Raimund Wieser

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

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 slight, but significant increase of the cdk4-inhibitor p16. In serum-deprived cells, the protein levels in p27 and p16 remained low. In contrast, we detected a rapid decrease of cyclin D1 and cyclin D3 which did not occur in contact-inhibited cells. These results indicate that serum-deprivation and contact-inhibition have different mechanisms although they affect the same pathway cyclin D – cdk4, pRB, cyclin E – cdk2.

p16INK4 mediates contact-inhibition of growth.

Growth of non-transformed cells in vitro is regulated by density-dependent mechanisms via cell-cell contacts, leading to arrest in late G1-phase at confluency (contact-inhibition of growth). In the present study it is shown that this results from p16INK4-mediated dissociation of the complex cdk4-cyclin D1, which is responsible for the inactivation of the gate keeper of G1-S transition, the retinoblastoma protein pRb. As a consequence of the inactivation of cdk4, downstream the activation of cdk2 and hyperphosphorylation and thus inactivation of pRb was impaired. Direct evidence for the central role of p16INK4 in growth control comes from the observation that a competitive inhibitor of p16INK4 repressed contact inhibition of growth. These findings provide an explanation for the high incidence of mutation or loss of INK4 in human tumours.

Involvement of plasma membrane glycoproteins in the contact-dependent inhibition of growth of human fibroblasts☆

The human embryonal lung fibroblasts used in this study showed a pronounced inhibition of growth when reaching a critical cell density. This effect has been mimicked by the addition of glutaraldehyde-fixed human fibroblasts to sparsely seeded growing cells. Inhibition of growth was not observed when glutaraldehyde-fixed cells were pretreated with galactosidase or with galactose-specific lectins, or when glutaraldehyde-fixed human or rabbit erythrocytes were added to the proliferating fibroblasts. In addition, glutaraldehyde-fixed mitotic cells were without effect on the proliferation, while cells prepared from sparse culture had lesser potency than cells prepared from confluent cultures. Plasma membranes, isolated from cells of confluent cultures, when added to growing cultures of human fibroblasts inhibited DNA synthesis in a concentration-dependent manner. On the other hand, plasma membranes isolated from sparsely seeded cells had only minor inhibitory potency. When the plasma membranes were isolated from cells treated previously with tunicamycin, an antibiotic which inhibits the synthesis of the oligosaccharide portion of asparagine-linked glycoproteins, the inhibitory effect was abolished. The same effect was observed when plasma membranes were pretreated with galactosidase. These data indicate that the growth of cells in vitro is regulated by specific cell-cell contacts. They also show that one of the molecular reactants in this process are membrane glycoproteins with asparagine-linked oligosaccharides.

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

There is evidence that protein kinase C δ (PKCδ) 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 PKCδ 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 α, δ, ε, and μ after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 μM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCδ-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCδ in contact-inhibition. In accordance, the PKCδ specific inhibitor Rottlerin (1 μM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCδ to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCδ in response to cell–cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCδ-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCδ induced a transformed phenotype. We conclude that PKCδ is involved in contact-dependent inhibition of growth.

Cell-contact mediated modulation of the sialylation of contactinhibin

Contactinhibin was found to be involved in contact-dependent inhibition of growth. The growth inhibitory activity of contactinhibin is mediated by N-linked oligosaccharides with desialylated β-glycosidically linked, terminal galactose residues. Here we show that in sparse human fibroblasts contactinhibin was expressed in a biologically inactive, highly sialylated form both on the plasma membrane and intracellularily, while in confluent cells plasma membrane localized contactinhibin was present in a biologically active, low sialylated form. Plasma membranes were shown to contain a glycoprotein sialidase which is suggested to be engaged in the activation of contactinhibin in a cell contact-dependent manner.

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

The treatment of human diploid fibroblasts with tumor necrosis factor (TNP)‐α and with lymphotoxin (LT) is associated with induction of interleuk‐in‐6 (IL‐6) transcripts with TNF‐α being 10‐fold more potent than LT. Here we report on the TNF‐α/LT‐induced signaling mechanisms responsible for the regulation of IL‐6 gene expression in these cells. Run‐on assays demonstrated that both TNF‐α and LT increase IL‐6 mRNA levels by transcriptional activation of this gene. Stability studies of IL‐6 transcripts in fibroblasts showed that TNF‐α delayed IL‐6 mRNA decay but not LT. The induction of IL‐6 transcripts by TNF‐α and LT was not inhibited by the isoquinoline sulfonamide derivative H7. Similarly, depletion of protein kinase C (PKC) by 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) did not change the ability of TNF‐α and LT to induce IL‐6 transcripts, demonstrating that stimulation by these agents may not be mediated by activation of PKC. Stimulation of IL‐6 transcripts in fibroblasts did also not require new protein synthesis as exposure to the protein synthesis inhibitor cycloheximide (CHX) enhanced accumulation of IL‐6 mRNA in the presence or absence of TNF‐α or LT.

