Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP

The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.     

Cyclic AMPand cytochalasin B-induced arborization in cultured aortic smooth muscle cells: its cytopharmacological characterization

Summary The present study analyzed effects of dibutyryl cyclic AMP (DB-cAMP) and cytochalasin B (CB) on the morphology of cultured aortic smooth muscle cells (SMC) from rat using phase-contrast microscopy, scanning electron microscopy, and fluorescence staining of actin filaments by the NBD-phallacidin method. The exposure of SMC to each of these agents led to rapid, extensive, and reversible (within 1–2 h of drug withdrawal) changes in their morphology including cytoplasmic arborization (stellation). The latter was preceded by (i) marginal membrane ruffles (DBcAMP) and (ii) increased zeiotic activity (CB), which were visible within 20 min of the exposure, followed (30–90 min incubation) by a centripetal retraction of the cytoplasm and progressive development of complete or partial arborization. Further, the effects of substances interfering with the assembly-disassembly of microtubules (colchicine, taxol, lidocaine) on DB-cAMPand CB-induced arborization were studied. None of these agents antagonized CB-induced morphological changes. Colchicine, but not lumicolchicine, taxol, or lidocaine (in a short-term study) prevented DBcAMP-induced arborization. Taxol added to cell cultures for 24 h promoted DB-cAMP-induced arborization. Both DB-cAMP and CB resulted in the disintegration of actin filaments. The present data suggest that the arborization of cultured aortic SMC is a cytoskeleton-based process involving stabilization of microtubules and disintegration of actin filaments. Our study also suggests that the SMC arborization may represent an in vitro case of SMC stellation found in situ.     

Drug-induced alterations in morphology and level of cAMP in cultured human glioma cells

Human glioma cells (138 MG) in serum-free medium within 1–3 h obtained a stellate astrocyte-like morphology after exposure to adrenalin (0.1 mM) or isoproterenol (0.1 mM). The changes, which were preceded by an increase in the cAMP content of the cells, could be antagonized by sotalol. The induced morphological alterations reversed on prolonged incubation (24 h). Two phosphodiesterase inhibitors, papaverine (0.2 mM) and RO 20 1974 had similar effects. Prostaglandin e₁ caused the highest increase in the cAMP level, followed by morphological changes which persisted for at least 72 h. A positive correlation between the level of cAMP and the duration of the astrocyte-like morphology was found. The N⁶-substituted adenine derivatives, zeatin and dimethylaminopurine induced a persistent stellate morphology without affecting the cAMP content. These substances possibly act as cAMP agonists.     

Cyclic AMP induces morphological changes of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway

Cyclic AMP (cAMP) is a pleiotropic second messenger that regulates numerous cellular processes. In vascular smooth muscle cells (VSMCs), these include cell proliferation, migration, and contractility. Here we show that cAMP-elevating agents induce dramatic morphological changes in VSMCs, characterized by cell rounding and formation of long branching processes. The stellate morphology is associated with disassembly of actin stress fibers and lamellipodia, loss of focal adhesions, and the formation of small F-actin rings. Because of the importance of Rho family GTPases in regulating actin dynamics, we analyzed their individual roles in the cAMP phenotype. We found that pharmacological or genetic inhibition of Rac mimics cAMP effect in inducing a stellate morphology of VSMCs. Expression of activated Rac1 prevents forskolin-induced cAMP stellation, suggesting that cAMP affects cell morphology by inhibiting Rac function. Consistent with this, treatment with forskolin inhibits agonist-stimulated Rac activation in VSMCs. We further show that activated Rac1 containing the F37A effector loop substitution fails to rescue the cAMP phenotype. Our results suggest that cAMP modulates the morphology of VSMCs by inhibiting a Rac-dependent signaling pathway.     

