Interferon-gamma synergises with tumour necrosis factor and lymphotoxin-alpha to enhance the mRNA and protein expression of adhesion molecules in mouse brain endothelial cells

Changes to the cerebral microvasculature are evident during cerebral malaria (CM). Activation of the endothelium is likely to be due to the actions of cytokines, circulating levels of which are elevated during CM. Endothelial cells are known to up-regulate the expression of cellular adhesion molecules, which can lead to cellular sequestration and obstruction of vessels. However, it is unknown whether cytokines synergise in the up-regulation of the adhesion molecules involved in CM. In this study, the mRNA and/or protein expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin and E-Selectin were examined in a mouse brain endothelial cell line. Endothelial cells were stimulated with interferon-gamma (IFN-gamma), tumour necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha), alone or in combination. The expression of ICAM-1, VCAM-1, P-selectin and E-Selectin mRNA in mouse brain endothelial cells by TNF and/or LT-alpha was found to be significantly enhanced in the presence of IFN-gamma. The same synergistic effect was found when analyzing ICAM-1 protein expression in cytokine stimulated mouse brain endothelial cells. The findings show that cytokines can synergise to influence gene expression and protein expression in a mouse brain endothelial cell line.     

Integrity of actin fibers and microtubules influences metastatic tumor cell adhesion

Tumor cell adhesion within host organ microvasculature, its stabilization and invasion into host organ parenchyma appear to be important steps during formation of distant metastasis. These interactions of circulating tumor cells with the host organs occur in the presence of fluid shear forces and soluble and cellular environmental conditions of the blood that can modulate their cellular responses and possibly their metastatic efficiency. Cytoskeletal components, such as actin filaments and microtubules, can regulate biophysical characteristics and cellular signaling of the circulating cells. Therefore, we investigated the role of these cytoskeletal structures for early steps during metastasis formation in vivo and in vitro. Using an intravital observation technique, tumor cell adhesion of colon carcinoma cells within the hepatic microcirculation of rats and their invasion into liver parenchyma was observed. Disruption of actin filaments increased cell adhesion, whereas tubulin disruption inhibited adhesive interactions in vivo. The impairment of the cytoskeleton modulated adhesion-mediated cell signaling via focal adhesion kinase (FAK) and paxillin under flow conditions in vitro. In the presence of fluid flow, focal adhesions were enlarged and hyperphosphorylated, whereas stress fibers were reduced compared to static cell adhesion. Disruption of microtubules, however, partially inhibited these effects. Combining the in vivo and in vitro results, our study suggested that changes in cell rigidity and avidity of cell adhesion molecules after disruption of cytoskeletal components appear to be more important for initial adhesive interactions in vivo than their interference with adhesion-mediated cellular signal transduction.     

Overexpression of the myristoylated alanine-rich C-kinase substrate inhibits cell adhesion to extracellular matrix components.

Mice lacking the myristoylated alanine-rich C-kinase substrate, or MARCKS protein, exhibit abnormalities consistent with a defect in the ability of neurons to migrate appropriately during forebrain development. To investigate the possibility that this phenotype could be due to disruption of normal cellular adhesion to extracellular matrix, an assay was developed in which 293 cells co-expressing MARCKS and green fluorescent protein were tested for their adhesion competence on various substrates. Fluorescence-activated cell sorting of adherent and non-adherent green fluorescent protein-expressing cells demonstrated that wild-type MARCKS inhibited adhesion of cells to fibronectin, whereas a non-myristoylated mutant did not inhibit adhesion of cells to a variety of substrates. The fibronectin competitive inhibitor RGD peptide inhibited adhesion of cells expressing all MARCKS variants equally. Cytochalasin D inhibited the adhesion of cells expressing non-myristoylated MARCKS, but did not further decrease the adhesion of cells expressing adhesion-inhibitory proteins. Confocal microscopy demonstrated the presence of inhibitory, myristoylated MARCKS at the plasma membrane, suggesting that localization at this region might be important for MARCKS to inhibit cellular adhesion. These data suggest a possible myristoylation-dependent function of MARCKS to inhibit cellular adhesion to extracellular matrix proteins, indicating a potential mechanism for the cell migration defects seen in the MARCKS-deficient mice.     

Effect of E-cadherin on activation of MAP-kinase by growth factors in human carcinoma cells

Met and EGF receptors can induce a decrease in intercellular adhesion and an increase in cell motility, which is a cause of metastatic progressions. Therefore, mechanisms of interaction in receptor tyrosine kinase and proteins of intercellular contacts attract the attention of researchers. The main protein that provides cellular adhesion is E-cadherin. Earlier, we have shown that the intracellular Met localization was dependent on function of E-cadherin. In the present work, we have found that localization of the EGF receptor also was determined by adhesion stability. Loss of intercellular contacts in HBL-100 cells leads to the EGF receptor being not stabilized at the cell membrane. A comparative study of MAP kinase activation by growth factors was carried out in cells differing by their intercellular adhesion states. It has been established that E-cadherin is able to modulate level and duration of activation of ERK kinase. The presented results allow for the suggestion to be made that not only intracellular localization, but also the intracellular signal pathway activated by Met and EGF receptors, depend on the E-cadherin function, which in turn can determine the specificity of cellular response.     