Identification of plasma membrane glycoproteins involved in the contact-dependent inhibition of growth of diploid human fibroblasts

Growth of normal, nontransformed cells is regulated by the interplay between growth stimulating compounds and growth inhibiting cell-cell contacts. We have previously shown that the growth of normal diploid human fibroblasts is mainly regulated by a specific class of plasma membrane glycoproteins (R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361-367). Because it was found that immobilization of the glycoproteins involved in contact-dependent inhibition of growth is an essential step in the recovery of the biological activity of the glycoproteins, we developed a technique for a first characterization of the active compounds. After SDS-PAGE separation of plasma membrane glycoproteins, they were transferred onto nitrocellulose. The nitrocellulose was cut along the separation track into circles which fit into wells of a 96-well microtiter plate. Culturing human diploid fibroblasts on the nitrocellulose circles resulted in characteristic growth patterns, which were dependent upon the source and the treatment of the plasma membrane proteins which had been separated. Five major inhibitory fractions with apparent molecular masses of 300, 170, 90, 50, and 25 kDa have been identified in plasma membranes from confluent fibroblast cultures.

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

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 not been fully characterized, our studies give evidence for the involvement of a plasma membrane glycoprotein with an apparent molecular weight of approximately 80 kDa in the growth regulation of diploid human fibroblasts. The important characteristic of this glycoprotein is: the biologically active determinant resides in terminal, beta-glycosidically linked galactose residues on N-glycosidically linked glycans. From our studies, a receptor has to be postulated which, in addition to the galactose residues, has additional structural requirements for the specific binding of this glycoprotein, since other glycoproteins carrying terminal, beta-glycosidically linked galactose-residues are without biological activity. The postulated receptor is suggested to be defective in tumor cells, since these cells are no longer able to respond to cell-cell contacts with stopped proliferation, although they are able to inhibit growth of non-transformed cells. The inability of a tumor cell to recognize and to bind to the specific glycoprotein would result in a release from growth inhibition, leading to clonal growth of these cells. Further detailed studies on the structure and the regulation of the glycoprotein, as well as an attempt to isolate the postulated receptor, should lead to a better understanding of the complex pattern of growth regulation of normal cells.

Immobilized glycolipids from human diploid fibroblasts inhibit DNA synthesis of cultured human fibroblasts.

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 gangliosides were isolated and on the pretreatment of the immobilized gangliosides: Preincubation with DMEM without FCS of immobilized gangliosides, isolated from confluent cultures, resulted in a 75% inhibition of growth rate of embryonal human lung fibroblasts (FH109) cultured on immobilized gangliosides. Under the same conditions, gangliosides from sparse cultures reduced the growth rate by about 30%. On the other hand, the degree of inhibition exerted by immobilized gangliosides isolated from confluent cultures was found to be greatly reduced by preincubation with DMEM with FCS, whereas the slight inhibition of growth rate, exerted by gangliosides from sparse cultures, was found to be reversed into a slight stimulation of growth rate after preincubation with complete medium. Concomitantly with the reduction of the inhibition of DNA synthesis, it was found that the complete medium, used for preincubation of the gangliosides, was no longer able to support DNA synthesis to the same extent as untreated complete medium. The data suggest that gangliosides bind growth-supporting factors of the serum, gangliosides isolated from sparse cultures being more potent in the binding of these molecules than gangliosides isolated from dense cultures.

Chemotactic migration of human diploid fibroblasts is inhibited by contactinhibin.

Dear Editor: Cellular migration is required for a variety of biological processes such as organ formation or tissue remedeling after tissue injury. In order to function as a controlled process, at least two prerequisites have to be fulfilled: i) migration must be directional and it) migration must be stopped when the original size of a tissue has been achieved. Directional migration or chemotaxis is induced by well defined chemoattractants such as lymphokines (7), collagenous peptides (8), fibronectin (3) and PDGF (9). In addition, only cells directly facing a free space or a wound are allowed to respond to chemoattraetants with migration, while cells surrounded by neighboring cells are migration inhibited (1,2,6). After rebuilding injured tissue by invasion and division of cells, a negative migration--and proliferation signal must be generated. It has long been accepted that cell-cell contacts are required for a stop of migration, although the molecular basis has since not yet been identified. We have shown previously that contactinhihin, a 60-70 kDa plasma membrane glycoprotein is responsible for contact-dependent inhibition of growth (11). Therefore, experiments were undertaken to address the question whether contactinhibin might also be involved in the inhibition of chemotactic migration. Table 1 shows, that the degree of chemotactic migration of human fibroblasts was inversely correlated to the cell density seeded in the upper compartment of the Boyden chamber. At 2 × 105 cells seeded onto the filter, the cell density most commonly used in similar studies, approximately 17% of the cells have migrated after 4 hours. This is in good agreement with experiments where human fibroblasts have been used (9). Chemically transformed mouse fibro-