Anchorage-dependent chick embryo fibroblasts synthesise DNA and proliferate when cultured in multilayered sheets suspended among glass fibres.

Chick embryo fibroblasts (CEFs) spontaneously form multicellular and multilayered sheets suspended on the network of glass fibres which are stabilized by fibronectin containing protein deposits located at cell-to-cell contacts. The cells situated within the sheets are surrounded by the neighbouring cells and their mechanical equilibrium is stabilised by intercellular "parabaric" effects. It was found that CEFs in the sheets retain relatively high mitotic activity corresponding to that observed in sparse monolayer cultures. These cells grew up to much higher local density than in confluent and contact-inhibited monolayer cultures and developed an abundance of microfilament bundles that terminated at vinculin-containing protein complexes. The results presented demonstrate that direct contact with solid substratum, cell-to-cell contacts, local cell density, and intercellular exchange of humoral factors are not directly involved in the density-dependent inhibition of growth observed in monolayer cultures. They also support the concepts concerning the role of mechanical equilibrium of cell membrane and sub-membranous cytoskeleton in the regulation of proliferation of non-transformed cells.

Directional movement of rat prostate cancer cells in direct-current electric field: involvement of voltagegated Na+ channel activity.

A two-part hypothesis has been tested, which proposes that (1) prostate cancer cells are galvanotactic (i.e. respond to an electric field by moving directionally) and (2) voltage-gated Na+ channel activity, which was shown previously to be expressed specifically by strongly metastatic cells, controls galvanotaxis. Two well-defined rat ('Dunning') cell lines, originally derived from the same prostate tumour but differing markedly in their metastatic ability, were used. Cells were exposed to exogenous direct-current electric fields of physiological strength (0.1-4.0 V cm(-1)), their reactions were recorded by light microscopy and analysed by a quantitative tracking method. Voltage-gated Na+ channel activity was modulated pharmacologically using a range of concentrations of a specific channel blocker (tetrodotoxin) or an opener (veratridine). The results showed that the highly metastatic MAT-LyLu cells responded to the application of the electric field strongly by migrating towards the cathode. By contrast, the weakly metastatic AT-2 cells gave no such response. Tetrodotoxin suppressed the galvanotactic response of the MAT-LyLu cells whereas veratridine enhanced it. Both compounds had little effect on the AT-2 cells. These results are consistent with functional voltage-gated Na+ channel expression occurring specifically in highly metastatic cells. This is also the first demonstration of control of galvanotaxis, in any cell type, by voltage-gated Na+ channel activity. The possible underlying mechanisms and the in vivo relevance of these findings are discussed.

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.

Locomotion of human skin keratinocytes on polystyrene, fibrin, and collagen substrata and its modification by cell-to-cell contacts.

Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.

Homotypic cell-cell contacts stimulate the motile activity of rat prostate cancer cells.

OBJECTIVE: To characterize the effect of homotypic cell-to-cell collisions upon the motile activities of two rat prostatic cancer cell lines of markedly different metastatic potential. MATERIALS AND METHODS: The movements of strongly and weakly metastatic MAT-LyLu and AT-2 cells, respectively, were recorded under an inverted microscope at 37 degrees C. The motile activities of the cells at various cell densities were characterized quantitatively by computer-aided tracking methods and image analysis. The following variables were assessed: speed of movement, final displacement, coefficient of movement efficiency, diffusion constant and positive flow. RESULTS: MAT-LyLu and AT-2 cells showed only limited motile activity in sparse cultures where there was little contact amongst the cells. However, under these and all other subsequent conditions tested, the motile activity of the MAT-LyLu cells was higher than the AT-2 cells. As the density of the cultured cells was increased (leading to more cell-to-cell contacts) there was a significant increase in motility. This effect was more pronounced for the AT-2 than for the MAT-LyLu cells, resulting in visible acceleration of movement by direct physical contact among the colliding cells. The motile activities of the tumour cells was only slightly affected by conditioned media. CONCLUSION: Homotypic collisions between migrating prostatic cancer cells can strongly stimulate their motility. The effect of increased contact is greater on the weakly metastatic cells, such that at high cell density, the difference in the motilities of weak and strong metastatic cells is greatly reduced.

Contractile elements of Lemna trisulca L. glycerinated cell models during chloroplast translocations.