A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor alpha in target tissues

An intrauterine pulse-stimulation with estradiol induced changes in the subcellular localization of estrogen receptor alpha in porcine endometrium, as detected with F(ab') fragments of various anti-receptor antibodies covalently linked to nanogold. The low-sterically hindered immunoreagents--recognizing different epitopes within the hormone binding domain--allowed for an efficient immunolabeling of estradiol receptor alpha, detecting it both in the cytoplasm and the nucleus of nonstimulated epithelium cells. In the cytoplasm, the receptor often seemed to be associated with actin filaments and the endoplasmatic reticulum. After the stimulation with estradiol, a predominantly nuclear localization and a labeling of nucleoli was observed. Our immunoelectron microscopy study demonstrates a localization of the receptor in cytoplasmic organelles that increased after the hormone pulse. These organelles exhibited the morphological properties of lysosomes and relocated to the perinuclear area. In analogous cytoplasmic organelles, the presence of cathepsin D was detected via indirect immunogold labeling, justifying their classification as lysosomes. Quantitative examinations revealed that not only the number of lysosomes in the proximity of the nucleus but also their immunostaining for estradiol receptor alpha increased significantly after the hormone pulse. Thus, estradiol induces both the rapid shift of receptor into the nucleus, a slower perinuclear accumulation of lysosomes and an increase of lysosomal ERalpha-immunoreactivity. These results suggest a role for lysosomes in the degradation of receptor shuttling out of the nucleus. This could serve as termination of the estradiol receptor alpha-dependent activation of target cells. This hypothesis is strengthened by the fact that the receptor content in uterine tissue declined drastically few hours after the hormone pulse.     

Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.     

Hormonally induced changes in the cytoskeleton of human thyroid cells in culture

Serially cultivated thyroid follicular cells are not active in hormone synthesis but retain a thyrotropin-responsive adenylate cyclase. The exposure of such cells to thyrotropin leads to an increase in the concentration of intracellular cAMP and a drastic change in morphology including a total cytoplasmic arborization. The present communication describes these changes at the cytoskeletal level using a cell line derived from a human functioning thyroid adenoma. Phase contrast microscopy showed that the cytoplasmic arborization was preceded by a total disappearance of stress fibers, visible within 20 min of exposure. Small marginal membrane ruffles could also be seen. These morphological changes could also be induced by the addition of dibutyryl cAMP. The action of both thyrotropin and dibutyryl cAMP was potentiated by theophylline. High voltage electron microscopy of whole mounted cells confirmed the loss of stress fibers (microfilament bundles). In addition, thyrotropin treatment led to an uneven redistribution of the cytoplasmic ground substance and to changes in the organization of the microtrabecular lattice. Stereo images demonstrated numerous minute surface ruffles. The thyrotropin-induced arborization was reversible even in the presence of thyrotropin. After 24 h of treatment, cells had flattened and then contained very straight and condensed microfilament bundles. The results thus demonstrate that thyrotropin induces a disintegration of microfilament bundles in human, partially dedifferentiated, follicular cells and that this effect to all appearances is caused by cAMP, the second messenger in thyrotropin action. The relation of this event in partially dedifferentiated cells to the effect of thyrotropin in the intact thyroid gland is unclear. The fact that several other cultured hormone-responsive cells round up or become arborized in conjunction with an increase in cAMP levels implies that cAMP may be a major factor in the disassembly of microfilament bundles in these cells.     

Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.     

Hepatic stellate cell activation in vitro: cell cycle arrest at G2/M and modification of cell motility

Hepatic fibrosis is a common response to chronic liver injury and is characterized by increased production of extracellular matrix components, whose major part is produced by hepatic stellate cells activated by inflammatory mediators to proliferate and migrate into the injured regions. GRX cells are a model of hepatic stellate cells characterized as myofibroblasts by morphological and biochemical criteria. We have recently shown that they respond to inflammatory mediators and cytokines present in the concanavalin A-activated spleen cell supernatant (SCS) by quantitative changes in the expression of intermediate filaments. The present study investigated the effects of SCS and TNF-alpha on the GRX cell proliferation and on the organization of the actin cytoskeleton. SCS and TNF-alpha diminished the culture cell density, with an increase of cell [(3)H]thymidine incorporation and of cellular protein content, indicating an arrest in the G2/M phase of the cell cycle, which was reversible 48 h after removal of SCS. This effect was abrogated by dibutiryl-cAMP. Actin cytoskeleton reorganization was observed after 24 h treatment, indicating increased cell motility. Our results suggest that inflammation-dependent activation of stellate cells occurs in ordered interaction and coordination of proinflammatory agents. The increase of cAMP levels activates the conversion of lipocytes into myofibroblasts and increases the number of cells that can participate in repair. Since cAMP retains cells in the G1 phase, cytokines of the TNF-alpha group are required for cell proliferation inducing the entry into the S phase. The progression through the G2/M checkpoint is mediated again by increased cAMP levels.     

