AN ADRENAL TUMOR: CYTOCHROME P-450 AND STEROIDOGENESIS

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Mouse adrenal tumor cells (Y-1) in monolayer culture is being used to study questions related to their invasive properties, to the regulation of their steroidogenic activity, and to the relationship between these two features of the cells as follows: What is the function of the newly synthesized proteins found associated with mitochondria after addition of ACTH to Y-1 cells? What effect do alterations in plasma membrane viscosity and relocation of membrane receptors in an electric field have on the initial events associated with responses of the cells to ACTH and cholera toxin? To what extent can the enhancement of growth and steroidogenesis produced by injection of cells into LAF1 mice be duplicated by addition of pituitary extracts to the cells in culture? The relationship between the new protein(s) and transport of cholesterol to mitochondria is studied by incubating [3H]cholesterol oleate--low density lipoprotein with cells in which side-chain cleavage is blocked and measuring mitochondrial [3H]cholesterol. Membrane receptors are labeled with fluorescent ligands (ACTH, cholera toxin, and Concanavalin A) after exposure to an electrical field to estimate membrane viscosity. Cholesterol content of cell membranes is altered by incubation with lipid-free plasma or cholesterol-containing liposomes. Steroidogenesis and growth of cells in the presence of pituitary extracts are compared with the response to animal passage. Nonionic detergents are used to improve extraction of mitochondrial P-450 from Y-1 cells and normal adrenal cells. The role of myosin in the response to ACTH is studied by measuring the extent of phosphorylation of the myosin light chain when cells are incubated without and with ACTH. A cyclic AMP-dependent protein kinase has been found closely associated with the cytoskeleton of Y-1 cells. The enzyme promotes phosphorylation of seven proteins. Protein kinase C has also been found in Y-1 cells which show increased steroidogenesis when incubated with phorbol esters. (D)