Desmosome Formation Research Articles

Ultrastructural observations on effects of different concentrations of calcium and thyroxine in vitro on larval epidermal cells of Rana catesbeiana tadpoles.

During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5 mM) calcium (Ca2+) in the absence or presence of thyroxine (10(-7) M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05 mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When tissues were exposed to high levels of calcium (0.5 mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca2+ concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca2+ leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca2+ concentrations (0.5 mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and differentiation in Rana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells.

Na,K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility.

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an alpha- and beta-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin-mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and beta(1)-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase beta(1)-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin-mediated cell-cell adhesion requires the Na,K-ATPase beta-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the beta(1)-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.

Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes.

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

New Function for NF1 Tumor Suppressor

The expression and subcellular localization of neurofibromatosis type 1 tumor suppressor was studied in keratinocytes induced to differentiate by increased Ca2+ concentration of the culture medium. Differentiating keratinocytes became intensely immunoreactive for neurofibromatosis type 1 protein, which was apparently associated with cellular fibrils. Double immunolabeling with antibodies to cytokeratin 14 and neurofibromatosis type 1 protein suggested an association of intermediate type cytoskeleton and neurofibromatosis type 1 protein. The presence of neurofibromatosis type 1 protein in cell preparations treated with cytoskeletal buffer indicated a high affinity interaction between intermediate filaments and neurofibromatosis type 1 protein. Further studies utilizing double immunolabelings revealed that the intense neurofibromatosis type 1 tumor suppressor signal on intermediate filaments was temporally limited to the period in keratinocyte differentiation in which the formation of desmosomes takes place. Keratinocytes were also cultured from nine patients with type 1 neurofibromatosis and were studied with respect to cell morphology, and association of neurofibromatosis type 1 protein with intermediate cytoskeleton. The results showed that keratinocytes cultured from patients with neurofibromatosis type 1 displayed a highly variable cell size and morphology compared to controls. The latter findings represent predicted alterations in a situation where cytoskeletal organization is disturbed. Furthermore, differentiating neurofibromatosis type 1 keratinocytes were characterized by a reduced number of cytokeratin bundles that were decorated neurofibromatosis type 1 protein. The results of this study suggest that neurofibromatosis type 1 tumor suppressor exerts its effects in part by controlling organization of cytoskeleton during the formation of cellular contacts.

Ultrastructural characterization of brain tumors using collagen gel culture

In an attempt to investigate the tumor type‐specific ultrastructure of cultured brain tumors, collagen gel culture was utilized instead of the conventional monolayer culture. In order to avoid intermingling of the normal brain cells, tumors with a clear margin were chosen. The tumors were cultured first in the monolayer culture for a few passages, and then tumor cell pellets were prepared and embedded in the collagen gel. In the event that tumors failed to grow in the monolayer culture, small fragments of tumor tissue were embedded. Ultrastructurally, the tumor cell processes of central neurocytoma contained numerous dense‐core vesicles. The formation of desmosomes was observed in the meningioma cell processes. The development of polypoid microvilli similar to those of the normal choroid plexus epithelium was observed in a case with ependymoma. A continuous basement membrane was seen surrounding the tumor cell processes facing the collagen gel in two ependymomas. These findings suggest the usefulness of the collagen gel culture in analyzing the tumor type‐specific ultrastructure of cultured brain tumors and possibly in studying cellular differentiation.

Transmembrane signaling for adhesive regulation of desmosomes and hemidesmosomes, and for cell-cell datachment induced by pemphigus IgG in cultured keratinocytes: involvement of protein kinase C.

We have investigated transmembrane signaling for the regulation of desmosomes and hemidesmosomes, using a human squamous cell carcinoma cell line (DJM-1) and normal human keratinocytes. This review discusses the involvement of protein kinase C (PKC) in regulation of these junctions, and signaling pathways involved in cell-cell detachment induced by pemphigus vulgaris (PV) IgG in a culture system. Cells grown in low-Ca++ conditions, which lack desmosomes, rapidly form desmosomes upon a low-normal Ca++-shift in association with PKC-activation and, in turn, PKC-activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces desmosome formation even in low-Ca++ conditions. TPA induces serine-phosphorylation of the 180 kDa-bullous pemphigoid antigen (BPAG2), generating 190 kDa-phosphorylated BPAG2, and dissociates BPAG2 from hemidesmosomes. TPA-treatment also causes secretion of urokinase-type plasminogen activator (uPA) and expression of its receptor (uPAR), which activates plasminogen to plasmin and may digest extracellular domains of desmosomes and hemidesmosomes. These results suggest that PKC may play a role in activation of desmosome turnover and dysfunction of hemidesmossomes, and thus a role in up-migration of keratinocytes. Binding of PV-IgG to Dsg3 induces activation of diverse isoenzymes of PKC, linked to uPA secretion and uPAR expression. Furthermore, PV-IgG binding alone induces the serine-phosphorylation of Dsg 3, associated with its dissociation from plakoglobin and its deletion from desmosomes. This PV-IgG-induced Dsg 3-phosphorylation and Dsg 3-deletion from desmosomes may impair desmosome formation, whereas PV-IgG-induced PKC signaling mediates the uPA secretion and uPAR expression leading to digestion of preexisting desmosomes from the outside of the cell. These two different PV-IgG-activated signaling pathways may play a key role in acantholysis in PV.