ON THE ROLE OF MICROTUBULES IN MOVEMENT AND ALIGNMENT OF NUCLEI IN VIRUS-INDUCED SYNCYTIA

Infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes rapid and extensive cell fusion. Time-lapse cinematography shows that when cells fuse, their nuclei migrate straight to the center of the syncytium at rates of 1–2 µ/min. Nuclei are often arranged in long, tightly packed, parallel rows in syncytia derived from the fibroblastic BHK21-F cells. Polarization microscopy shows birefringent material between and parallel to these rows of nuclei, and electron microscopy shows bundles of cytoplasmic microtubules, ~250 A in diameter, and filaments, ~80 A in diameter, parallel to and between the rows of nuclei. Colchicine treatment causes disappearance of microtubules from BHK21-F cells and an apparent increase in the number of 80-A filaments. Although colchicine-treated, SV5-infected cells fuse, their nuclei do not migrate or form rows but remain randomly scattered through the syncytial cytoplasm. Incubation at 4°C does not disrupt microtubules in BHK21-F cells. Rows of nuclei have been isolated from SV5-induced syncytia, and the nuclei in them have been found to be intimately associated with microtubules but not with other cytoplasmic structures. These results suggest that microtubules demarcate cytoplasmic channels through which nuclei migrate and that they may also be involved in the mechanism of nuclear movement.     

Possible involvement of protein phosphorylation in the regulation of cytoplasmic organization and streaming in root hair cells as revealed by a protein phosphatase inhibitor, calyculin A

Summary The effects of a protein phosphatase inhibitor, calyculin A (CA), on cytoplasmic streaming and cytoplasmic organization were examined in root hair cells ofLimnobium stoloniferum. CA at concentrations higher than 50 nM inhibited cytoplasmic streaming and also induced remarkable morphological changes in the cytoplasm. The transvacuolar strands, in which actin filament bundles were oriented parallel to the long axis, disappeared and spherical cytoplasmic bodies emerged in the CA-treated cells. In these spherical bodies, actin filaments were present and the spherical bodies were connected to each other by thin strands of actin filaments. Upon CA removal, transvacuolar strands, in which actin filament bundles were aligned, and cytoplasmic streaming reappeared. A nonselective inhibitor for protein kinases, K-252a, delayed the inhibitory effect of CA on cytoplasmic streaming and suppressed the CA-induced formation of the spherical bodies. From these results, it is suggested that phosphatases sensitive to CA regulate cytoplasmic streaming and are involved in the organization of the cytoplasm in root hair cells.Abbreviations APW artificial pond water - CA calyculin A     

The relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis

Summary The folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells.Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted.The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.     

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.     

The terminal web. A reevaluation of its structure and function

The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.     

Comparison of morphological effects of PGE2 and TGFβ on osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures

RANKL, in the presence of M-CSF, induces the development and fusion of TRAP+ osteoclasts in mouse bone marrow cultures at 3–5 days. Early during culture (day 3), most cells are small (up to six nuclei). At lower cell densities, these osteoclasts exhibit a rounded morphology with cytoplasm extending around the cells but, at higher densities, this changes to a stellate morphology with the cytoplasm being retracted around the nuclei with numerous localised cytoplasmic extensions. Under optimal conditions, osteoclast fusion results in conglomerates of many cells, which become large cytoplasmic masses on day 4. PGE2 and TGFβ have both been shown to increase osteoclast development in this model and their effects on the morphology of osteoclasts during fusion and differentiation have been compared under all these conditions. PGE2 or TGFβ increase osteoclast numbers and size and also the number of nuclei, indicating increased osteoclast development and fusion. TGFβ increases the size of rounded osteoclasts (with respect to the number of nuclei) more than PGE2, suggesting that TGFβ increases cytoplasmic extension. TGFβ increases the size and number of nuclei in stellate cells but particularly increases the number and length of the cytoplasmic extensions, in contrast to PGE2. Fusion of these extensions with other osteoclasts results in large networks of interconnected cells. On day 4, spreading cells develop but these are still interconnected by cytoplasmic links, a phenomenon not seen in control wells or after treatment with PGE2. TGFβ is more effective than PGE2 in increasing fusion in the formation of cell conglomerates and cytoplasmic masses. PGE2 decreases overall cell density resulting in additional indirect effects on osteoclast numbers and morphology. However, PGE2 particularly promotes the formation of large mature spreading osteoclasts later during culture.     

Staurosporine-induced morphological changes in the rat osteoblasts.