Electron microscopy of Lemna glycerinated cell models depicts contractile elements during chloroplast translocations. One contractile element, the thin ectoplasmic layer, is < or = 0.4 microm thick, pressed against plasma membrane-cell wall. Thin ectoplasmic layer contains numerous oriented filaments and some appear to be actin and myosin. Another contractile element is the outer chloroplast membrane which envelops each chloroplast and joins or fuses with the thin ectoplasmic layer. Chloroplast interconnections are formed between two or more chloroplasts by outer chloroplast membranes; they enhance chloroplast communications, translocations, and molecular exchanges.

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields.

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300-600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10-40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs.

Contact guidance of chick embryo neurons on single scratches in glass and on underlying aligned human skin fibroblasts.

The influence of substratum topography on the morphology and orientation of neurites of chick embryo neurons was studied. Two series of experiments are reported. One concerned the behaviour of growth cones when the axons become contact-guided by the surface texture. The second studied contact guidance of neurites extending on a compact layer of fixed aligned human skin fibroblasts (HSF). It was observed that when the growth cones of sensory neurons isolated from dorsal root ganglions encountered a single scratch in a glass surface (0.1-2 microm in depth and diameter) they turned and continued movement following the axis of the scratch. These neurons became contact-guided as a result of the sequence of events. The growth cone filopodia recognized the irregularity in the substratum surface, whereas the growth cone lamella stabilized contact with the scratch and moved forward along the scratch axis. Scanning electron microscope revealed that the single scratches 150 nm in width and ca. 100 nm deep growth cone filopodia less than 200 nm in diameter could detect and react by turning into them. These filopodia extensions followed the edge of scratches. However, phase contrast and Nomarski's differential interference contrast appeared insufficient for analysis of primary contact guidance of fine growth cone filopodia which themselves are often less than 200 nm. In neuron cultures on fixed aligned HSF, the neuron aggregates assumed spindle-like shapes, and sparsely seeded individual neurons extended axons along the long axes of the fibroblasts. The axons extended significantly further on the fixed underlying fibroblasts than on collagen-covered glass. In crowded cultures of neurons, the cells extended neurites ignoring both the surface anisotropy (the scratches) and the orientation of the aligned fibroblasts. Immunofluorescence staining of neurons with antibodies against neurofilaments made it possible to analyse their shape and orientation on the fibroblasts. Computer-assisted image analysis permitted the observed alignment of the neurites to be characterized quantitatively.

Effects of cyclosporin A on contractile activity and cytoskeleton in chick embryo cardiomyocytes.

The effects of cyclosporin A (CsA), a clinically used immunosupressive drug, on contractile activity of chick cardiomyocytes grown as small aggregates or explants suspended on a network of elastic glass fibres or cultured in a monolayer were analysed in vitro with computer-aided image cytometry methods. At therapeutic concentrations (200-1500 ng/mL), CsA induced changes in the frequency and amplitude of the beating activity of cardiomyocytes 15 min after application. Longer treatment of cardiomyocytes, for 20-24 h, additionally induced changes in their shape and cytoskeleton organization (F-actin and alpha-actinin distribution). These results indicate that CsA is able to affect directly the contractile activity, morphology, and cytoskeleton architecture of heart cells.

Changes in morphology of human skin fibroblasts induced by local anaesthetics: role of actomyosin contraction.

Local anaesthetics block action potentials in the membranes of excitable cells but their effects on non-excitable cells are less well known. Some local anaesthetics are applied directly onto the skin, and for this reason the effect of procaine (p-aminobenzoic acid diethylamino-etyl ester hydrochloride) and tetracaine (4-[butylamino]benzoic acid 2-[dimethylamino]ethyl ester) upon the morphology and cytoskeleton organisation of human skin fibroblasts was investigated. The time lapse video recording of fibroblasts cultured in serum-enriched medium revealed that the cells rapidly change shape after the addition of the anaesthetic. These effects were fully reversible. The microscopic observations were confirmed by quantitative analysis of projected cell area and cell shape parameters. Local anaesthetics significantly changed the actin cytoskeleton organisation, inducing total disappearance of stress fibres. Serum-starvation or myosin light chain kinase inhibitors, KT 5926 inhibitor (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b,11a-triazadibenzo[a,g]c ycloocta[cde] trinden-1-one or wortmannin, which induce the 'relaxed' morphology of the cells, prevent both the anaesthetic-induced changes in cell shape and the disassembly of stress fibres. Together, the observations suggest that local anaesthetics affect the actomyosin system, inducing contraction.

