Growth factor-induced proliferation of human fibroblasts in serum-free culture depends on cell density and extracellular calcium concentration

Human neonatal skin fibroblasts plated sparsely in MCDB 105 traversed a complete cell cycle in the absense of serum or serum-derived proteins. Addition of pure PDGF did not significantly increase entrance into S phase as revealed by ³H-thymidine labeling index or clonal growth on palladium islands. In subphysiologic Ca²⁺ concentrations or in the presence of a calmodulin inhibitor, W7, proliferation in the absence of growth factors ceased and PDGF became mitogenic. In contrast, confluent fibroblast cultures were stimulated by PDGF in physiologic Ca²⁺ concentrations. This was also the case with sparse adult skin fibroblast cultures while a fetal strain entered S in the absence of PDGF even in low extracellular Ca²⁺ concentrations. EGF gave similar results as PDGF in all experiments performed. This proposes a similar role for the two growth factors in the cell cycle. However, a difference in the mechanisms of action of PDGF and EGF is indicated by the fact that PDGF and EGF were additive at optimal concentrations when maximal growth response by a single growth factor was restricted by a subphysiologic extracellular Ca²⁺ concentration.     

Binding of thrombin to normal and transformed fibroblasts depends on cell density

Binding of biologically active [¹²⁵I]thrombin to several normal and transformed human and chicken cell lines was found to depend on cell density; more [¹²⁵I]thrombin per cell was bound to sparse than to dense cultures. When normal and transformed cells were compared at equal densities the previously reported difference in [¹²⁵I]thrombin binding was not evident.     

The influence of virus transformation and cell population density on some membrane properties of mouse fibroblasts in culture

The influence of cell population density and simian virus 40 transformation on the activity of the Na-K pump was studied in mouse fibroblasts cultured in medium supplemented with fetal bovine serum. The activity of the Na-K pump was determined from K+ influx, ethacrynate-sensitive K+ influx, (Na+ + K+)-ATPase assay, and the determinations of intracellular potassium and sodium ion concentrations in these cells. The activity of the Na-K pump was found to decrease in density-inhibited cultures of normal fibroblasts (designated as 3T3 cells), while in the virus-transformed cells (SV3T3) the activity remained fairly constant at all cell population densities.     

Growth inhibition of mitogen-stimulated fibroblasts induced by double-stranded RNA depends on cell density

Polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA, is an inhibitor of mitogen-induced proliferation of normal fibroblasts. We show that this inhibition depends strongly on cell density. While cultures with densities at or above confluence are completely inhibited by poly(I:C) in their proliferative response to epidermal growth factor (EGF), the proliferation of sparse (subconfluent) cultures is only delayed. Conditioned medium from dense fibroblasts exposed to poly(I:C) inhibits EGF stimulation of sparse cells, indicating that the inhibition is, at least in part, mediated by a factor released from the cells. Preincubation of quiescent cultures with poly(I:C) renders the cells refractory to the inhibitory effects of poly(I:C). This desensitization correlates with a decreased production of the inhibitor. Since the inhibition of mitogenic stimulation by poly(I:C) is completely overcome by antisera recognizing interferon-beta (IFN-beta) and interleukin-6 (IL-6), we tested the effect of IL-6 and IFN-beta on EGF mitogenicity. None of the available IL-6 preparations had any effect on cell cycle entry. IFN-beta caused a dose-dependent delay of cell division but did not affect the density-dependent proportion of cells entering the cell cycle in response to EGF. Thus, IFN-beta cannot be the sole mediator of the poly(I:C)-induced inhibition. In the presence of dexamethasone, poly(I:C) did not inhibit EGF mitogenis. Indeed, the combined presence of poly(I:C) and dexamethasone did more than just restore the density-dependent control levels of EGF stimulation; most cells entered the cell cycle even at extremely high cell densities. Thus, poly(I:C) in combination with dexamethasone could deactivate the cell density-dependent negative control of proliferation.     

Intracellular compartmentation of deoxycytidine nucleotide pools in S phase mouse 3T3 fibroblasts

We labeled mouse 3T3 fibroblasts, synchronized in G0 or S phase, from [3H]cytidine or [3H]deoxycytidine and measured the flow of isotope into and through deoxycytidine nucleotide pools, including the two deoxyliponucleotides dCDP choline and dCDP ethanolamine. Compared to G0 cells, S phase cells had much larger pools with a 20-40-fold faster turnover. The dCTP pool of S phase cells during steady state conditions attained a 6-fold higher specific activity than the pool of G0 cells when labeled from cytidine but a 10-fold lower specific activity when labeled from deoxycytidine. The dCTP pool of G0 cells showed a slow but measurable turnover indicating a limited amount of de novo synthesis also in resting cells. The labeling pattern of dCTP and deoxyliponucleotides of G0 cells was compatible with a simple precursor-product relationship. In S phase cells, however, dCDP choline had a 4-6 times higher specific activity during steady state conditions than dCTP and dCMP when the cells were labeled with [3H]deoxycytidine. We suggest that 3T3 cells contain two distinct intracellular dCTP pools, one labeled preferentially from cytidine and used for DNA replication, the other labeled from deoxycytidine and used for deoxyliponucleotide synthesis. We speculate that the latter pool during S phase may be temporarily sequestered in the cell's membrane fraction before equilibration with the much larger dCTP pool originating in S phase cells from the reduction of CDP.     