Treatment of cultured rat osteoblasts with staurosporine caused a rapid outgrowth of long slender cellular processes and the formation of stellate cells. The number of stellate cells increased with higher concentrations of and longer incubation with staurosporine. Scanning electron microscopy (SEM) revealed that the smooth surface of control polygonal cells became ruffled with many long slender cellular processes, thus increasing the cell surface area. Transmission electron microscopy (TEM) of the stellate cells showed a rich accumulation of large lipid droplets in the cytoplasm. Some lipid droplets had coalesced under the cytoplasmic membrane. We suggest that staurosporine has an effect on the differentiation of cultured rat osteoblasts.     

Schistosoma japonicum soluble egg antigens induce apoptosis and inhibit activation of hepatic stellate cells: a possible molecular mechanism

Hepatic stellate cells play a key role in the development of hepatic fibrosis. Activated hepatic stellate cells can be reversed to a quiescent-like state or apoptosis can be induced to reverse fibrosis. Some studies have recently shown that Schistosoma mansoni eggs could suppress the activation of hepatic stellate cells and that soluble egg antigens from schistosome eggs could promote immunocyte apoptosis. Hence, in this study, we attempt to assess the direct effects of Schistosoma japonicum soluble egg antigens on hepatic stellate cell apoptosis, and to explore the mechanism by which the apoptosis of activated hepatic stellate cells can be induced by soluble egg antigens, as well as the mechanism by which hepatic stellate cell activation is inhibited by soluble egg antigens. Here, it was shown that S. japonicum-infected mouse livers had increased apoptosis phenomena and a variability of peroxisome proliferator-activated receptor γ expression. Soluble egg antigens induce morphological changes in the hepatic stellate cell LX-2 cell line, inhibit cell proliferation and induce cell-cycle arrest at the G₁ phase. Soluble egg antigens also induce apoptosis in hepatic stellate cells through the TNF-related apoptosis-inducing ligand/death receptor 5 and caspase-dependent pathways. Additionally, soluble egg antigens could inhibit the activation of hepatic stellate cells through peroxisome proliferator-activated receptor γ and the transforming growth factor β signalling pathways. Therefore, our study provides new insights into the anti-fibrotic effects of S. japonicum soluble egg antigens on hepatic stellate cell apoptosis and the underlying mechanism by which the liver fibrosis could be attenuated by soluble egg antigens.     

Hormonally induced cell shape changes in cultured rat ovarian granulosa cells

Cultured rat ovarian granulosa cells undergo a dramatic morphological change when exposed to follicle-stimulating hormone (FSH). Exposure to FSH causes the flattened epithelioid granulosa cells to assume a nearly spherical shape while retaining cytoplasmic processes which contact the substrate as well as adjacent cells. This effect of FSH is preceded by a dose-dependent increase in intracellular cAMP, is potentiated by cyclic nucleotide phosphodiesterase inhibitors, and is mimicked by dibutyryl cAMP. Prostaglandins E1 or E2 and cholera enterotoxin also cause the cells to change shape. A subpopulation of the cells responds to luteinizing hormone. These morphological changes, which are blocked by 2,4-dinitrophenol, resemble those produced by treating cultures with cytochalasin B. Electron microscopy shows that the unstimulated, flattened cells contain bundles of microfilaments particularly in the cortical and basal regions. After FSH stimulation, microfilament bundles are not found in the rounded granulosa cell bodies but they are present in the thin cytoplasmic processes. These data suggest that the morphological change results from a cAMP-mediated, energy-dependent mechanism that may involve the alteration of microfilaments in these cells.     

The effects of 5-bromodeoxyuridine on the growth and morphology of transformed rat liver cells

The effects of bromodeoxyuridine (BrdUrd) on the growth, morphology, and tumorigenicity of the spontaneously transformed rat liver cell line R72/3 were studied. These cells grow either in suspension or in a monolayer and are tumorigenic. In monolayer cultures, cells treated with low concentrations (2.5 μg/ml) of BrdUrd were larger, more spread out, and more firmly attached to the substratum than were untreated controls. Treated cells failed to grow in suspension or on confluent monolayers of 3T3 cells and did not form colonies in soft agar. Scanning electron microscopy revealed extensive flattening of treated cells and a dramatic reduction in the number of microvilli on the cell surface. Transmission electron microscopy showed an increase in polyribosomes and rough endoplasmic reticulum, as well as an enlargement of endoplasmic reticulum cisternae and a complete absence of the bundles of intermediate size filaments that were conspicuous in untreated cells. The persistence of these changes required the continuous presence of BrdUrd in the medium. The effects of BrdUrd were readily reversed by withdrawal of BrdUrd and were not expressed in the presence of excess thymidine.     

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.