The objectives of this research are to elucidate the mechanism(s) of chronic (long term) endocrine, paracrine and autocrine regulation of steroidogenic enzyme synthesis and activity in gonad alpha tumor cells in monolayer culture. Specifically, the processes that regulate the steroid 17 alpha-hydroxylase/C-17,20- lyase (17 alpha-hydroxylase cytochrome P-450 P-450, 17 alpha) of rat Leydig tumors cells will (cAMP-independent) agents (e.g., transforming growth factors, EGF and estrogens) on the synthesis of cytochrome P-450 17 alpha and the level of translatable mRNA that encodes this enzyme in cultured rat Leydig tumor cells will be determined. The rate of synthesis of P-450 17 alpha will be estimated after its specific immunoprecipitation from cell extracts following radiolabeling of cells with (35S)methionine, and also form an in vitro translation system programmed with cellular RNA. The levels of total mRNA that encodes P-450 17 alpha after the various treatments will be quantified by Northern blotting using a radiolabeled nick-translated specific cDNA probe. After the elucidation of the regulatory (untranscribed) 5'-flanking sequence of the rat P-450 17 alpha gene, """"""""foot-printing"""""""" studies of this region of the gene will be conducted using nuclei isolated from tumor cells before and after cAMP treatment, to determine whether the altered expression of this gene involves a tissue-specific trans-acting factor that interacts with a cAMP-responsive element. Further delineation of regulatory sequences will be achieved with deletion analysis of regulatory gene sequence constructs with the bacterial chloramphenicol acetyl transferase reporter gene. Utilizing a polyclonal antibody directed against rat cytochrome b5' the role of cytochrome b5 to alter the activity and selectivity of 17 alpha-hydroxylase/C-17,20-lyase of Leydig tumor microsomes and the nature of factors that regulate the synthesis of cytochrome b5 in tumor cells will be evaluated. The rat Leydig tumor R2C cell line expresses steroid aromatase in addition to cholesterol side chain cleavage enzyme, but demonstrates low expression of steroid 17 alpha-hydroxylase/C-17,20-lyase. We will determine endocrine paracrine, autocrine and tumor/tissue specific factors that alter the levels of expression of 17 alpha-hydroxylase and aromatase in the R2C cell line. These findings will provide insight into the interdependent actions and mechanisms of endocrine paracrine, and autocrine factors to alter differentiated cell function.
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