This application is for continuation of a project now in its eleventh year. Our long term goals are to elucidate cellular and molecular mechanisms by which environmental signals regulate the differentiation and function of adrenal medullary cells. In order to understand regulatory derangements which occur during the development and progression of adrenal medullary tumors. During the previous funding period. we found that sympathoadrenal precursor cells undergo neoplastic transformation in vitro after combined transfection with the v-myc and v-raf oncogenes to generate cell lines which appear to be poorly differentiated neuroblastomas. This finding is of considerable interest because naturally occurring neuroblastomas are infrequent in rats, and because myc is frequently overexpressed in human neuroblastomas. Raf is an important downstream component of signal transduction pathways in normal cells, and our findings suggest that oncogenes or other factors which affect signal transduction may cooperate with myc to generate or modify the phenotype of adrenal tumors of neuronal lineage. We now propose to extend these studies with four Specific Aims: (1) To establish and characterize adrenal medullary cell lines derived from fetal, neonatal and adult rats expressing v-myc alone, v-raf alone, or v-myc plus v-raf, in order to determine the effects of these oncogenes on the expression of neuronal and chromaffin cell traits, and to determine whether the age of sympathoadrenal cells at the time of transfection limits the ability of cell lines to be generated or influences the traits expressed in vitro or in vivo, (2) To determine whether cell lines expressing v-myc, v-raf, or v-myc plus v-raf give rise to neuroblastomas, pheochromocytomas or compound tumors when injected into fetal, neonatal and adult rats, and to determine whether the age of animals into which cell lines are injected influences tumor formation, patterns of metastasis or expression of neuronal and chromaffin cell traits, (3) To evaluate the roles of v-myc and v-raf in establishing sympathoadrenal cell lines of differing phenotypes and to determine whether c-fos induction is involved in the mechanisms underlying the v-raf phenotypes. and (4) To determine whether lines of human chromaffin cells can be obtained by Introduction of v-myc, v-raf or v-myc plus v-raf in vitro. The proposed studies may provide important insights into the clinical characteristics of neuroblastomas and other adrenal medullary tumors by directly comparing the contributions of cell factors versus host factors to their development and behavior in vivo and in vitro. They might also provide new understanding of the contributions of basic mechanisms which regulate cell lineage commitment In the adrenal medulla to the pathobiology of adrenal neoplasia.
