Translocation of cdk2 to the nucleus during G1-phase in PDGF-stimulated human fibroblasts.

1997

We studied the subcellular distribution of cdk2 in synchronized, PDGF-stimulated human fibroblasts (FH109). After contact inhibition and serum depletion, more than 95% of FH109 cells were arrested in G0/G1-phase. PDGF-AB led to a 16-fold increase in proliferation compared with untreated cells. Cell cycle progression was studied by flow cytometric analysis, [3H]thymidine incorporation, and phosphorylation of the retinoblastoma gene product, pRB. Using Western blot analysis after subcellular fractionation, we revealed that after PDGF stimulation the phosphorylated (Thr 160), i.e., activated, form of cdk2 (33 kDa) first appeared in the nucleus at late G1-phase and persisted throughout until to…

Reduction of glutathione content by 12-O-tetradecanoylphorbol-13-acetate in confluent, but not in sparse cultures of human diploid fibroblasts.

1990

Treatment of confluent cultures of human diploid fibroblasts with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-7) M) resulted in a 70% reduction of the glutathione (GSH) content, compared with untreated controls. The effect, which was dose-dependent, was observed 8 h after the beginning of the treatment could be followed for up to 72 h. On the other hand, GSH reduction was specific for confluent cultures, as the level of glutathione remained unchanged by TPA treatment of sparse cultures. The addition of immobilized plasma membrane proteins to sparsely seeded cells has been shown previously to induce cellular reactions which are characteristic for confluent cultures. It was shown that TPA…

Loss of Contact-Dependent Inhibition of Growth in Chemically Transformed Fibroblasts

1988

The plasma membrane has been recognized as an important regulatory unit of mammalian cells during determination, differentiation, and social behaviour of individual cells within various tissues (1–4). On the molecular level, plasma membrane glycoproteins and glycolipids have been shown to be involved in these processes (1–4). Density-dependent growth of non-transformed cells in vitro has been proposed to be regulated by secreted inhibitory compounds (5–7), by the cell’s shape (8) or by diffusion boundary layers (9). On the other hand, specific cell-cell interactions via cell membrane molecules were found to be of great importance for the contact-dependent inhibition of growth (10–16) and co…

Chemotactic migration of human diploid fibroblasts is inhibited by contactinhibin.

1992

Control of Behaviour and Growth by Imitating the Contact Environment

1983

The behaviour of cells in vitro is the result of all the influences exerted by the actual microenvironment which, in a simplified form, is composed of two main compartments (Fig. 1): (i) the diffusive environment, (ii) the contact environment. These two compartments regulate the cell behaviour by distinct mechanisms induced by molecules which, beside being compartment-specific, differ in their mobility.

Immobilized glycolipids from human diploid fibroblasts inhibit DNA synthesis of cultured human fibroblasts*1

1987

Several previous studies have shown that glycolipids isolated from plasma membranes of cultured cells and added to cells in culture inhibit the growth rate in a concentration-dependent fashion. In order to investigate the possible involvement of glycolipids in the growth regulation of normal cells by cell-cell contacts, we tested the effect of immobilized glycolipids, isolated from human fibroblasts, on the DNA synthesis of freshly seeded fibroblasts. Gangliosides inhibited DNA synthesis to a great extent, whereas neutral glycolipids had only a minor effect. The degree of inhibition of DNA synthesis by immobilized gangliosides depended both on the cell density of the cultures from which the…

Isolation and characterization of a 60-70-kD plasma membrane glycoprotein involved in the contact-dependent inhibition of growth

1990

Previous studies have shown that plasma membrane compounds are involved in the contact-dependent inhibition of growth of human diploid fibroblasts. The purification of the active plasma membrane glycoprotein is described in this report. The glycoprotein has an apparent molecular mass of 60-70 kD and, due to differential sialylation, isoelectric points between pH 5.5. and 6.2. Treatment with sialidase yielded one spot in two-dimensional gel electrophoresis with an isoelectric point of 6.3. After removal of the N-glycosidically linked oligosaccharide chains, the apparent molecular mass is reduced by approximately 22 kD. Treatment was diluted NaOH, which removes the O-glycosidically linked por…

Contact-dependent inhibition of growth of normal diploid human fibroblasts by plasma membrane glycoproteins.

1988

Homeostasis in vivo is maintained by a highly complex network of positive and negative signals. At the cellular level, this regulatory microenvironment can be divided, in a simplified fashion, into two major compartments: the humoral compartment, including compounds such as hormones, growth factors and nutrients, and the contact-environment compartment, including cell-cell and cell-matrix interactions. At least in cultures of diploid, non-transformed cells, cell-cell and cell-matrix interactions have been shown to be of major importance for the regulation of growth as well as of differentiation. Although until now the glycoprotein involved in the contact-dependent inhibition of growth has n…

Plasma membrane glycoproteins covalently bound to silica beads as a model for molecular studies of cell-cell interactions in culture.

