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Response to ACTH and dibutyryl cyclic AMP by nucleated and enucleated adrenocortical tumor cells

University of British Columbia

The objective of this study was to determine conclusively whether or not the nucleus is required for the cyclic AMP-mediated steroidogenic response of adrenocortical cells to adrenocorticotropic hormone (ACTH). Enucleated cells are ideal for studying various aspects of cellular metabolism without the added complexity of superimposed transcriptional control. Cytochalasin B can trigger the enucleation of several types of cultured cells. Therefore, by modifying a procedure which has been described in the literature, cultured adrenocortical cells were enucleated with cytochalasin B, and the response of these enucleated cells to ACTH and dibutyryl cyclic AMP was investigated. The type of adrenocortical cells chosen for this study was the Y-1 functional mouse adrenocortical tumor cell line. However, it was found that the Y-1 cells could not be enucleated efficiently, thus, during the course of this study, a subline denoted Y-1-L cells was selected from the Y-1 cell line. These cells were later passaged in isogeneic animals. Tumor #2 cells were tumor cells arising from a LAF₁ mouse which had received a subcutaneous inoculum of Y-1-L cells. Similar to the Y-1 cells, Y-1-L cells and Tumor #2 cells did not require ACTH for growth or maintenance of a basal steroid output, but would respond to added ACTH or dibutyryl cyclic AMP by a change in morphology and by an increase in steroidogenesis. In contrast to the Y-1 cells, Y-1-L cells and Tumor #2 cells could be efficiently enucleated by the enucleation procedures developed and used. The morphology of Y-1-L cells and Tumor #2 cells in control medium, in medium containing ACTH, and in medium containing dibutyryl cyclic AMP was characterized by light and electron microscopy, and the steroid outputs of these cells under various incubation conditions were characterized by standard biochemical methods. Enucleated Y-1-L cells and Tumor #2 cells were viable as shown by dye exclusion for a minimum of 60 hours, and synthesized proteins as indicated by ³H-leucine incorporation into acid-insoluble material for at least 48 hours. Enucleated adrenocortical cells responded to either ACTH (10 mU/ml) or dibutyryl cyclic AMP (1 mM) by a change from flat to rounded cell shape, and by increased steroidogenesis as determined by spectrofluorometric assay of the culture medium. The steroidogenic response of enucleated cells during the first three hours after enucleation was comparable in magnitude to that of the nucleated cells, and persisted in diminishing degrees for an additional 9 hours. On the other hand, the morphologic change can be induced by ACTH even at 33 hours following enucleation. The results of this study show that the nucleus is not required for the expression of the acute effects of ACTH, and that the cytoplasmic components necessary for cell "rounding" and steroidogenesis are stable for at least 36 and 12 hours respectively.

Text

2010-01-28

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http://hdl.handle.net/2429/19254

Zoology

University of British Columbia

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