Morphometric analysis of pancreatic carcinoma by computer-assisted image analysis.

This report describes the results of applying the interactive image analysis system for the measurement of some cytological parameters corresponding to features of adenocarcinoma of the pancreas. The present experiments were carried out by means of the digital cell image analysis of haematoxilyn and eosin stained archival standard glass slides of cancer bearing and healthy patients. Four different parameters describing the morphology of nuclei and nucleoli were selected to quantitate the differences between control and malignant tissues: area, perimeter, elongation, and extension. The parameters that showed the greatest differences between cancerous and normal pancreas were: area and elongation in the case of nuclei as well as area and perimeter for nucleoli. However, the results of this study suggest that none of the four analysed parameters can be selected alone to discriminate neoplastic from normal cells, but could be used all together in diagnosis of pancreatic cancer.

Morphometric analysis of LPS-stimulated human monocytes by computer-assisted image analysis.

This report describes the results of applying the computer-assisted image analysis system for the measurement of some cytological parameters of LPS-stimulated and nonstimulated human monocytes. The experiments were carried out by means of the digital cell image analysis of haematoxilyn stained monocytes. Five different parameters describing the morphology of monocytes and their nuclei were selected to quantitate the differences between control and activated cells area, perimeter, elongation, dispersion, and extension of images of cell projections. The results suggest that all of the analysed parameters can be used to discriminate stimulated from nonstimulated monocytes which permits detailed monitoring of the changes in cell morphology during monocyte activation.

Contact of sarcoma cells with aligned fibroblasts accelerates their displacement: computer-assisted analysis of tumour cell locomotion in co-culture.

The shape and locomotion of rat sarcoma XC cells on glass, polystyrene, and confluent monolayer cultures of aligned human skin fibroblasts were studied with quantitative, computer-assisted methods. The cell shape depended upon the substratum; the sarcoma cells seeded on fibroblasts assumed polarized shapes. The tumour cells emigrating from aggregates and in sparse cultures showed random locomotion when plated on glass or on the polystyrene surface of tissue culture dishes in isotropic conditions. However, when sarcoma cell aggregates were plated onto underlying aligned fibroblasts, the sarcoma cells showed contact guidance, migrating along the long axes of fibroblasts. Simultaneously, suppression of migration normal to the axis of fibroblasts orientation was observed. The sarcoma cells displaced a few times faster on aligned fibroblasts than under isotropic conditions in control cultures. This fast displacement was found to result from the less frequent cell turnings and straightening of cell trajectories (i.e., from klinokinesis), and not from an acceleration of cell movement and the longer cell tracks (i.e., not from orthokinesis). The presented results support the suggestion of Abercrombie (M. Abercrombie. 1979. Nature (London). 281: 259-262.) that tumour cells may be guided by the underlying normal cells when invading surrounding tissues and forming metastases.

Isolation, spreading, locomotion on various substrata, and the effect of hypotonicity on locomotion of fish keratinocytes.

The paper describes improved methods for the isolation of fish skin keratinocytes, which spread and locomote 15 min after trypsinization, in the absence of extracellular matrix proteins. The random locomotion of these keratinocytes under isotropic conditions on glass, plastic (polystyrene), and glass covered with poly-L-lysine or collagen IV was studied with computer-aided methods. Several methods for quantitative description of random cell locomotion were compared. The values of some parameters commonly computed showed non-Gaussian distribution. A comparison of keratinocyte locomotion under isotonic and hypotonic conditions revealed that the hypotonic conditions increased cell displacement (net migration) owing to the klinokinetic and not the orthokinetic effect.

Chemotaxis of Amoeba proteus in the developing pH gradient within a pocket-like chamber studied with the computer assisted method.

A new "U" shaped, pocket-like chamber was used to observe the chemotactic responses of individual cells. This method permits monitoring of both the development of the concentration gradient of a tested substance and cell locomotion. We investigated the chemotactic responses of Amoeba proteus and observed that the amoebae moved in positively and negatively developing [H+] gradients towards the solution of lower pH in a pH range 5.75-7.75. The chemotactic response of amoebae to [H+] gradients required the presence of extracellular calcium ions. It was blocked and random locomotion was restored by the replacement of calcium with magnesium in the cell medium. Time-lapse video recording and data processing were accomplished with computer-assisted methods. This made it possible to compare selected methods of data presentation and analysis for cells locomoting in isotropic and anisotropic conditions. The cell trajectories were determined and displayed in circular diagrams, lengths of cell tracks and final cell displacements were estimated and a few parameters characterizing cell locomotion were computed.

