We propose to examine and compare the biochemical and molecular basis of retinoid action in normal human prostate and in prostate tumors. Retinoic acid (RA), the most potent natural retinoid currently known binds to specific retinoic acid receptors (RARs) located in the cell's nucleus. Upon ligand binding, RARs turn into potent transcription factors that regulate the expression of specific target genes. There are six retinoid receptors in humans (known as alphaRAR, betaRAR, gammaRAR, alphaRXR, betaRXR, and gammaRXR). In addition to RA, tissues contain other retinoids including precursor molecules (e.g. retinol), physiologically active analogs of RA (e.g. didehydroretinoic acid) and a yet unknown retinoid """"""""X"""""""", the putative ligand of the RXRs. Many cells express cytosolic binding proteins (CRAPBs and CRPBs) to which various retinoids will selectively bind. Cells also contain a yet unknown number of enzymes critical for synthesis and metabolism of retinoids. To investigate the biochemistry and molecular biology of retinoid action in prostate we will, as a first step, explore the following issues. (1) We will examine by Northern blotting the expression of retinoid receptors in normal and tumor prostate tissue. The expression pattern of those receptors that are expressed will further be studied by in situ hybridization. This will reveal at cellular resolution the expression pattern of RARs and RXRs in normal and tumor tissues. (2) We will compare retinoid metabolism in normal and tumor prostate using previously established enzyme assays. In these assays subcellular fractionations of prostate tissue are incubated with radiolabeled retinoids. Metabolites formed are identified and quantified by high performance liquid chromatography (HPLC). These studies will reveal whether and how prostate tissue metabolizes retinoids. (3) We will identify endogenous retinoids using HPLC and gaschromatography/massspectroscopy. (4) We will examine by Northern blotting the expression of the retinoid binding proteins CRAPB and CRPB, in normal and tumor prostate tissue. Expression will be further characterized by in situ hybridization. Taken together, such studies will provide a solid basis to evaluate the significance of retinoids for prostate physiology and pathophysiology and if successful, will allow more efficient clinical testing of natural and synthetic retinoids for their therapeutic potential.
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