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Biomedical subjects

R Bravo

Publications and source records attributed to R Bravo.

At least 253 records · Page 14Linked to original sources
PubMed1986

Expression of c-fos in NIH3T3 cells is very low but inducible throughout the cell cycle.

It has previously been shown that the c-fos proto-oncogene is rapidly and transiently induced following growth factor stimulation of quiescent NIH3T3 mouse fibroblasts. To investigate a possible role of c-fos in growth control mechanisms we have studied its expression and inducibility during the NIH3T3 cell cycle. Two major conclusions can be drawn from this analysis. First, expression of c-fos is not cell cycle-regulated, and is barely detectable in all phases of the cycle. Second, cells at different stages of the cell cycle (except for mitosis) are as sensitive to c-fos induction by growth factors as quiescent cells. These observations suggest that induction of the c-fos gene does not play a role during the continuous cycling of NIH3T3 cells, but they are fully compatible with the hypothesis that a function of c-fos may be associated with the induction of competence in fibroblasts. Through such a function c-fos may contribute to moving cells out of the quiescent state.

Animals↗
PubMed1986

A basic cytoplasmic protein (p27) induced by serum in growth-arrested 3T3 cells but constitutively expressed in primary fibroblasts.

Serum stimulation of quiescent 3T3 cells immediately induces the synthesis of a set of basic proteins that are absent in growing cells. The induction of some of these polypeptides p27 (27 kd), p35 (35 kd), p38 (38 kd) and p69 (69 kd) can be 'superinduced' in the presence of cycloheximide and completely blocked by actinomycin D. In vitro translation experiments show that the levels of mRNA coding for these proteins in serum-stimulated cells are several fold higher than in non-stimulated cells. Induction of p35 and p38 is transient (4 h); in contrast, p27 and p69 are induced for a longer period (8 h). Platelet-derived growth factor and fibroblast growth factor strongly induce p35 and p69 but weakly induce p27 and p38. Cultures of primary mouse fibroblasts express p27 but not the other polypeptides at levels similar to those found in serum-stimulated quiescent 3T3 cells. Enucleation and Triton extraction of cells show that p27 is a soluble cytoplasmic protein. The synthesis of this protein in density-arrested or serum-deprived primary cultures is only 20% reduced showing that the expression of p27 in these cells is independent of cell proliferation.

Animals↗
PubMed1986

Wounding a fibroblast monolayer results in the rapid induction of the c-fos proto-oncogene.

The c-fos gene has previously been shown to be transiently induced within minutes after the stimulation of mouse fibroblasts with growth factors. Induction of c-fos was observed specifically with competence factors (e.g., platelet-derived growth factor), not with progression factors (e.g., platelet-poor plasma), suggesting a role for c-fos in conferring competence on fibroblasts. To test this hypothesis we have analyzed c-fos expression in NIH 3T3 cells that were made competent in a different way, namely by wounding a confluent monolayer of cells. Using antibodies raised against either a synthetic fos peptide or a beta-galactosidase--fos fusion protein, we show in this study that in the majority of cells lining the wound c-fos protein is rapidly and transiently induced to high levels. No induction is observed in cells at a distance from the wound greater than approximately 5 cell layers. Induction is equally efficient in both serum-containing and serum-free medium, and is similar in cells that were deprived of fetal calf serum for 40 h prior to making the wound. Our observations support the hypothesis that c-fos may be involved in inducing the 'competent state' in fibroblasts and suggests an early role for c-fos in wound healing and tissue regeneration.

Animals↗
PubMed1986

Synthesis of the nuclear protein cyclin (PCNA) and its relationship with DNA replication.

Synthesis of the nuclear protein cyclin (MW 36 000) and DNA in quiescent mouse fibroblasts is coordinately induced by serum and purified growth factors. Inhibition of DNA synthesis by hydroxyurea or aphidicolin in serum-stimulated quiescent cells does not affect the induction of cyclin. The levels of cyclin synthesis decrease rapidly at the end of the S phase. Immunofluorescence studies reveal that there are dramatic changes in the nuclear distribution of cyclin during S phase and that these depend on DNA synthesis or events during S phase. These observations strengthen the notion that cyclin is an important component of the events leading to DNA replication.

