3T3 Fibroblast Cells Research Articles

Modulation of mitogenic stimuli by angiogenin correlates with in vitro phosphatidylinositol bisphosphate synthesis.

125I-labeled angiogenin binds rapidly to the plasma membrane of several cell lines at 37 degrees C (t1/2 less than 1 min) but in comparatively small amounts. Competition with unlabeled angiogenin varies markedly with different cell lines, being most effective in vascular smooth muscle and fibroblast cells. Angiogenin modulates mitogenic stimuli in bovine adrenal capillary endothelial (BACE), rat aortic smooth muscle (RASM), and fibroblast (3T3) cells. Thus, it enhances the mitogenic effect of certain conditioned media on RASM and 3T3 cells, but it inhibits the mitogenic effect on BACE cells. In RASM and 3T3 cells, mitogenesis is increased at low (less than 5 ng/ml) and high (greater than 100 ng/ml) but not at intermediate concentrations of angiogenin. Plasma membranes from RASM and 3T3 cells that have been treated with angiogenin show an enhanced in vitro synthesis of phosphatidylinositol bisphosphate (PtdInsP2) with an angiogenin concentration dependence similar to that of enhanced mitogenesis. PtdInsP2 synthesis arises by activation of a fatty acid (arachidonyl) coenzyme A synthetase and either a plasma membrane fatty acid acyltransferase or phosphatidylinositol kinase(s), or both. Increased PtdInsP2 or the derived second messengers (e.g., diacylglycerol) may mediate modulation of the mitogenic stimulus. The differential mitogenic interaction of angiogenin with several cell types, either stimulation or inhibition, probably reflects the multistep nature of angiogenesis.

Cell cycle expression of RNA duplex unwindase activity in mammalian cells.

An RNA duplex unwindase activity has been found by using an in vitro assay with various types of mammalian, somatic cells, including HeLa, mouse plasmacytoma, and Burkitt lymphoma. The unwindase activity is very low in mouse fibroblast 3T3 cells arrested into quiescence, but increases when the cells are released into renewed growth by serum. In addition, a gel retardation assay proved to be specific and sensitive for detection of RNA duplex-unwindase complexes.

Cell Cycle Expression of RNA Duplex Unwindase Activity in Mammalian Cells

An RNA duplex unwindase activity has been found by using an in vitro assay with various types of mammalian, somatic cells, including HeLa, mouse plasmacytoma, and Burkitt lymphoma. The unwindase activity is very low in mouse fibroblast 3T3 cells arrested into quiescence, but increases when the cells are released into renewed growth by serum. In addition, a gel retardation assay proved to be specific and sensitive for detection of RNA duplex-unwindase complexes.

Partial purification of an adrenal growth factor produced by normal bovine anterior pituitary cells in culture.

An adrenal growth factor (AGF) was partially purified from the conditioned medium of cultured bovine anterior pituitary cells. This 900-fold purification was accomplished by gel filtration, ammonium acetate precipitation, cation exchange fast protein liquid chromatography, and C8 reverse phase fast protein liquid chromatography. The AGF had an apparent mol wt of 25,000-30,000, as determined by gel filtration chromatography at acid pH. It was tested on mouse fibroblasts (3T3 cells), rat fibroblasts (NRK cells), mouse adrenocortical cells (Y1 cells), and normal bovine adrenocortical cells (BAC cells) and found to be a relatively potent mitogen for BAC cells only, contrasting with fibroblast growth factor, which could stimulate all of these cell types. It is sensitive to reducing agent, alkaline pH, and boiling. Purification steps were carried out at acid pH, suggesting some degree of stability to acidic pH. The mitogenic activity in the conditioned medium could be blocked by ACTH, and the purified AGF contained no ACTH immunoactivity. The AGF appears distinct from epidermal growth factor, since it could not bind to epidermal growth factor receptors, and platelet-derived growth factor, which could not stimulate BAC cells. Purification to homogeneity was not accomplished, because very broad bands of mitogenic activity were observed with all of the chromatographic methods tried. The AGF appears to be distinct from any of the known growth factors.

Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor

Two mitogenic peptides in bovine liver extract were purified to apparent homogeneity by monitoring the purification steps with two in vitro bioassays; one based on stimulation of adult bovine aortic arch endothelial cell proliferation and the other incorporation of [ 3H]thymidine to mouse fibroblast 3T3 cells. The purification procedure involved cation-exchange chromatography followed by affinity chromatography on heparin-Sepharose and two steps of reversed-phase HPLC. The purified material showed the same biological activity as pituitary basic fibroblast growth factor (FGF). Amino acid analyses of the purified mitogen yielded a similar, but not identical composition to that of bovine pituitary basic FGF(1–146) reported previously. Gas-phase microsequencing identified two sequences in equal amounts in the purified preparation. Furthermore, the sequencing results are in accord with the theoretical data obtained when two truncated forms of basic FGF, corresponding to FGF(12–16) and (16–146), are being sequenced simultaneously. Basic FGF(12–146) is a novel truncated form of basic FGF which has not been isolated before although the (16–146) fragment has been found previously in kidney, corpus luteum, and adrenal. SDS-PAGE analysis could not separate the two forms and showed that both migrated as a protein of about 15 100 daltons, which is slightly smaller than intact basic FGF(1–146) (16 200 daltons). These results, taken together, indicate that at least some of the mitogenic activity in liver may be derived from basic FGF-related polypeptides.

Extraction of glycolytic enzymes: myo-inositol as a marker of membrane porosity.

Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X-100 and Brij 58, led to similar results for extraction of myo-inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X-100 in isotonic phosphate-buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with non-extractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X-100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X-100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X-100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent-mediated alterations in membrane porosity than glycolytic enzyme leakage.(ABSTRACT TRUNCATED AT 250 WORDS)

Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.

Interaction between the tyrosyl free radical and the antiferromagnetic iron center in ribonucleotide reductase

Ribonucleotide reductase catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides. The enzymes from E. coli, from E. coli after infection with bacteriophage T4, and from mouse fibroblast 3T6 cells have all been shown to be of similar type, containing a tyrosyl free radical essential for enzyme activity [1–5]. The free radical is situated in one of the two subunits of the enzyme, denoted B2 in the bacterial enzyme, β 2 in the T4-induced enzyme, and M2 in the mammalian enzyme. Proteins B2 and β 2 have in addition been shown to contain an antiferromagnetically coupled pair of high-spin ferric iron atoms exhibiting a temperature-dependent paramagnetism [4]. A similar iron pair stabilizing the M2 radical is postulated also for the mammalian enzyme. The EPR spectra of the radicals at 77 K and below show slight differences between the various enzyme species. These differences have been ascribed to small differences in the angle of the aromatic ring in relation to the β methylene group of tyrosine [2]. The variations probably reflect differences in the polypeptide chain around the radical site. However, the differences observed are small and the major geometrical properties of the protein around the radical are obviously conserved, which may be important for the radical stability and enzyme function. The temperature dependence of the EPR spectra of the radicals was studied from 10 K up to room temperature. With increasing temperature gradual changes were observed in spectral linewidth (broadening), signal amplitude (decrease), double integral of the spectrum, representing apparent signal intensity (decrease), and microwave saturation. The temperature dependences of these changes were significantly different for the radicals residing in proteins of different origins (B2, β 2 or M2). Taken together the results strongly indicate a significant interaction between the tyrosyl free radical and the temperature-dependent magnetic moment of the antiferromagnetically coupled iron pair. A tentative evaluation is presented using the dipolar coupling model proposed by Leigh for the EPR line shape in a system of two interacting spins [6]. This has to be combined with results of microwave saturation of the radicals showing that the product T 1·T 2 is smaller in M2 than in B2. The influence of a weaker antiferromagnetic coupling in the M2 iron pair and a shorter iron pair-radical distance will be discussed.

The effect of the plant hormone indole-3-acetic acid and chemically related compounds on the growth of mouse fibroblast 3t3 cells

1. The effect of the plant hormone indole-3-acetic acid (IAA) and the chemically related compounds: serotonin, tryptophan, I. pyruvic, I. butyric, I. propionic and tryptamine on the growth of 3T3 cells has been studied. 2. Each of these compounds increases the cell number in a variable degree. The increase is related to the presence of the serum in the medium. 3. The kinetic relation between IAA, foetal calf serum and cell growth was studied. 4. It may be that IAA and related compounds affect the cell growth through adenylate cyclase and cAMP-cGMP systems.

Characterization of the free radical of mammalian ribonucleotide reductase.

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.

