Retinoids (vitamin A and its derivatives) can profoundly influence vertebrate differentiation and development. Retinoids have also been implicated in the inhibition of carcinogenesis. Retinoic acid (RA) can induce differentiation of F9 mouse teratocarcinoma stem cells into endoderm, an epithelial cell type of the late mouse blastocyst. The intracellular mechanism of action of RA is not understood, although experimental evidence supports the role of the cellular retinoic acid binding protein (CRABP) in this mechanism. Our objective is to determine what role the CRABP plays in the differentiation of F9 cells in response to RA. Our first goal is to obtain a full length mouse CRABP cDNA. The CRABP cDNA identity will be verified by DNA sequencing. The CRABP cDNA will be inserted into either procaryotic or eucaryotic expression vectors to produce large quantities of the protein for biochemical analyses and anti-body production. We will then assay for the ability of this recombinant CRABP to bind to DNA by using DNA-cellulose chromatography, DNA:protein:CRABP antibody binding assays, or gel electrophoresis DNA-binding assays. The question of whether the CRABP (+RA) binds DNA has not been answered previously. Another area of investigation involves the question of how the level of CRABP expression influences differentiation. First, we want to either increase, or decrease CRABP gene (in the sense, or anti-sense orientation, respectively) into F9 wild type stem cells, to determine whether cell growth and RA induced differentiation are altered in these stably transfected cells. Second, we plan to attempt to restore the ability to differentiate in response to RA in a functionally CRABP negative F9 mutant cell line (RA-3-10) by transfection of the wild type CRABP gene. Finally, we plan to generate mutations in the CRABP gene in vitro; some of these mutated CRABPs will have alterations in RA binding. These mutated CRABP proteins will be characterized biochemically, and then some of the mutated CRABP genes, in expression vectors, will be transfected into the RA-3-10 CRABP- mutant cells to assay for alterations in in vivo function (eg. ability to differentiate) resulting from the various CRABP mutations. The proposed experiments should allow us to determine the function of the CRABP in F9 cells, and in so doing, provide significant information about the intracellular mechanism of action of RA.
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