Human keratinocytes cultured without a feeder layer undergo progressive loss of differentiation markers.

Culture of keratinocytes in conventional medium without a mesenchyme-derived feeder layer leads to poor growth and impaired differentiation; however, the exact pathway and degree of differentiation achieved in such conditions is unclear. We have cultured normal human keratinocytes in Rheinwald and Green's medium, on plastic without a feeder layer, in order to investigate the degree of differentiation that they achieve in these conditions. Intermediate filament proteins, tonofibrils and desmosomes were assumed as markers of differentiation and their expression was analyzed by immunohistochemistry and electron microscopy. Before reaching confluence, keratinocytes expressed keratin molecules, as well as vimentin, and formed tonofibrils and desmosomes. The expression of these markers was progressively reduced until confluence and was totally lost thereafter, while cultures could be propagated for at least six passages. On the contrary, reseeding on a feeder layer after the first passage led to rapid cell death. It could be concluded that signals from a feeder layer are relevant to support continuous synthesis of intermediate filaments proteins and formation of tonofibils and desmosomes, and that the derangement of the cytoskeleton in these conditions leads to altered, not simply defective, response to delayed stimulation by a feeder layer.

Pemphigus Vulgaris-IgG Causes a Rapid Depletion of Desmoglein 3 (Dsg3) from the Triton X-100 Soluble Pools, Leading to the Formation of Dsg3-Depleted Desmosomes in a Human Squamous Carcinoma Cell Line, DJM-1 Cells

In this study, we examined desmoglein (Dsg) 3 and other desmosomal molecules after pemphigus vulgaris (PV)-immunoglobulin G (IgG) binding to the Dsg3 on the cell surface in DJM-1 cells, a human squamous cell carcinoma cell line. After cells were incubated with PV-IgG for various time periods (0, 5, 10, 20, 30, 60 min, or 30 h), cells were fractionated into phosphate-buffered saline soluble (cytosol), phosphate-buffered saline insoluble-Triton X-100 soluble (membrane), and Triton X-100 insoluble (cytoskeleton) fractions, and subjected to immunoblotting and immunofluorescence microscopy using antibodies against Dsgl, Dsg3, plakoglobin, desmoplakin 1, and cytokeratins. Immunoblot analysis with PV-IgG revealed that Dsg3 was already dramatically depleted from the membrane fraction 20 min after PV-IgG treatment, whereas no reduction of Dsg3 was detected in the cytoskeleton fraction as examined by immunoblotting. A 30 h incubation with PV-IgG, however, caused a marked disappearance of Dsg3, but not other desmosomal molecules, from cytoskeleton fractions. Furthermore, double-staining immunofluorescence microscopy revealed that Dsg3 was depleted from the desmosomes whereas Dsg1, desmoplakin 1, plakoglobin, and keratin filaments were bound to desmosomes. These results provide a novel interpretation for a better understanding of mechanisms for blistering in PV; i.e., a possibility that PV-IgG generates the formation of aberrant desmosomes, which are lacking in Dsg3, but not other desmosomal constituents.

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

H-7 stimulates desmosome formation and inhibits growth in KB oral carcinoma cells

Protein kinase inhibitor H-7 was reported to stimulate desmosome formation in normal keratinocytes and to inhibit proliferation of neural cell lines. In the present study, the effects of this inhibitor on adhesion and growth of KB human oral carcinoma cells were investigated. H-7 was found to enhance desmosome assembly, as evidenced by an increased punctate labeling for the major desmosomal markers. Immunogold labeling confirmed the formation of desmosomes both at the cell surface and in the cytoplasm. In order to assess cell proliferation and possible correlation with adhesion, confluent cultures were treated and both adherert and detached cell fractions were counted. Under serum-free conditions, H-7 significantly reduced cell detachment. In contrast, EGF stimulated cell detachment, and this effect was abolished when cells were simultaneously treated with both EGF and H-7. Total cell counts were also significantly reduced by H-7, both in the presence and absence of EGF. Using the TUNEL technique, labeled cells were increased after H-7 treatment, thus implicating protein kinase inhibition in cell death. These results indicate that H-7 inhibits growth and stimulates adhesion of KB carcinoma cells.