Studies on plasminogen activator and other proteases in subcultured human vascular cells

Plasminogen activator activity (PAA) of human vascular cells in culture was quantitated in an assay system in which the conversion of purified plasminogen to plasmin was measured by activity against a soluble low-molecular-weight plasmin substrate. Reliable detection of PAA required cell lysis and use of membrane-disrupting detergents. After such treatment, PAA was found only in the 100,000 g subcellular fractions of human aortic smooth muscle and mixed populations of rabbit aortic cells. No corresponding activity was found in any fraction from human umbilical vein endothelial cells. In contrast, PAA was detected in significant amounts in intact mouse embryo fibroblasts (3T3 cells) used as controls. Very low levels of enzymatic activity against a spectrum of substrates preferentially amidolyzed by serine proteases were also demonstrable in human aortic smooth muscle or 3T3 fibroblast subcultures. Neither whole cell homogenates nor subcellular fractions demonstrated the previously described acid-labile inhibitor of PAA when assayed in this system. The above findings suggest that significant differences in PAA exist between various cell types and vascular segments. In addition, the subcellular localization and quantitatively low levels of PAA and other serine proteases found in human arterial and venous cells may reflect the presence of membrane-associated enzymes whose biological role is restricted to local homeostasis.

Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells.

Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxy-ribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in bacteria and mammalian cells. The reductase from Escherichia coli consists of two nonidentical subunits (B1 and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required for enzyme activity. The mammalian enzyme also consists of two nonidentical subunits (M1 and M2), only one of which (M1) has been obtained in pure form. By continuous culture at stepwise increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea. Extracts from resistant cells showed a 3- to 15-fold increase in reductase activity. The amount of M1 protein was not increased. The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells (but not normal cells) showed an EPR spectrum very similar to that of the tyrosine radical of the bacterial B2 subunit. We propose that resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein. It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase.

Independent Regulation of the Nuclear and Mitochondrial DNA Synthesis Induced by either Simian Virus 40 or Serum in Mouse Fibroblast 3T3 Cells

Resting cultures of 3T3 cells (an established line of mouse fibroblasts) were released from density inhibition by either infection with Simian virus 40 or addition of serum. The increased rate of thymidine incorporation into DNA, induced by these two agents, was measured in the presence and in the absence of three inhibitory conditions (cycloheximide or dibutyryladenosine 3':5'-monophosphate added to the medium, or lack of anchorage). The inhibition was found to be quite similar in cultures stimulated by virus or serum; under the same conditions, however, the incorporation into mitochondrial DNA was much less inhibited than that into nuclear DNA. The experiments also suggest that new protein synthesis may not be necessary, for either virus or serum, to start the inductive mechanism.

De novo pyrimidine nucleotide biosynthesis in synchronized rat hepatoma (HTC) cells and mouse embryo fibroblast (3T3) cells

Activity of carbamyl phosphate synthetase, aspartate transcarbamylase, and orotate phosphoribosyl transferase was measured in cultures of rat hepatoma (HTC) cells synchronized using Colcemid inhibition. All three enzymes had distinct peaks of activity during the S phase of the cell cycle and had very little activity during the G 2 and M phases. Whereas carbamyl phosphate synthetase and aspartate transcarbamylase activities increased rapidly during early G 1, orotate phosphoribosyl transferase activity did not increase rapidly until the end of G 1. The incorporation of 14CO 2 into acid-soluble pyrimidine nucleotides and RNA followed a similar pattern to that obtained for enzymes of de novo uridylic acid biosynthesis, with the maximum incorporation occurring during the S phase in HTC cells. 3T3 cells, synchronized by release from contact inhibition, also had the maximum incorporation of 14CO 2 into acid-soluble pyrimidine nucleotides and RNA during the S phase, although there was also a lesser peak of incorporation early in the G 1 phase. Neither culture incorporated 14CO 2 into DNA to any significant extent.

Differences in membrane fluidity and structure in contact-inhibited and transformed cells.

Studies on contact-inhibited mouse embryo fibroblast 3T3 cells and 3T3 cells transformed by oncogenic RNA and DNA viruses and by a chemical carcinogen have demonstrated differences in plasma membrane architecture. Spin-label and freeze-fracture ultrastructural studies have shown that contact-inhibited cells have ordered membrane lipids and aggregated intramembranous particles, whereas transformed cells have fluid membrane lipids and randomly distributed intramembranous particles. These findings suggest a model for how changes in the cell membrane may account for some of the characteristic differences observed between contact-inhibited and transformed cells.

Ganglioside accumulation by transformed murine fibroblasts (3T3) cells and canine erythrocytes.

Ganglioside accumulation by transformed murine fibroblasts (3T3) cells and canine erythrocytes.

Effect of concanavalin A on interferon induction by poly IC

Concanavalin A, a plant lectin having specific membrane properties, has an inhibitory effect on interferon induction by poly IC, both in human fibroblast and mouse 3T3 cells. In the studied systems, the inhibition is independent of an effect on polynucleotide binding.

