Our laboratory has made the initial observations suggesting that endogenous neuropeptides are related to neuro-oncogenicity, and that alterations of endogenous opioid/opioid receptor interactions influence the course of neoplasia (Science 221:671, 1983). During the first 2 years of this grant, we have carefully defined the role of endogenous opioid systems in neural cancer. A major discovery was that endogenous opioids and opioid receptors are present in all types of human and animal tumors, neural and non-neural in origin. Our hypothesis is that endogenous opioids serve to regulate neoplasia through interaction with opioid receptors associated with tumor cells; this may reflect an autocrine mechanism. In this grant proposal, we continue to explore our thesis with a rigorously-defined murine neuroblastoma model. Prototypic opioids related to growth will be identified through drug displacement studies, and structure-function experiments in tissue culture. Binding assays will be used to establish opioid/receptor interaction, and will include assessment of saturability, binding affinity and capacity. Information as to precursors of growth-related opioids will be gained by immunocytochemical procedures. In vitro investigations will be correlated with physiological/pharmacological responsiveness to opioids in mice with transplanted neuroblastoma. The cellular mechanism of neuropeptide regulation will be studied biochemically and structurally, by opioid transport experiments using opioids conjugated to gold and examined by electron microscopy, and with flow cytometry. Finally, the opioid receptor associated with cell growth will be isolated and identified. Receptor characteristics including size and subunit composition, peptide maps, and binding function as studied by reconstitution experiments, will be explored. Receptors will be quantitated by immunodot assays, and receptor distribution assessed by immunocytochemistry and immunoelectron microscopy. Our research efforts will contribute to comprehending the etiology and pathogenesis of neural neoplasia, and will provide strategies for prevention and therapeutic intervention of neuro-oncogenesis. This research is part of a long-range program in cellular and molecular neurobiology which seeks to understand the fundamental principles underlying normal growth and abnormal growth of the nervous system.
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