Desmosome formation by KB cells after treatment with H-7

Desmosome formation by KB cells after treatment with H-7

Stem cells and rat liver carcinogenesis: contributions of confocal and electron microscopy.

Microscopic analysis in combination with cytochemistry and immunocytochemistry has revealed the presence of four cell types not previously described in the portal area and parenchyma of the liver from an experimental rodent hepatocarcinogenic rat model. Within the intrahepatic bile ductules, which proliferate after administration of chemical carcinogens and partial hepatectomy, small, undifferentiated nonpolarized, nonepithelial cells with a blast-like phenotype and polarized epithelial cells different from the polarized epithelial cells that typically line the walls of the bile ductules were found. In the connective tissue stroma surrounding the bile ductules, nonpolarized epithelial cells with hepatocyte phenotype were found. In the parenchyma, subpopulations of bile ductule epithelial cells that established ATPase-positive bile canalicular structures, including the formation of desmosomes and tight junctions, with parenchymal hepatocytes within the hepatic lobule were found. These observations raise the following questions in this model. Are there undifferentiated progenitor cells with stem cell-like properties within bile ductules? What are the interrelations of the newly described cell types with each other, with parenchymal hepatocytes, with preneoplastic nodules, and with hepatomas? Do the heterogeneous cell types within the bile ductules, in the surrounding connective tissue, and within the hepatic cords represent intermediate stages of single or multiple cell lineage pathways leading to hepatocyte differentiation, liver regeneration, and/or preneoplastic nodule formation?

Desmosome formation in cultured Hailey-Hailey keratinocytes cannot be impaired by UV-B radiation

Desmosome formation in cultured Hailey-Hailey keratinocytes cannot be impaired by UV-B radiation

Plakoglobin induces desmosome formation and epidermoid phenotype in N‐cadherin‐expressing squamous carcinoma cells deficient in plakoglobin and E‐cadherin

Pg is a homologue of β-catenin and Armadillo, the product of the Drosophila segment polarity gene and has been shown to have both adhesive and signaling functions. It interacts with both classic and desmosomal cadherins. Pg interaction with the desmosomal cadherins is essential for desmosome assembly. Its precise role in the classic cadherin complexes is unclear, although Pg-E–cadherin interaction appears to be necessary for the formation of desmosomes. In addition to cadherins in adhesion complexes, Pg interacts with a number of proteins involved in regulation of cell differentiation and proliferation such as Lef-1/Tcf-1 transcription factors and the tumor suppressor protein APC. In this study, we have introduced Pg cDNA into SCC9 cells, a Pg- and E-cadherin-deficient squamous cell carcinoma line, which also lacks desmosomes. These cells have both α-catenin and β-catenin, display unusual expression of N-cadherin, and have the typical fibroblastic phenotype of transformed cells. Pg-expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation coincided with the appearance of an epidermoid phenotype, with increased adhesiveness and a contact-dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of N-cadherin and a decrease in level and stability of β-catenin, without any apparent effects on α-catenin. These results show that, in the absence of E-cadherin, Pg can efficiently use N-cadherin to induce desmosome formation and epidermoid phenotype. They also suggest a role for Pg as one of the regulators of the intracellular β-catenin levels and underscore the pivotal role of this protein in regulating cell adhesion and differentiation. Cell Motil. Cytoskeleton 40:87–100, 1998. © 1998 Wiley-Liss, Inc.

Plakoglobin induces desmosome formation and epidermoid phenotype in N-cadherin-expressing squamous carcinoma cells deficient in plakoglobin and E-cadherin.

