The objective of this research is to characterize phenomenologically and mechanistically the complex steroid-mediated response of rat hepatoma tissue (HTC) cells to identify possible relationships to hepatic processes and to identify the factors modulating the glucocorticoid action. The overall goal is to assemble a comprehensive picture of hormonal regulation in one system that will serve as a valid and experimentally manipulatable tissue culture model for liver-specific events. Dexamethasone-mediated alterations in plasma membrane properties of HTC cells are assessed by a combination of immunoelectrophoretic analyses, and in situ labeling of externally exposed proteins, followed by two-dimensional electrophoretic separation and measurements of the uptake of metabolites. The effect on the secretion program is determined by metabolic labeling of secretory glycoproteins. To relate these effects to events in the liver, primary hepatocytes have been prepared from animals of different developmental stages and analyzed under culture conditions that maintain the phenotype at the time of isolation. To understand what factors in vivo or in tissue culture can modify the phenotypic pattern of dexamethasone action, HTC and liver cells have been tested in reconstituted culture systems or treated with possible effectors, such as interleukin-1, glucagon, or cAMP. It was possible to correlate some of the dexamethasone responses of HTC cells to specific events in the liver during acute phase reaction. At the molecular level, it was found that concentrations of several mRNAs were increased in HTC cells by glucocorticoids. Two mRNAs encoded for abundant secretory glycoproteins, one of which corresponds to gammal-acid glycoprotein (AGP), the major acute phase plasmaprotein of the rat liver. DNAs complementary to mRNAs of some dexamethasone-inducible proteins, including AGP, were cloned. These have been used as hybridization probes to measure kinetics and specificity of hormone induction in both HTC and liver cells. (A)
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