The long-term objective of this project is to understand the nature and significance of human neurophysin (HNP) production by small-cell carcinoma (SCCL), and to use this understanding as an insight into tumor cell pathobiology, and develop rational approaches to treatment. HNP production is a persistent characteristic of most SCCL tumors and leads to the formation of a number of abnormal proteins, to HNP-related surface markers (NPRM), and to peptide hormones that my be involved in cell autocrine function.
Specific Aims are directed towards (i) evaluating SCCL biosynthesis of HNPs at the cellular level. This involves characteristics normal and abnormal products, and identifying factors controlling their formation and secretion; (ii) determining the relative importance of extragranular and intragranular processing, and examining the integrity of tumor secretory granules with respect to their content of enzymes (required for post-translational modification of precursors) and their membrane composition; (iii) evaluating the binding of polyclonal and monoclonal antibodies to NPRM on SCCL cells in vitro, and on tumor xenografts in athymic mice, and establishing methods for maximizing this binding; and (iv) assessing the influence that vasopressin and other secreted products of HNP biosynthesis have on tumor cell growth and division. Our examination of HNP biosynthesis will include pulse-chase studies with radiolabelled amino acids and fucose, performed on short-term and long-term cultures of HNP-producing SCCL cells. A highly refined HPLC procedure, affinity chromatography, micro-sequencing and radioimmunoassay will be employed to define and characterize synthesized products. Secretory granules from cultured cells and tumor specimens will be isolated by sucrose-gradient ultracentrifugation. The enzyme activities of these secretory granules will be evaluated using synthetic peptides and available bovine and rat neurophysin precursors. Cytoflurographic and radiometric analyses will be employed to quantitate the binding of antibodies (and their derivatives) to SCCL cells in vitro and in vivo. The mitogenic activities of peptides will be indexed by 3H-thymidine incorporation and colony formation in agar matrices. Short-term goals are focused on identifying key elements in tumor generation of HNP-related products and establishing methods to control formation of these products. The formation gained could possibly by applied to antibody-directed therapy, and the proposed studies should lead to more effective treatments for SCCL.
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