Retinoic acid, a teratogenic morphogen metabolite of vitamin A (retinol), influences a multiplicity of biological processes including the growth and differentiaton programs of a diverse array of cell-types. Empirically, the focus of the present research proposal will be to gain a deeper understanding of the molecular genetic actions and role (s) of retinoic acid in the processes that regulate the growth and differentiation of neuroblastoma cells. Investigations will initially focus on a developmentally-regulated, retinoic acid responsive gene, known to be highly expressed in rat and human neuroblastoma cells, namely, thymosin B-10 (B10). Specific cDNA probes (encoding both the rat and human thymosin (B10), will be used to isolate clones from rat and human genomic libraries. Promoter/enhancer elements will be identified and chimeric thymosin B10-CAT constructs used in cell transfection expression experiments to characterise the nucleotide sequences that confer retinoid sensitivity upon the thymosin B10 gene. These experiments will furnish information regarding DNA sequences that may interact with the occupied retinoic acid receptor (RAR). Additional investigations will involve the differential screening of cDNA libraries constructed from neuroblastoma cells cultured in the presence or absence of retinoic acid so as to facilitate identification of other as of yet unidentified genes sensitive to this morphogen and whose expression profile is altered upon acquisition of a differentiated cell state. In order to gain more insights into the molecular mechanisms of action of retinoic acid, specific RAR cDNA clones will be used to investigate the regulation of the retinoic acid receptor gene during the process of cell growth and differentiation using rat and human neuroblastoma cells. Collectively, these studies will lead to a better understanding of the molecular mechanisms of action of retinoids at the level of the gene in normal and neoplastic cell growth.
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