Effects of Cytochalasin B on Unstimulated and Adrenocorticotropin-Stimulated Adrenocortical Tumor Cells in Vitro*

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Abstract
We have employed cytochalasin B (CB) to investigate the possibility that the acute steroidogenic response of cultured adrenocortical tumor cells to ACTH may be mediated by microfilaments. The steroid production and ultrastructure of cultures incubated separately with ACTH and CB for 20 min to 2 h, or simultaneously with these agents, were recorded. At levels below 10 μ/ml, CB either stimulated a slight increase in basal steroid levels or had no effect on this output. Higher doses were progressively inhibitory. When CB and ACTH were added to cultures simultaneously, drug doses of 10–100 μg/ml produced progressively increasing inhibitions of the ACTH response. At 50–100 μg/ml CB, inhibition reached 80%. All inhibitions were reversible within 1–2 h after the removal of CB. Our electron microscopic studies indicate that although CB and ACTH caused a loss of stress fibers and produced cell rounding, the effects of each agent on microfilament-associated structures were unique. In ACTH-treated cells, there were increased numbers of microvilli and filopodia, and stress fiber remnants were surrounded by disarranged microfilaments; in contrast, in CB-treated cells, microvilli were sparse, and microfilaments were converted to felt-like masses. The stress fiber loss in response to CB was dose dependent, as were the rate and extent of cell shape changes. At low CB doses (1–3 μg/ml), bouquets of organelles protruded from cell surfaces, and a few cells containing cytoplasmic tunnels and dilated intracellular membranes were present. As CB doses were raised from 10 to 100 μg/ml, the number of tunneled cells increased substantially. When cultures were incubated with ACTH and CB simultaneously, as doses of the drug were increased, there were associated increases in the number of cells with CB-type features. In cultures treated with ACTH or CB, the loss of a stress fiber cage may enhance the uptake of lipoproteins, providing additional substrate for steroidogenesis. The rearrangement of microfilaments in ACTH-stimulated cells may promote organelle associations that facilitate the steroidogenic action of the peptide. The effects of low concentrations of CB have no apparent impact on the steroidogenic action of ACTH; however, the increased disruption of microfilaments at higher doses of CB, leading to the production of larger quantities of an amorphous felt, could prevent these organelle associations and thereby inhibit steroidogenic activity. The rapid reversibility of this inhibition strongly indicates that it is not due to a toxic effect of CB on the cells.