Impedance studies of bio-behavior and chemosensitivity of cancer cells by micro-electrode arrays

Impedimetric analysis on adherently growing cells by micro-electrodes provides information related to cell number, cell adhesion and cellular morphology. In this study, cell-based biosensor with micro-electrode arrays (MEAs) was used to monitor the culture behavior of mammalian cancer cells and evaluate the chemosensitivity of anti-cancer drugs using electrochemical impedance spectroscopy. The platinum electrode arrays were fabricated by semiconductor technology to a 10 x 10 pattern, with diameter of 80 microm of each electrode. The human oesophageal cancer cell lines (KYSE 30) were cultured on the surface of the electrodes with the pre-coated fibronectin, the connecting protein for tumor cells metastasis and adhesion in extracellular matrix. Morphology changes during cells adhesion, spreading, and proliferation can be detected by impedimetric analysis in a real time and non-invasive way. Cisplatin was added to cells for potential drug screening applications. The experimental results show that this well-known anti-cancer drug has characteristic chemosensitivity effects on KYSE 30 cells which can be detected by MEA. Thus, this cell-based chip provides a useful analytical method for cancer research.     

Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion

The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice.     

Specificity of intercellular adhesion mediated by various members of the immunoglobulin supergene family

The immunoglobulin supergene family members have been shown to be involved in cell-cell recognition and interaction during cell growth and differentiation. Neural cell adhesion molecule, myelin-associated glycoprotein, and carcinoembryonic antigen (CEA) are immunoglobulin supergene family members which can mediate cell adhesion. We show here that nonspecific cross-reacting antigen (NCA), a closely related CEA family member, is found on the surface of rodent cells transfected with functional NCA complementary DNA in different glycosylated forms, all of which can be deglycosylated to an Mr 35,000 core protein. Furthermore, NCA can mediate Ca2(+)-independent, homotypic aggregation of these NCA-producing transfectant cells. Since CEA has three internal repeated C2-set, immunoglobulin-like domains, whereas NCA has one, only one such domain is required for the intercellular adhesive function. We also demonstrate that NCA- and CEA-producing transfectants can form heterotypic aggregates, whereas mixtures of CEA or NCA transfectants and neural cell adhesion molecule or long form-myelin-associated glycoprotein transfectants sort themselves out into homotypic aggregates. The results suggest that subsets of the immunoglobulin superfamily, such as the CEA family, can be used in both homotypic and heterotypic cellular interactions, whereas less closely related members of the family can be used to separate different cell types by strictly homotypic interactions.     

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.     

A novel role for atypical MAPK kinase ERK3 in regulating breast cancer cell morphology and migration

ERK3 is an atypical Mitogen-activated protein kinase (MAPK6). Despite the fact that the Erk3 gene was originally identified in 1991, its function is still unknown. MK5 (MAP kinase- activated protein kinase 5) also called PRAK is the only known substrate for ERK3. Recently, it was found that group I p21 protein activated kinases (PAKs) are critical effectors of ERK3. PAKs link Rho family of GTPases to actin cytoskeletal dynamics and are known to be involved in the regulation of cell adhesion and migration. In this study we demonstrate that ERK3 protein levels are elevated as MDA-MB-231 breast cancer cells adhere to collagen I which is concomitant with changes in cellular morphology where cells become less well spread following nascent adhesion formation. During this early cellular adhesion event we observe that the cells retain protrusive activity while reducing overall cellular area. Interestingly exogenous expression of ERK3 delivers a comparable reduction in cell spread area, while depletion of ERK3 expression increases cell spread area. Importantly, we have detected a novel specific endogenous ERK3 localization at the cell periphery. Furthermore we find that ERK3 overexpressing cells exhibit a rounded morphology and increased cell migration speed. Surprisingly, exogenous expression of a kinase inactive mutant of ERK3 phenocopies ERK3 overexpression, suggesting a novel kinase independent function for ERK3. Taken together our data suggest that as cells initiate adhesion to matrix increasing levels of ERK3 at the cell periphery are required to orchestrate cell morphology changes which can then drive migratory behavior.     

