Colorectal carcinoma is the second prevalent cause of cancer death in the United States. Recurrence and/or metastasis account for almost all the clinical failures following successful resection of the primary tumors. The goal of the present proposal is to establish the molecular basis for major changes occurring at the surface of human colorectal carcinoma cells during progression and metastasis. We have found that a family of sulfomucins inversely, and a Mr900,000 sialomucin directly correlate with progression and metastasis. We have purified normal colon sulfomucins and Mr900,000 sialomucin from human colon carcinoma cells cultured from metastases and generated monoclonal antibodies (MAbs) with specificities for these molecules. We will use clinical specimens (normal mucosa and carcinoma tissues) and colon carcinoma cell lines to purify sulfomucins and sialomucins, which were previously defined by sulfate incorporation, cationic dye binding or lectin reactivity, using MAb-affinity chromatography. We will elucidate how the sulfate groups and underlying structures (penultimate carbohydrates and polypeptides) are involved in the antigenic epitope of anti- sulfomucin MAb 91.9H, and why the reactivity of this MAb with carcinoma mucins are lower than mucins in normal mucosa. Attempt will be made to isolate oligosaccharides recognized by MAb 91.9H and to determine their structures Differences among Mr900,000 sialomucins from cultured cells and sialomucins from colorectal carcinoma tissues will be examined, for their carbohydrate profiles, core polypeptide differences and immunochemical reactivities with various MAbs and lectins. Special attention will be paid on wheat germ agglutinin-binding carbohydrate chains (and peanut lectin binding after removal of sialic acid), because differential expression of sialomucins on metastatic cells were previously demonstrated by these methods. The questions of how sulfomucins and sialomucins (defined by Mr, MAb-reactivity and carbohydrate structures) are structurally related will be answered using these molecules purified from colonic mucosa and colon carcinoma tissues by: (a) determining their structural similarities and differences in carbohydrate and core polypeptide structures and (b) testing if these two types of mucins can be interconverted with respect to their immunochemical nature by chemical, enzymatic or biosynthetic modifications. Finally, differences in localization of these molecules in normal and carcinoma colon tissues will be studied by a combination of immunohistochemistry and enzymatic/chemical modifications of carbohydrate chains in situ. Subcellular localization of Mr900,000 sialomucin will also be studied with human colon carcinoma cells growing in cultures.
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