interruptions or a complete lack of this extracellular structure. Our long term goal is to test the hypothesis that the invasive cells are impaired in their ability to biosynthesize and deposit the basal lamina. The specific objectives of this proposal are (i) to compare malignant and nonmalignant human cells in culture in terms of the biosynthesis of the basal lamina components laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan, (ii) to compare the biosynthesis of these basal lamina components by a series of paired squamous cell lines derived from both the primary tumor and from a metastatic site in the same patient, and (iii) to isolate and biochemically characterize human laminin, employing as a source a high laminin-producing cell line (JAR) derived from choriocarcinoma. Classification of human tumors in terms of the basal lamina-like matrix they deposit may have potential for cancer diagnosis. The proposed studies will be carried out principally with cell and organ cultures of human tumors. The biosynthesis of the basal lamina components will be assessed by incubating the cell cultures with radioactive amino acid or sugar precursors and then isolating the radioactively labeled proteins or proteoglycans by selective extraction, immunoabsorption, affinity chromatography, gel filtration chromatography and ion exchange chromatography. The concentrations of basal lamina components in culture fluids and cell extracts will also be measured by enzyme-linked immunosorbent assay (ELISA) employing specific antibodies directed against each basal lamina component. The carbohydrate structure of human laminin will be determined by carbohydrate compositional analysis, glycosidase digestion, lectin affinity chromatography, and methylation analysis. The polypeptide portion of human laminin will be characterized by amino acid compositional analysis, peptide mapping, and N-terminal amino acid sequencing.
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