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 pathobiology, and to develop of more rational approaches to treatment. HNP production is a persistent characteristic of most small-cell tumors, and appears to result from the expression of an abnormal vasopressin-mRNA. It gives rise to Neurophysin-Related cell-Surface Antigen (NRSA) that can be targeted with antibodies.
Specific Aims are directed towards (i) determining the base sequence for tumor vasopressin-mRNA, and evaluating its incidence in small-cell tumors; (ii) characterizing the multiple forms of HNPs produced by small-cell tumors, and studying the dynamics of their production; (iii) assessing the relationships between intragranular versus extragranular post-translational processing and the formation of NRSA, and; (iv) evaluating the binding of different antibodies to tumor cell surfaces and relating this to the surface density and composition of NRSA present on cells. Our approach to acquiring a knowledge of the sequence for tumor vasopressin-mRNA involves performing reverse transcription on poly A+ RNA from mouse tumor xeonografts, amplifying specific cDNAs by PCR, cloning the products, and then subjecting them to nucleic acid sequencing using the dideoxy chain-termination procedure. The screening of tumors for the presence of vasopressin-mRNA will employ sequences complimentary to unique regions of tumor vasopressin-mRNA. A knowledge of the mRNA sequence is expected to lead to the rapid identification of HNP forms when this information is coupled with end-group analysis of these proteins. The sub-cellular localization of HNPs will be investigated through the application of immunogold electron microscopy and of biochemical methods for characterizing the different forms of HNPs in sub-fractions from cells. Cytoflourographic and radiometric analyses will be employed to quantitate the binding of antibodies to cultured SCCL cells that have undergone treatments with different drugs. Short term goals are focused on identifying the key elements involved in tumor generation of HNPs, and revealing those aspects of protein biosynthesis in SCCL cells that lead to the formation of NRSA. The information obtained is expected to result in more effective chemotherapeutic and immunotherapeutic interventions.
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