A duplexed microsphere-based cellular adhesion assay

Interactions of integrin cellular adhesion molecules with matrix proteins play important roles in complex bidirectional signaling pathways. To investigate these interactions, a novel flow-cytometry-based cellular adhesion assay has been developed. Based on the concept of microcarrier cell culture, derivatized polystyrene microspheres (9.6 microm) are used as a substrate for the immobilization of type I collagen to which cells then adhere. Using cytometric detection, the extent of cellular adhesion can be precisely determined by comparison of adhered and nonadhered populations based on the side scatter properties of the microspheres. In combination with immunostaining, the novel format of this assay enables the correlation of adhesive function to other cellular characteristics such as surface expression. In this work, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to stimulate increased adhesion in Chinese hamster ovary cells stably transfected with the collagen receptor integrin alpha2beta1. Multiple clones of varying expression distributions were analyzed, and correlations of adherent populations versus receptor distributions show a threefold increase in functional cellular adhesion to collagen upon treatment with TPA. Probability binning analysis of duplexed data revealed subtle changes in adhesion versus receptor distribution mediated by TPA which otherwise would not have been detectable.     

Developmentally regulated cell-cell adhesion in Dictyostelium purpureum is mediated by a glycoprotein synthesized in nonadhesive cells

Upon starvation the cellular slime mold, Dictyostelium purpureum, develops a form of cell-cell adhesion aiding in the formation of large multicellular aggregates, which are capable of further differentiation. The molecule that mediates this adhesion is a glycoprotein of Mr approximately 40,000. The protein shares a common carbohydrate epitope with another well-characterized cell adhesion molecule from Dictyostelium discoideum, contact sites A, but the polypeptides to which it is attached differ for each species. Although mediating a developmental form of adhesiveness, the protein is synthesized in vegetative cells at a time when they do not adhere. Most of the vegetative protein is associated with cell membranes and appears to be on the surface of these cells. The protein is compared to other cell adhesion molecules from other species of cellular slime molds, and possible explanations for its inability to function in vegetative cells are discussed.     

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).     

Guidance of Mesenchymal Stem Cells on Fibronectin Structured Hydrogel Films

Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.     

Cell surface adhesion molecules and adhesion-initiated signaling: understanding of anoikis resistance mechanisms and therapeutic opportunities

Cells express various cell surface adhesion molecules (receptors) that not only mechanically serve as contacting sites between the cell and extracellular matrix (ECM) or adjacent cells, but also initiate intracellular signaling pathways modulating important cellular events including survival and proliferation. Normal cells undergo apoptosis when lacking ECM attachment. This type of cell death has been termed anoikis. Anoikis can be viewed as a normal process which ensures tissue homeostasis and failure to execute the anoikis program or resistance to anoikis could result in adherent cells surviving under suspension condition and proliferating at ectopic sites where the matrix proteins are different from those the cells originally contact. Resistance to anoikis is emerging as a hallmark of metastatic cancers which enables cancer cells to disseminate to distant organs through systemic circulation. In this review, we will discuss the molecular basis of adhesion-initiated signaling, the impact of loss of cell-ECM adhesion on normal cell survival, the role of cancer cell aggregate formation via intercellular adhesion under non-adherent condition, and mechanisms of anoikis resistance developed in metastatic cancer cells. Understanding of these aspects will provide opportunities to find new potential molecular targets, and therapeutic strategies based on these findings will likely prove to be more specific and effective.     

Sensing scaffolds to monitor cellular activity using impedance measurements

Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.     

A Computational Model for Collective Cellular Motion in Three Dimensions: General Framework and Case Study for Cell Pair Dynamics

Cell migration in healthy and diseased systems is a combination of single and collective cell motion. While single cell motion has received considerable attention, our understanding of collective cell motion remains elusive. A new computational framework for the migration of groups of cells in three dimensions is presented, which focuses on the forces acting at the microscopic scale and the interactions between cells and their extracellular matrix (ECM) environment. Cell-cell adhesion, resistance due to the ECM and the factors regulating the propulsion of each cell through the matrix are considered. In particular, our approach emphasizes the role of receptors that mediate cell-cell and cell-matrix interactions, and examines how variation in their properties induces changes in cellular motion. As an important case study, we analyze two interacting cells. Our results show that the dynamics of cell pairs depends on the magnitude and the stochastic nature of the forces. Stronger intercellular stability is generally promoted by surface receptors that move. We also demonstrate that matrix resistance, cellular stiffness and intensity of adhesion contribute to migration behaviors in different ways, with memory effects present that can alter pair motility. If adhesion weakens with time, our findings show that cell pair break-up depends strongly on the way cells interact with the matrix. Finally, the motility for cells in a larger cluster (size 50 cells) is examined to illustrate the full capabilities of the model and to stress the role of cellular pairs in complex cellular structures. Overall, our framework shows how properties of cells and their environment influence the stability and motility of cellular assemblies. This is an important step in the advancement of the understanding of collective motility, and can contribute to knowledge of complex biological processes involving migration, aggregation and detachment of cells in healthy and diseased systems.