Animals↗
PubMed1986

Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis.

The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various times postinfection were also analyzed. At least 13 proteins labeled with [3H]glucosamine were detected in vaccinia-infected HeLa cells.

Cycloheximide↗
PubMed1985

Regulation of c-fos transcription in mouse fibroblasts: identification of DNase I-hypersensitive sites and regulatory upstream sequences.

In quiescent mouse fibroblasts, the c-fos gene is expressed at very low levels, but is rapidly and transiently inducible by peptide growth factors. In this study, we have identified in quiescent cells five DNase I-hypersensitive sites located -1700, -290, +10, +240 and +700 bp relative to the 5' cap site. After serum stimulation, the distinct nuclease hypersensitive site at position +10 rapidly disappeared, and instead a broad region of DNase I accessibility between positions 0 and +250 occurred. Nucleotide sequence analysis of the 5'-flanking region of the mouse c-fos gene showed that the hypersensitive site around position -290 is located in a region that is highly conserved between mouse and human, and that contains an enhancer-like structure. When the mouse c-fos promoter and 351 bp of 5'-flanking sequences were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into NIH3T3 cells efficient, constitutive expression of CAT activity was observed, even in unstimulated, quiescent cells. However, removal of a 256-bp stretch upstream from position -95 completely abolished CAT expression, indicating that sequences within a region of approximately 350 bp upstream from the cap site are indispensable for c-fos transcription. In addition, our findings point to the existence of other sequence elements that exert negative regulation in the absence of growth factor stimulation. Such sites may be found around the growth factor-responsive nuclease hypersensitive sites in the vicinity of the cap site.

Acetyltransferases↗
PubMed1985

Changes in the nuclear distribution of cyclin (PCNA) during S-phase are not triggered by post-translational modifications that are expected to moderately affect its charge.

Indirect immunofluorescence studies of HeLa cells using PCNA autoantibodies specific for cyclin have revealed striking changes in the nuclear localization of this protein during S-phase. Two-dimensional gel electrophoretic analysis of the [32P]orthophosphate and [35S]methionine labelled proteins from synchronized cells showed that phosphorylation, or other post-translational modifications that are expected to moderately affect the charge of cyclin (acetylation, glycosylation, sialylation, etc.) are not likely part of the mechanism(s) triggering the migration of this protein.

Cell Nucleus↗
PubMed1985

An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments.

The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.

Animals↗
PubMed1985

Changes in the nuclear distribution of cyclin (PCNA) but not its synthesis depend on DNA replication.

Synthesis of cyclin in serum-stimulated quiescent 3T3 cells increases shortly before DNA synthesis after 10 h of stimulation, reaching a maximum after 16 h. Inhibition of DNA synthesis by hydroxyurea does not affect the increase of cyclin following stimulation, as determined by quantitative two-dimensional gel electrophoresis. The levels of cyclin decrease dramatically at the end of the S-phase. Cells kept in the presence of hydroxyurea (G1/S boundary) do not show this decrease in cyclin, indicating that its amounts are regulated by events occurring during the S-phase. Immunofluorescence studies of serum-stimulated quiescent cells in the presence of hydroxyurea, using proliferating cell nuclear antigen (PCNA) autoantibodies, confirm the results obtained by protein analysis. They also reveal that there are dramatic changes in the nuclear distribution of cyclin and that these depend on DNA synthesis or events occurring during the S-phase. Cyclin (PCNA) is no longer detectable at the end of the S-phase. However, pulse-chase experiments indicate that this protein is very stable, suggesting that it possibly interacts with other macromolecules rendering it inaccessible to the antibody. These results strengthen the notion that cyclin is an important component of the events leading to DNA replication and cell division.

Animals↗
PubMed1985

Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes.

Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.

Calcimycin↗
PubMed1985

Persistence of the competent state in mouse fibroblasts is independent of c-fos and c-myc expression.

Induction of competence in quiescent fibroblasts by platelet-derived growth factor (PDGF) is accompanied by a dramatic increase in the expression of c-fos and c-myc genes. However, the maintenance of the competent state and progression through G1 does not require high expression of these proto-oncogenes. These results suggest that the induction of c-fos and c-myc by growth factors in quiescent fibroblasts may be required to render the cells competent for progression.