1987

Abstract In previous studies, we have shown that plasma membrane glycoproteins are of major importance in the density-dependent regulation of growth of normal diploid fibroblasts. Due to the hydrophobic portions of these molecules, functional studies in cell culture are often diffucult to perform and to interpret. Specially, the addition of these molecules in soluble form to cell culture, after depletion of detergents needed for their solubilization, leads to aggregation and internalization. Therefore, we developed a method for the covalent immobilization of the solubilized plasma membrane proteins to derivatized silica beads for further investigations on the molecular nature of the active …

Differential regulation of interleukin-6 expression in human fibroblasts by tumor necrosis factor-alpha and lymphotoxin.

1990

The treatment of human diploid fibroblasts with tumor necrosis factor (TNF)-alpha and with lymphotoxin (LT) is associated with induction of interleukin-6 (IL-6) transcripts with TNF-alpha being 10-fold more potent than LT. Here we report on the TNF-alpha/LT-induced signaling mechanisms responsible for the regulation of IL-6 gene expression in these cells. Run-on assays demonstrated that both TNF-alpha and LT increase IL-6 mRNA levels by transcriptional activation of this gene. Stability studies of IL-6 transcripts in fibroblasts showed that TNF-alpha delayed IL-6 mRNA decay but not LT. The induction of IL-6 transcripts by TNF-alpha and LT was not inhibited by the isoquinoline sulfonamide de…

Involvement of protein kinase Cdelta in contact-dependent inhibition of growth in human and murine fibroblasts.

2001

There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryosta…

Differences in the mechanisms of growth control in contact-inhibited and serum-deprived human fibroblasts

1997

In the present work we studied mechanisms of growth control in contact-inhibited and serum-deprived human diploid fibroblasts. The observation that the effects on [3H]thymidine incorporation and reduction of retinoblastoma gene product-phosphorylation were additive when contact-inhibition and serum-deprivation were combined led us to the conclusion that the underlying mechanisms might be different. Both contact-inhibition and serum-deprivation led to a strong decrease of cdk4-kinase-activity and cdk2-phosphorylation at Thr 160, while the total amounts of cdk4 and cdk2 remained constant. In contact-inhibited cells, we revealed a strong protein accumulation of the cdk2-inhibitor p27 and a sli…

Rottlerin induces a transformed phenotype in human keratinocytes.

2001

PKCdelta plays a fundamental role in cell cycle control. Consistent with its proposed tumour suppressor function, ras transfection of the human keratinocyte cell line HaCaT results in a loss of PKCdelta expression mediated by TGFalpha (Exp. Cell Res., 219, 299, 1995). To get more insight into the role of PKCdelta in keratinocytes, we investigated the effects of Rottlerin, a specific inhibitor of protein kinase Cdelta, in HaCaT cells. After Rottlerin treatment, HaCaT cells lost their cobble-stone morphology and displayed a spindle-shaped, fibroblastic phenotype. Additionally, the establishment of cell-cell contacts was prevented. This was caused by an internalization of E-cadherin and beta-c…

Separation of plasma membrane proteins of cultured human fibroblasts by affinity chromatography on bonded microparticulate silicas.

1984

Abstract Adsorbents for high-performance affinity chromatography were prepared by bonding proteins and reactive Procion triazine dyes to 3-isothiocyanatopropyl- and 3-aminopropylsilicas. The materials prepared were used successfully in the separation of hydrophobic plasma membrane proteins of cultured human fibroblasts. The data obtained show that the reaction of 3-isothiocyanatopropyltriethoxysilane (ITCPS) with the surface hydroxyl groups of silica yields a new and convenient route to preparing an “activated carrier” that is capable of coupling with potential affinity ligands containing amino functional groups. The reaction and bonding procedures of 3-isothiocyanatopropyltriethoxysilane a…

Induction of Cell Differentiation in Transformed Keratinocytes by Synthetic (Glyco)peptides from the Homophilic Recognition Domain of E-Cadherin

2002

Synthetische (Glyco)peptide aus der homophilen Erkennungsregion von E-Cadherin zur Induktion von Zelldifferenzierung in transformierten Keratinocyten

2002

Growth control in mammalian cells by cell-cell contacts.

1990

Growth of normal diploid mammalian cells in vitro is strongly regulated by the actual cell density. Cell-cell contacts via specific plasma membrane glycoproteins whose glycan moieties interact with specific receptors has been found to be a main growth regulatory principle. Malignant growth is suggested to result from impaired function of these receptors.

Subcellular distribution of ras in human and murine fibroblasts.

1996

Abstract Ras proteins play a significant role in signal transduction in response to growth factors and in cell transformation. To be active, ras has to be translocated to the cell membrane. Since subcellular distribution has been mainly studied in vector-transformed cells which highly express ras proteins, and it has been difficult to detect ras in cells expressing the protein at physiological levels, we studied subcellular distribution in human and murine fibroblasts. Here we show for the first time that a significant amount of ras is associated with the membrane skeleton and the cytoskeleton.