Pg is a homologue of beta-catenin and Armadillo, the product of the Drosophila segment polarity gene and has been shown to have both adhesive and signaling functions. It interacts with both classic and desmosomal cadherins. Pg interaction with the desmosomal cadherins is essential for desmosome assembly. Its precise role in the classic cadherin complexes is unclear, although Pg-E-cadherin interaction appears to be necessary for the formation of desmosomes. In addition to cadherins in adhesion complexes, Pg interacts with a number of proteins involved in regulation of cell differentiation and proliferation such as Lef-1/Tcf-1 transcription factors and the tumor suppressor protein APC. In this study, we have introduced Pg cDNA into SCC9 cells, a Pg- and E-cadherin-deficient squamous cell carcinoma line, which also lacks desmosomes. These cells have both alpha-catenin and beta-catenin, display unusual expression of N-cadherin, and have the typical fibroblastic phenotype of transformed cells. Pg-expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation coincided with the appearance of an epidermoid phenotype, with increased adhesiveness and a contact-dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of N-cadherin and a decrease in level and stability of beta-catenin, without any apparent effects on alpha-catenin. These results show that, in the absence of E-cadherin, Pg can efficiently use N-cadherin to induce desmosome formation and epidermoid phenotype. They also suggest a role for Pg as one of the regulators of the intracellular beta-catenin levels and underscore the pivotal role of this protein in regulating cell adhesion and differentiation.

Membranous Ultrastructure of Human Arachnoid Cells

The ultrastructure of arachnoid cell membranes was investigated by conventional transmission EM and by freeze-fracture techniques in human arachnoid granulations. Arachnoid cells showed widespread membrane specialization in the granulations including the formation of desmosomes, gap junctions, tight junctions, intermediate junctions, hemidesmosome-like structures, and micropinocytotic vesicles. However, the extent of the specialization varied from portion to portion; this was clearly shown on freeze-fracture replicas. Numerous extracellular cisterns were separated by cytoplasmic bodies or slender processes, joined by these junctional complexes. Uncoated and coated vesicles were abundant along the surface of extracellular cisterns representing the pathway of CSF. Complexes of branching tight junctions were comprised of 1-50 particle strands, which formed elaborate meshworks accompanied by numerous gap junctions and desmosomes. Micropinocytotic vesicles were often concentrated in the arachnoid cell cluster up to 40 per microm2, which is equivalent to the concentration in brain capillary endothelial cells. The results of this study clearly suggest that arachnoid cells in arachnoid granulations are not only tightly adherent to form a firm structure for the passage of CSF, but that the arachnoid cells lining the CSF pathway show intense cell-cell communication and pinocytotic activity. This high transcellular activity probably reflects active transports or secretion of certain molecules by arachnoid cells.

Cross-talk between adherens junctions and desmosomes depends on plakoglobin.

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either beta-catenin or plakoglobin, which is linked to alpha-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell-cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of beta-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.

Regulation of desmosomal cell adhesion in human tumour cells by polyunsaturated fatty acids.

Desmosomes are key structures in cell-cell adhesion. In this study we examined the effect of n-6 essential fatty acids on the expression of desmoglein (Dsg), desmosomal cadherin and the formation of desmosomes in E-cadherin negative human breast, colon and lung cancer cells and melanoma cells. Electron microscopy revealed that cells cultured with gamma linolenic acid (GLA) showed increased cell-cell adhesion together with an increase in the formation of desmoglein-containing desmosomes. Western blotting studies of cellular proteins demonstrated that, following culture with fatty acids, Dsg expression was modified, with the greatest increase seen after GLA treatment. Other fatty acids increased Dsg expression, but to a lesser extent. It is concluded that GLA regulates desmosome-mediated cell-cell adhesion in human cancer cells, particularly in cells without E-cadherin.

Effect of suramin on differentiation of human stomach cancer cell lines.

This study was designed to demonstrate that differentiation of stomach cancer cells can be modified by microenvironmental change and to look for a method inducing or promoting tumor cell differentiation. To evaluate the biomorphological characterization of tumor cell differentiation in suramin-containing in vitro culture of human stomach cancer cell lines, inverted phase-contrast microscopic examination, analysis of growth curves and BrdU-positive S-phase fraction, immunocytochemical study, radioimmunoassay for CEA, transmission electron microscopic examination, DNA flow cytometry, and heterotransplantation in SCID mice were performed. Suramin inhibited tumor cell growth. Development of intracytoplasmic lumina and intercellular lumina was noted in suramin-containing culture with formation of numerous microvilli and frequent desmosomes. The amount of CEA released by a cell was increased in suramin-containing culture. Suramin inhibited heterotransplantation, and a transplant from suramin-containing culture revealed a much higher degree of differentiation than that from suramin-absent culture. Suramin induced no change in DNA ploidy pattern. Elimination of suramin from the culture medium did not reverse the tumor cell differentiation. Each stomach cancer cell line showed a different degree of responsiveness to suramin. In conclusion, this study shows that suramin inhibits growth of SNU-5 and SNU-16 cells and that suramin induces differentiation of SNU-16 cells.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes.

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.