Okadaic acid co-induces vimentin expression and cell cycle arrest in MPC-11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10^?9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid. © 1995 Wiley-Liss, Inc.     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Milk growth factor (MGF) induces transformation into ATDC5 cells, prechondrocytes, and cooperates with retinoic acid to transform the cells into new forms

The effects of milk growth factor (MGF) showed the transformation of ATDC5 prechondrocytes and differed from that of retinoic acid (RA) as follows. MGF (200 ng/ml) did not suppress the proliferation of ATDC5 cells, though RA (1 x 10(-7) M) suppressed the cell proliferation. However, MGF showed the result as RA, which was verified to suppress the production of proteoglycan. The synthesis of vimentin in ATDC5 cells was slightly induced by RA, but its withdrawal induced the large-scale induction and the fibril formation of vimentin, which may indicate that the cells became fibroblastic cells, namely dedifferentiation. MGF, which hardly induced the vimentin synthesis in ATDC5 cells, induced its synthesis under control by the withdrawal. MGF suppressed the synthesis of alpha-smooth muscle actin (alpha-SM-actin), which was apt to reverse in its withdrawal. However, RA did not affect this synthesis of ATDC5 cells. The combination of MGF and RA enlarged the cells and enhanced the synthesis of vimentin due to RA under control, however, almost terminated alpha-SM-actin-synthesis in the cells. And its effect is almost irreversible. Furthermore, the combination of MGF and RA prevented the induction of fibroblasts due to RA in the cells. And the withdrawal of the mixture transformed prechondrocytes into hypertrophic cells. Then, MGF contributes to bone metabolism in prechondrocyte.     

Induction of vimentin synthesis in a murine myeloma cell line by TPA is strongly dependent on the composition of the cell culture medium

MPC-11 mouse plasmacytoma cells virtually lacking intermediate filament (IF) proteins can be induced to synthesize and accumulate the IF protein vimentin by treatment with the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Like MPC-11 cells, X63-Ag8.6.5.3 mouse myeloma cells (Ag8) proved to be vimentin-negative, as assayed by immunoblotting of whole cellular protein using goat antiserum to vimentin and [125I]protein A. Vimentin synthesis could be elicited by a TPA concentration as low as 10(-9) M in cells grown in HB-102 serum-free medium. Transfer of these cells to medium containing 15% fetal calf cerum (FCS) greatly reduced the ability of these cells to synthesize vimentin upon TPA treatment. After 50 generations of culture in the presence of FCS, induction of vimentin synthesis was barely detectable even at a TPA concentration of 10(-6) M. Addition of FCS to cells grown in serum-free medium partially suppressed vimentin induction by TPA. This suppression seems to be due, at least in part, to nondialyzable, heat-sensitive components of FCS, since the dialyzable fraction even enhanced vimentin induction by TPA. When cells grown in the presence of FCS were transferred back to serum-free medium, their ability to synthesize vimentin in response to TPA treatment was readily restored. The individual components of serum-free medium which proved to support vimentin induction by TPA were insulin and the unsaturated fatty acids oleic acid and linoleic acid. An even stronger TPA response could be elicited by a combination of these components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of Cyclic AMP-Dependent Changes in Intermediate Filament Protein Phosphorylation and Cell Morphology in Cultured Astroglia

Receptor agonists that increase cyclic AMP levels in cultured astroglia have been shown to increase 32P-labeling of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin in these cells. Experiments were designed to determine if the increase in 32P-labeling resulted from either an increase in the turnover or net number of phosphates associated with the intermediate filament proteins and if the phosphorylation of these proteins causally affected astroglial morphology. Time course experiments indicated that 6-8 h were required to reach steady-state 32P-labeling of both GFAP and vimentin. Treatment with forskolin (10 microM) after steady-state 32P-labeling increased GFAP and vimentin phosphorylation fourfold and twofold, respectively, and also induced a morphological change from polygonal to process-bearing cells within 20-30 min of drug addition. Cells incubated in media containing brain extract (30%) for 24 h at 37 degrees C and then 3 h at 23 degrees C underwent changes from polygonal to process-bearing cells with no apparent increase in the phosphorylation of either GFAP or vimentin. Treatment of process-bearing cells (induced by brain extract) or polygonal cells with 10 microM forskolin at 23 degrees C resulted in a three- to fourfold increase in GFAP phosphorylation without significant morphological changes. These results suggest that forskolin stimulation of GFAP and vimentin increases net number of phosphates associated with these intermediate filament proteins and that the resulting increase in phosphorylation can be dissociated from morphological changes.     