Bradykinin-mediated cell proliferation depends on transactivation of EGF receptor in corneal fibroblasts

In previous studies, bradykinin (BK) has been shown to induce cell proliferation through BK B2 receptor (B2R) via p42/p44 MAPK in Statens Seruminstitut Rabbit Corneal Cells (SIRCs). In addition to this pathway, EGFR transactivation pathway has been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we further investigate whether these transactivation mechanisms participating in BK-induced cell proliferation in SIRCs. Using an immunofluorescence staining and RT-PCR, we initially characterize that SIRCs were corneal fibroblasts and predominantly expressed B2R by BK. Inhibition of p42/p44 MAPK by the inhibitors of Src, EGFR, and Akt or transfection with respective siRNAs prevents BK-induced DNA synthesis in SIRCs. The mechanisms underlying these responses were mediated through phosphorylation of Src and EGFR via the formation of Src/EGFR complex which was attenuated by PP1 and AG1478. Moreover, BK-induced p42/p44 MAPK and Akt activation was mediated through EGFR transactivation, which was diminished by the inhibitors of MMP-2/9 and heparin-binding EGF-like factor (HB-EGF). Finally, increased nuclear translocation of Akt and p42/p44 MAPK turns on early gene expression leading to cell proliferation. These results suggest that BK-induced cell proliferation is mediated through c-Src-dependent transactivation of EGFR via MMP2/9-dependent pro-HB-EGF shedding linking to activation of Akt and p42/p44 MAPK in corneal fibroblasts.     

Shedding of gangliosides from tumor cells depends on cell density

The ganglioside composition of mouse ascites hepatoma ( MAH ) cells, the ascites fluid and cell-conditioned media were determined and found to be qualitatively identical, but quantitatively different. The ganglioside content of the ascites fluid and the medium conditioned by MAH -cells at the native cell concentration (10(8) cells/ml) comprised respectively 74.9% and 23% of the cell-associated gangliosides. When incubated at lower cell-density (10(6) cells/ml) the cells were found to be release about three-times higher amounts of ganglioside per cell than during incubation at the native concentration. Centrifugation of the dense-cell-conditioned medium revealed the major part of the released gangliosides to be associated with a 150000 X g pellet that probably contains shed plasma membrane fragments. In the 150000 X g pellet of the extracellular fluids the relative content of the most polar cell ganglioside corresponding chromatographically to GT1b was about ten-times higher than in the cells. The possibility is raised that the more intense shedding of gangliosides from less crowded MAH cells may play a role in the self protection of the tumor from host immune rejection during initial stages of growth.     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

Cellular density regulation of plasminogen gene expression in mouse hepatocytes

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.     

Regulation of collagen VI expression in fibroblasts. Effects of cell density, cell-matrix interactions, and chemical transformation

Collagen VI expression was studied in cultured human skin fibroblasts and mouse 3T3 cells using cDNA probes specific for alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. A 2-3-fold increase of these mRNAs was observed when fibroblasts were grown at increasing densities while only minimal changes occurred for the mRNA levels of collagens I and III, fibronectin, and beta-actin. Changes in mRNA correlated well with an increased production of corresponding proteins as determined by immunological assays. A comparable increase of alpha 1(VI) and alpha 2(VI) but not of alpha 3(VI) chain mRNAs was found for fibroblasts grown in a three-dimensional collagen gel after gel contraction. These conditions resulted, however, in a decrease of steady-state levels of collagens I and III and actin mRNAs. Transformation of 3T3 cells by phorbol ester did not change collagen VI mRNAs but caused a 3-5-fold reduction in mRNA levels for the other extracellular matrix proteins. These data strongly imply different regulatory mechanisms for the expression of collagen VI compared with collagens I and III and fibronectin. The differences may be correlated to changes in cell shape and reflect the requirement for collagen VI as a cell-binding protein.     

Cellular autophagocytosis in mouse seminal vesicle cells in vitro

Cellular autophagocytosis was observed in mouse seminal vesicle cells incubated in vitro up to 8 h in medium 199 or Krebs-Ringer bicarbonate buffer. During the first 2 h of incubation, early forms of autophagic vacuoles were seen in the cells, advanced forms containing degraded material began to cumulate later. After 6--8 h, early vacuoles occurred sparsely, while advanced forms were detected in a great number. During the first 2 h of incubation, we often observed smooth surfaced membrane pairs between the cisternae of rough surfaced endoplasmic reticulum resembling isolating membranes of autophagic vacuoles. They varied in size and shape from short, straight cisternae to long, curved ones, almost completely encircling areas of the cytoplasm. Based on these observations, we propose a tentative scheme of the formation of autophagic vacuoles, viz., the short, straight cisternae would represent the first stage in the development of an autophagic vacuole, while the curved sack-like forms are interpreted as successive steps leading to the complete sequestration of an area of the cytoplasm.     

Effects of sera types and cell density on the concentration of cyclic nucleotides in normal and simian virus transformed mouse fibroblasts

The effects of serum and cell density on the concentration of cyclic AMP, cyclic GMP in normal mouse fibroblasts cells (3T3 cells) and their Simian Virus 40 transformed derivative (SV3T3 cells) were studied. 3T3 cells grown in 10% foetal bovine serum exhibit density dependent inhibition of growth and associated with this in an increase in the concentration of cyclic AMP, a decrease in the concentration of cyclic GMP and an increase in the ratio (cyclic AMP/cyclic GMP) of the cyclic nucleotides. 3T3 cells grown in 10% newborn calf serum exhibit a higher saturation density and this is associated with a low concentration of cyclic AMP and a high concentration of cyclic GMP. SV3T3 cells grown in either 10% foetal bovine serum or 10% newborn calf serum show high saturation densities and this is associated with a low and decreasing concentration of cyclic AMP and a high concentration of cyclic GMP. When the level of the cyclic AMP in both cell lines was artificially raised by adding dibutyryl cyclic AMP and theophylline to the growth media, the cells grew to low densities.