cytochalasin-b and Adrenal-Cortex-Neoplasms

In addition to the well-characterized surface gap junctions expressed at contact sites between cells, annular gap junction profiles have been localized within the cytoplasm of some cell populations. To study and characterize these annular profiles, gap junction protein type was demonstrated with Western blot and immunocytochemistry. The distribution of annular gap junctions and the relationships to cytoskeletal elements were demonstrated with immunocytochemical, transmission electron microscopic, or image analysis with confocal microscopy techniques. SW-13 adrenal cortical tumor cells expressed alpha1 gap junctions at areas of cell to cell contact. In addition, alpha1 gap junction annular profiles were seen within the cytoplasm. Actin and myosin II were found closely associated with these annular gap junctions, while no physical association between tubulin- or vimentin-containing fibers and gap junction protein could be established. Disruption of microfilaments with cytochalasin B treatment (10 microg/ml, 1 h) resulted in a decrease in the average number and an increase in the average size of annular gap junctions compared to control populations. The results are consistent with a role for cytoskeletal elements containing actin and myosin II in annular gap junction turnover.

Topics: Actin Cytoskeleton; Actins; Adrenal Cortex Neoplasms; Cell Communication; Connexins; Cytochalasin B; Gap Junctions; Humans; Myosins; RNA, Messenger; Tumor Cells, Cultured; Vimentin

Human adrenal cortical tumor cells (SW-13) grow into a typical epithelial cell monolayer when seeded onto culture dishes. The cells of the SW-13 population monolayer appear flattened with few conspicuous surface features. The cells are attached to one another at their lateral borders and are arranged in a cobblestone-like manner. Following Triton X-100 extraction, the distribution of the cytoskeletal elements was observed with scanning electron microscopic techniques to correspond to the shape of the non-extracted cell. Changes in the distribution and morphology of projections on the cell surface as well as changes in cell shape were revealed after treatment of the cultures with compounds which bring about microtubular and microfilament disruption. Following 60 minute treatment of the cell population with cytochalasin B (10 micrograms/ml), 90% of the cells became round while remaining attached to neighboring cells and to the substrate by slender cell processes and filopodia. Some bleeding could be seen on the cell surfaces of cytochalasin B treated cultures and an increase in the number of microvilli was evident. When the cytoskeletal elements were observed with scanning electron microscopic techniques after Triton X-100 extraction, the amount of peripheral cytoskeletal elements was decreased and only slender projections of the microfilaments and microtubules were evident. Colchicine (0.06 micrograms/ml) treatment of the SW-13 adrenal cell population resulted in the appearance of surface blebs within 10 minutes of the initiation of treatment. The changes in surface projections are discussed in relationship to the loss of microtubules and microfilaments from the cytoplasm of the cell.

Topics: Adrenal Cortex Neoplasms; Cell Line; Cell Membrane; Colchicine; Cytochalasin B; Cytoskeleton; Humans; Microscopy, Electron, Scanning

ACTH increases the basal steroidogenic activity of cultured adrenocortical tumor cells, whereas moderate-high doses of cytochalasin B (CB) inhibit both basal and ACTH-induced steroidogenesis. Previous ultrastructural studies have revealed that ACTH rearranges microfilaments in these adrenal cells, whereas CB causes microfilaments to aggregate into felt-like masses. It has been postulated that the ACTH effects may facilitate organelle motility and increase organelle interactions that are required for steroid biosynthesis, and that the CB-created "foci" may impede or prevent the organelle meetings. To shed light on these possibilities, we have employed 16 mm cinemicrography of unstimulated adrenal tumor cells and cells incubated for 1-2 h with ACTH (10 mU/ml), or low (10 micrograms/ml), or high (50 micrograms/ml) doses of CB. ACTH caused initial increases in membrane ruffling and a "flurry" of particle (organelle) activity above that seen in unstimulated cells. The stimulated cells then retracted from each other and began their characteristic "rounding up" in response to the hormone. Particles appeared to move towards the nucleus, and in fully-rounded cells were extremely congested. Steroid production rose several fold above basal levels. CB10 produced slight-marked cell convexities, nearly stopped particle motility and inhibited steroid production moderately. CB50 produced an asymmetrical, spidery cell form, stopped membrane ruffling and particle motility and abolished steroidogenesis. After a washout of CB50, particle motility resumed nearly immediately. Our CB data indicate that associations between particles, presumably between mitochondria and various sources of cholesterol, are prerequisite for basal steroidogenesis in the adrenal tumor cells. In ACTH-stimulated cells, increases in steroid output correspond with increased opportunities for particle associations. These opportunities appear to arise directly or indirectly from ACTH effects on microfilaments. The responses of microfilaments to the hormone may be particularly intense in tumorous forms. By these means, the cells may express their differentiated function, although their cytoplasm has a distinctly unspecialized appearance.