Cytoskeletal Alterations in Human Fetal Astrocytes Induced by Interleukin-1β

Abstract: Previous studies in this and other laboratories have shown that interleukin-1β (IL-1β) is a selective and potent activator of human astrocytes with respect to induction of cytokines and hematopoietic growth factors. To study the effect of recombinant human IL-1β (rhIL-1β) on astrocyte morphology, glial fibrillary acidic protein (GFAP) and vimentin expression, and actin organization, we conducted a systematic survey using dissociated human fetal astrocyte cultures. Within hours of stimulation with IL-1β, the majority of astrocytes converted from flat, polygonal cells to small, contracted, highly branched cells. This change in morphology was more striking when serum was eliminated from the medium. Complete dissolution of filamentous actin occurred simultaneously with the change in cell shape, as demonstrated by fluorescein-phalloidin binding. These “activated” astrocytes displayed intense GFAP and vimentin immunoreactivity in the small perikarya and processes. In contrast, the large, flat astrocytes in control cultures showed diffuse pale immunoreactivity for GFAP and vimentin. To quantify the changes in GFAP and vimentin content with IL-1β stimulation, densitometric analyses of northern and western blots were performed. Northern blot analysis of IL-1β-stimulated astrocytes revealed a transient, marked decrease in steady-state levels of mRNA for GFAP, vimentin, and microtubule-associated protein 4. The decrease in mRNA levels was evident by 4–8 h and fell to the lowest level at 16–24 h (80–98% decrease by densitometry) with partial recovery by 72 h. By immunoblotting, a significant decrease in both GFAP and vimentin protein content was observed after IL-1β stimulation. Furthermore, metabolic labeling studies revealed an almost total loss of GFAP synthesis following stimulation with IL-1β for 16 h. These observations are consistent with the idea that increases in immunoreactivity were related to factors such as redistribution of epitope, rather than increases in total protein content. We hypothesize that in IL-1β-stimulated astrocytes, synthesis of other proteins, e.g., inflammatory cytokines, occurs at the expense of structural proteins and that the decrease in content of cytoskeletal proteins may reflect an “activated” state of astrocytes.     

Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis : Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.     

Ca2(+)-calmodulin-dependent protein kinase II phosphorylates various types of non-epithelial intermediate filament proteins

We have investigated the actions of Ca2(+)-calmodulin (CaM)-dependent protein kinase II on various types of non-epithelial intermediate filament proteins, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament triplet proteins. Most of these filament proteins could serve as substrates. The effects of phosphorylation on the filamentous structure of vimentin were investigated in sedimentation experiments and by using electron microscopy. The amount of unassembled vimentin increased linearly with increased phosphorylation. However, the extent of the effect of phosphorylation on the potential to polymerize was also affected by the MgCl2 concentration, under conditions for reassembly. The actions of Ca2(+)-CaM-dependent protein kinase II on non-epithelial intermediate filaments under physiological conditions are given attention.     

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina

By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells. Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days. For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP). For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ. The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP. The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum. In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP. These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells. The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma. Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.     

Effect of media composition on the induction of chorionic gonadotropin by sodium butyrate in HeLa cells

Summary Production of the glycoprotein hormone α-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis in the presence of butyrate was highest in RPMI 1640, lowest in Medium 199 (M199), and intermediate in minimum essential medium (MEM) and Waymouth's MB 752/1. Cell growth was similar in all media examined and was retarded in the presence of butyrate. Alkaline phosphatase activity was also lower in M199 than in RPMI 1640, although, in general, the magnitude of this difference was less than that for the hormone subunit. Incorporation of [1-¹⁴C]butyrate by HeLa cells was simimar in both M199 and RPMI 1640, indicating that uptake and metabolism of the fatty acid were not significantly different under these conditions. In the presence of 3 mM butyrate, mixtures of RPMI 1640 and M199 gave intermediate levels of α-subunit and alkaline phosphatase compared to each medium alone. Intracellular levels of α-subunit as well as that of the culture medium were reduced in M199 compared to RPMI 1640 indicating that synthesis rather than secretion was altered. This work was supported by Grant CA 21534 from the National Institutes of Health, Bethesda, MD.     