Replication of polyoma DNA in isolated nuclei: I. Characterization of the system from mouse fibroblast 3T6 cells

Nuclei isolated from polyoma virus-infected mouse fibroblast 3T6 cells incorporated radioactive [ 3H]dTTP into the replicative intermediate of polyoma DNA. The replicative intermediate was characterized by centrifugation through neutral and alkaline sucrose gradients, isopycnic banding in a propidiumiodide/cesium chloride gradient, by chromatography on benzoylated naphthoylated DEAE-cellulose and DNA-DNA hybridization. A small but significant amount of radioactivity was incorporated into mature polyoma DNA. The incorporation of radioactivity into polyoma DNA required the presence of dATP, dCTP and dGTP and was stimulated by ATP, Mg 2+ and monovalent cations. A substantial part of the radioactivity was incorporated into short chains during the first five minutes of incubation while after 30 minutes most of the incorporation had occurred into longer chains which, in an alkaline sucrose gradient, sedimented close to 16 s, the sedimentation coefficient of unit-length linear polyoma DNA. The time-course of the reaction was studied at 25, 30 and 35 °C. The incorporation was non-linear at all temperatures but reached a similar plateau value in each case. It is proposed that the nuclei in vitro retained the capacity to elongate replicative intermediate molecules present at the time of their isolation but only had a very limited ability to initiate the replication of new polyoma DNA molecules.

Replication of polyoma DNA in isolated nuclei: II. Evidence for semi-conservative replication

Nuclei from polyoma-infected mouse fibroblast 3T6 cells catalysed the incorporation of [ 3H]dTTP into the replicative intermediate of polyoma DNA. The nature of this process was studied by the introduction of a density label into the viral DNA. For this purpose dBrUTP replaced dTTP in the in vitro system and the product formed in the presence of [ 14C]dATP was analysed by equilibrium centrifugation in neutral CsCl or alkaline Cs 2SO 4 gradients. The results suggest that the density label was incorporated into newly synthesized progeny strands and that the incorporation process represented semi-conservative replication. In a second type of experiment the replicative intermediate was first pulselabelled in vivo with [ 3H]thymidine. The isolated nuclei were then incubated in vitro with dBrUTP and [ 14C]dATP. From an analysis of the distribution of the two isotopes in buoyant density gradients we propose that in vitro synthesis represented elongation of essentially all replicative intermediate molecules present in the nuclei at the time of isolation. From the observed density shifts we could calculate that the initial rate of the in vitro elongation process corresponded to a replication time of about 20 minutes and thus may amount to about 20% of the reported in vivo rate.

The fluid mosaic model of the structure of cell membranes.

A fluid mosaic model is presented for the gross organization and structure of the proteins and lipids of biological membranes. The model is consistent with the restrictions imposed by thermodynamics. In this model, the proteins that are integral to the membrane are a heterogeneous set of globular molecules, each arranged in an amphipathic structure, that is, with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipid. The bulk of the phospholipid is organized as a discontinuous, fluid bilayer, although a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent. Recent experiments with a wide variety of techniqes and several different membrane systems are described, all of which abet consistent with, and add much detail to, the fluid mosaic model. It therefore seems appropriate to suggest possible mechanisms for various membrane functions and membrane-mediated phenomena in the light of the model. As examples, experimentally testable mechanisms are suggested for cell surface changes in malignant transformation, and for cooperative effects exhibited in the interactions of membranes with some specific ligands. Note added in proof: Since this article was written, we have obtained electron microscopic evidence (69) that the concanavalin A binding sites on the membranes of SV40 virus-transformed mouse fibroblasts (3T3 cells) are more clustered than the sites on the membranes of normal cells, as predicted by the hypothesis represented in Fig. 7B. T-here has also appeared a study by Taylor et al. (70) showing the remarkable effects produced on lymphocytes by the addition of antibodies directed to their surface immunoglobulin molecules. The antibodies induce a redistribution and pinocytosis of these surface immunoglobulins, so that within about 30 minutes at 37 degrees C the surface immunoglobulins are completely swept out of the membrane. These effects do not occur, however, if the bivalent antibodies are replaced by their univalent Fab fragments or if the antibody experiments are carried out at 0 degrees C instead of 37 degrees C. These and related results strongly indicate that the bivalent antibodies produce an aggregation of the surface immunoglobulin molecules in the plane of the membrane, which can occur only if the immunoglobulin molecules are free to diffuse in the membrane. This aggregation then appears to trigger off the pinocytosis of the membrane components by some unknown mechanism. Such membrane transformations may be of crucial importance in the induction of an antibody response to an antigen, as well as iv other processes of cell differentiation.