Animals↗
PubMed1985

Induction of the nuclear protein cyclin in serum-stimulated quiescent 3T3 cells is independent of DNA synthesis.

Inhibition of DNA synthesis and cell proliferation of mouse 3T3 cells by aphidicolin did not affect the expression of cyclin, a nuclear protein whose synthesis correlates with cell proliferation, as determined by quantitative two-dimensional gel electrophoresis analysis. Serum stimulation of quiescent 3T3 cells revealed that cyclin synthesis increases shortly before DNA synthesis. Inhibition of DNA synthesis by aphidicolin in serum-stimulated quiescent cells did not affect the increase of cyclin following stimulation. These results demonstrate that cyclin synthesis is not coupled to DNA synthesis and that it is one of the latest events before DNA replication.

Animals↗
PubMed1985

Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK.

Secretion of a foreign protein--chicken oviduct lysozyme--and of endogenous proteins was studied in the polarized epithelial Madin-Darby Canine Kidney (MDCK) cell line. Cell clones that secrete enzymatically active chicken lysozyme were generated by transforming the cells with lysozyme cDNA inserted in a SV40-pBR322 recombinant vector and a dominant selectable marker gene. The kinetics and polarity of lysozyme secretion from one transformed cell clone were studied using cell monolayers grown on nitrocellulose filters. Lysozyme was secreted into the apical and the basolateral medium, demonstrating the existence of direct transport pathways to each cell surface. Control experiments excluded the effects of monolayer leakiness, reabsorption, transepithelial transport, and depolarization. In contrast, the secretion of a set of endogenous proteins of MW 30-40 kd was found to be strictly apical showing that polarized secretion also occurs in this cell line. The latter group of proteins appear to be generated from larger precursor molecules by intracellular cleavage.

Animals↗
PubMed1985

Transient induction of c-fos and c-myc in an immediate consequence of growth factor stimulation.

Treatment of serum-deprived fibroblasts with serum or growth factors results in an immediate induction of the c-fos and c-myc proto-oncogenes. Maximal levels of c-fos mRNA are detected 30 minutes after treatment and maximal levels of c-myc mRNA are detected 60 minutes after treatment. The c-fos protein is expressed at high levels for about two hours following induction, yet the cell morphology remains normal. Thus, either an extended period of c-fos expression is required for cellular transformation, or the highly modified form of the fos protein, present in stimulated cells, is biochemically different from the transforming protein and is therefore not capable of inducing transformation. In this system, growth factor treatment results in mitogenesis. However, c-fos and c-myc are also induced in A431 cells, and in subclones derived from A431 cells, treated with epidermal growth factor (EGF). No correlation was found between the effects of EGF on A431 cell proliferation and the induction of c-fos and c-myc. Interestingly, the strongest inducer of c-fos in A431 cells was the calcium ionophore A23187. Induction occurred in almost 100% of the treated cells without prior serum deprivation or growth arrest. Treatment of HL60 cells with 12-0 tetra decanoylphorbol-13-acetate (TPA), which promotes macrophage-like differentiation, also induced c-fos with a time course similar to that observed in mitogen-treated fibroblasts. Thus, in HL60 cells, c-fos induction is associated with differentiation. In normal macrophages c-fos and c-myc can also be induced by CSF-1. However, the kinetics of induction are entirely different from those in growth factor-stimulated fibroblasts. Taken together, the data suggest a more general role for c-fos and c-myc in the transduction of growth factor signals received at the cell membrane, within the nucleus.

Animals↗
PubMed1984

Induction of the nuclear protein 'cyclin' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis.

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.

Animals↗
PubMed1984

Synthesis of the nuclear protein cyclin in growing, senescent and morphologically transformed human skin fibroblasts.

Quantitative two-dimensional gel electrophoretic analysis (IEF) of the nuclear polypeptide cyclin in normal human skin biopsies, growing and senescent fibroblasts and morphologically transformed skin fibroblasts (limited life span) has revealed a direct correlation between the levels of this protein and proliferative state of the cells. These results strengthen the notion that cyclin may be a key component of the pathway(s) that control cell proliferation.

Cell Division↗