A new protein (J1-31 antigen, 30 kD) is expressed by astrocytes,Müller glia and ependyma

Monoclonal antibody (MAb) J1-31 raised using human brain homogenate as immunogen in mice can be used as a cell type marker for certain types of CNS macroglia, namely astrocytes, Müller cells and tanycytes as well as ciliated ependymal cells. Except for the ciliated ependymal cells, these types of macroglia express glial fibrillary acidic protein (GFAP). J1-31 antigen is an intracellular protein which has a MW of 30 kD under reducing conditions for gel electrophoresis (Singhet al., 1986). This protein is distinct from GFAP (MW 50 kD) and vimentin (MW 55 kD), the two core proteins of 10 nm IFs known to be expressed in the above types ofmacroglia. This conclusion is based on several criteria including temporal differences in the onset of expression of GFAP and J1-31 antigen during development of the rat cerebellum. Also, there is no detectable (by immunofluorescence microscopy) expression of J1-31 antigen in the prenatal CNS or outside the CNS where vimentin has been reported to be abundant. The most direct evidence that J 1-31 antigen and GFAP are distinct proteins comes from studies on the mature ciliated ependymal cells which do not express GFAP and yet show intense immunostaining for J1-31 antigen.     

Growth requirements of ferret tracheal epithelial cells in primary culture

In mass cell culture conditions, protease dissociated ferret tracheal epithelial cells (FTE) proliferated in growth factor-supplemented F12 medium to high cell densities (0.5 X 10(5) cells/cm2) with an average population doubling time of 24 hr. The growth factor constituents of the F12 medium included epidermal growth factor (25 ng/ml), insulin (1 microgram/ml), transferrin (10 micrograms/ml), hydrocortisone (18 ng/ml), hypothalamus extract (30-100 micrograms/ml), and conditioned medium from mouse 3T3 fibroblasts. Growth of these cells under clonal conditions was achieved by the partial replacement of F12 medium with M199 medium which was attributed, in part, to the presence of vitamin A in M199 medium. Serum did not stimulate the growth of FTE cells. The epithelial cell nature of these cells in culture was confirmed by ultrastructural features and by immunofluorescent staining for fibronectin.     

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.     

Isolation and Characterization of a Subsurface Bacterium Capable of Growth on Toluene, Naphthalene, and Other Aromatic Compounds

A bacterium, designated F199, utilized toluene, naphthalene, dibenzothiophene, salicylate, benzoate, p-cresol, and all isomers of xylene as a sole carbon and energy source. This bacterium was isolated from Middendorf sediments, a Cretaceous age formation that underlies the Southeast Coastal Plain in South Carolina, at a depth of approximately 410 m. F199 is a gram-positive, irregular-shaped bacterium that has a varied cell morphology that is dependent on culture medium type and growth stage. F199 required microaerobic conditions (40 to 80 μM O₂) for growth on hydrocarbons, glucose, acetate, and lactate in mineral salts medium but not for growth on rich media. [¹⁴C]naphthalene mineralization by F199 was induced by either naphthalene or toulene; however, [¹⁴C]toluene mineralization by this strain was induced by toluene but not naphthalene. F199 was also found to harbor two plasmids larger than 100 kb. Restricted F199 plasmid and genomic DNA did not hybridize with toluene (pWW0) or naphthalene (NAH7) catabolic plasmid DNA probes. The presence in the Middendorf formation of bacteria with the capacity for degrading a variety of aromatic compounds suggests that indigenous microorganisms may have potential for in situ degradation of organic contaminants.     

Induction of acute phase proteins by dexamethasone in rat hepatocyte primary cultures

The effect of dexamethasone on the synthesis of acute phase proteins has been studied in primary cultures of rat hepatocytes. In the absence of dexamethasone no detectable amounts of alpha 2-macroglobulin were synthesized by hepatocytes cultured for 1 day. alpha 2-Macroglobulin synthesis was induced by dexamethasone concentrations of 10(-8) M or higher with a maximum at a concentration of 10(-7) M. alpha 1-Acid glycoprotein was synthesized in the absence of dexamethasone; however, its synthesis was also greatly stimulated by dexamethasone concentrations of 10(-8)-10(-6) M. Synthesis of alpha 1-proteinase inhibitor was stimulated only 1.4-fold at a dexamethasone concentration of 10(-7) M. The kinetics of induction of alpha 2-macroglobulin and alpha 1-acid glycoprotein were studied at a dexamethasone concentration of 10(-7) M. After an initial lag phase of 3 h the synthesis of both proteins showed a steady increase during 2 days. Synthesis of albumin remained unchanged under these experimental conditions. Unlike alpha 2-macroglobulin and alpha 1-acid glycoprotein tyrosine aminotransferase activity increased already during the first 3 h of induction by dexamethasone with a maximum at 12 h followed by a slight decrease.