A new model for the research into rhythmic contraction activity of cardiomyocytes in vitro.

Heart cells continue to contract rhythmically after isolation and in culture in vitro. We describe a model of heart preparation in vitro that permits quantitative research on the frequency of contractions of cardiomyocytes. The chick embryo heart explants placed on a network of elastic glass fibers continued beating for months, recorded and analyzed with the methods of computer-assisted image analysis. The efficacy of this experimental model for the screening of effects of various agents on the frequency of contractions was examined by following the effects of nifedipine, caffeine, ethanol, and benzamide. The reversibility of the effects and the reproducibility of results were demonstrated quantitatively. The significance of a mechanical elastic load provided by glass fibers for the preservation of long-lasting contractile activity of cardiomyocytes is discussed and the common occurrence of oscillatory contraction processes in various eucaryotic cells is noted.

Activation of macrophage-like cells by multiple grooved substrata. Topographical control of cell behaviour.

We studied the influence of substrata topography on the behaviour of murine P388D1 macrophage cell line. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves of varying depth and width. Cell spread area, elongation, orientation and F-actin content were measured on plain substratum and 6 sets of gratings. The speed and persistence of cell movement were also studied. We found that patterned substrata substantially activated cell spreading and elongation and significantly increased the persistence and speed of cell movement, shallow grooves being more effective than deep ones. The contact of cells with micropatterned substrata significantly increased the F-actin content in cells. The sensitivity of LPS (lipopolisaccharide) stimulated and unstimulated macrophages to topographical cues was also compared.

Benzamide-induced changes in the actin cytoskeleton in human skin fibroblasts.

The incubation of human skin fibroblasts in the presence of 10 mM benzamide in Joklik's modification of Eagle's Minimal Essential Medium caused an extensive reorganization of actin filaments. The disappearance of stress fibers and changes in cell morphology were observed, whereas no changes in the microtubule architecture were noticed. The observed effects appeared fully reversible within 3 hours after the removal of benzamide. The results are discussed in relation to the two known activities of benzamide as an anaesthetic and an inhibitor of ADP-ribosylation.

An improved method for the isolation of cells from tissues: prevention of cell rounding enhances spreading and locomotion of keratinocytes (epitheliocytes).

Benzamide (10mM) in Ca-free solution anesthetizes small cold blooded animals such as tadpoles of Rana temporaria, Xenopus laevis and an aquarium fish, guppy (Lebistes [Poecilia] reticulatus). Trypsinization of tails of tadpoles in Ca-free, benzamide-supplemented solution permits isolation of keratinocytes (epitheliocytes) maintaining polygonal shapes and prevents cell rounding. Such cells quickly attach to glass, spread and commence locomotion in contrast to those cells which assume spherical shape after standard trypsinization.

Efficacy of the Frame and Hu mathematical model for the quantitative analysis of agents influencing growth of chick embryo fibroblasts.

The experiments on the effect of various sera and substratum surface area upon growth of chick embryo fibroblast-like cells in secondary cultures are described and discussed on the grounds of a mathematical model for growth of anchorage-dependent cells proposed by Frame and Hu [14]. The model and presented results demonstrate the mutual independence of the effects of agents influencing the rate of cell proliferation (i.e. accelerating or retarding growth) and the agents that modify the limitation of cell proliferation (i.e. maximum cell density at confluence). The model proposed by Frame and Hu due to its relative simplicity offers an easy mode of description and quantitative evaluation of experiments concerning cell growth regulation. It is shown that various sera added at constant concentration significantly modify the rate of cell proliferation with little effect upon the maximum cell density attainable. The cells grew much more slowly in the presence of calf serum than in the presence of chick serum and the addition of iron and zinc complexes to calf serum significantly accelerated cell growth. An increase in the substratum surface area by the addition of glass wool to culture vessels significantly increased cell density per constant volume of medium even when retardation of growth was observed. The results presented point to the need of direct cell counting for estimation of cell growth curves and discussion of effects of agents influencing parameters characterizing cell proliferation.