Topics: Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Cells, Cultured; Cytochalasin B; Male; Mice; Motion Pictures; Steroids

Under lowered Ca2+ content and in the post-treatment state of low Ca2+, we studied the cellular structure and functioning in mouse adrenocortical tumor cells, Y-1. These cells had been maintained in Ham F12 medium containing 10% fetal calf serum. The Ca2+ present in this complete medium was 0.39 mM. Under a slightly lowered Ca2+ (0.29 mM) produced by EGTA, the cells had many blebs on their surfaces and specific functional activity decreased in steroidogenesis as did ACTH reactivity. In the post-treatment state of a low Ca2+, the cellular surface was covered with many short microvilli and there was greater cellular activity than in the control cells. When the Ca2+ concentration was below 0.17 mM, the cellular structure and functioning were disturbed, and there was no recovery even at the physiological Ca2+ after the removal of EGTA.

Topics: Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Calcium; Cell Line; Cell Membrane; Cytochalasin B; Egtazic Acid; Mice; Microscopy, Electron, Scanning; Receptors, Cell Surface; Receptors, Corticotropin; Time Factors

We have demonstrated previously that the steroidogenic activity of ACTH on cultured adrenal tumor cells is associated with cell rounding and a rearrangement of microfilaments. Cytochalasin B (CB) also induces cell rounding, but changes the conformation of microfilaments and severely inhibits steroidogenesis. ACTH and CB may have different modes of action on the contractile machinery which are related to their opposing actions on steroidogenesis. To investigate this possibility further, we have examined the reversibility of the morphological and functional effects of these agents. Cultures were incubated for 1 hr, with and without ACTH (10 microU/ml of media), or with CB (50 micrograms/ml), or with both agents simultaneously. After a media wash, the cultures were incubated for 1 hr, with and without ACTH. The steroid production of the cells during pre- and post-washout incubations was determined, and some cultures were fixed for electron microscopy at the end of both incubation periods. The three- to ten-fold increases in steroidogenic activity of ACTH-stimulated cells declined during recovery incubations, but remained well above basal values. These cells nearly reflattened and began to regain stress fibers which had been 'pulled apart'. The 'washed out' ACTH-stimulated cells were often refractory to restimulation. Cells recovering from CB also reflattened. Masses of filamentous felt induced by the drug disappeared from the cytoplasm, lost microvilli reappeared and stress fibers reformed. The 20-50% inhibition of basal steroidogenesis by CB was completely reversed. When ex-CB-treated cells were incubated with ACTH, their morphology and steroid production were typical of acutely stimulated cells. The recovery behavior of cells incubated with ACTH and CB simultaneously reflected the observation that there were cell-specific responses to one agent or the other during initial incubations. The persistence of heightened steroidogenic activity following a washout of ACTH and the rapid reversal of the effects of CB strongly support the concept that regulated actomyosin interactions are an integral part of the steroidogenic process.

Topics: Actomyosin; Adrenal Cortex Hormones; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Cell Line; Cytochalasin B; Cytoskeleton; In Vitro Techniques

Topics: Adrenal Cortex Hormones; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Cell Line; Cell Membrane; Cytochalasin B; Cytoplasm; Cytoskeleton; Dose-Response Relationship, Drug; Male; Mice; Microscopy, Electron; Microscopy, Electron, Scanning; Microvilli; Neoplasms, Experimental

Y-1-L cells, a subline of the Y-1 functional murine adrenocortical tumor cell line, were enucleated with cytochalasin B and the response of these enucleated cells to ACTH and dibutyryl cyclic AMP (dbcAMP) was examined. Enucleated Y-1-L cells maintained a basal steroid output for at least 24 h and responded to either ACTH (10 mU/ml) or dbcAMP (1 mM) by a change from flat to rounded cell shape, and by increased steroidogenesis. The steroidogenic response of enucleated cells during the first 3 h after enucleation was highly significant and, though it decreased rapidly thereafter, it persisted to a limited degree for up to 12 h. On the other hand, the morphologic change could be induced even at 33 h after enucleation. The results of this study show that the nucleus is not required for the expression of the acute effects of ACTH or dbcAMP and that the cytoplasmic components necessary for cell 'rounding' and a steroidogenic response are stable for 36 h and 12 h, respectively.

Topics: Adrenal Cortex Neoplasms; Adrenal Gland Neoplasms; Adrenocorticotropic Hormone; Bucladesine; Cell Line; Culture Media; Cytochalasin B; Cytoplasm; Leucine; Neoplasms, Experimental; Pregnenolone; Protein Biosynthesis; Steroids; Time Factors