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In order to understand the factors determining the length of fibroblasts, three cell lines (mouse embryonic fibroblasts plus human fibroblast lines AGO 1523 and M19) were cultivated on the usual planar substrate (glass) and on specially prepared narrow linear strips of the same substrate, where the cells could spread only linearly. Morphometric measurements showed that the average length of cells of each type on the 'unidimensional' strips was no different from that on the usual 'bidimensional' substrate. The addition of colcemid significantly decreased cell length on both substrates, whereas cytochalasin D increased the length. We concluded that fibroblasts have an intracellular mechanism maintaining a relatively constant average cell length. This mechanism may involve the dynamic balance of centripetal and centrifugal forces developed by two cytoskeletal systems: the microtubules and the actin-myosin cortex. Three epitheliocyte cell lines (rat IAR2, canine MDCK and bovine FBT) were tested but, in contrast to fibroblasts, they did not maintain similar cell lengths on the usual substrate and on the linear strips, suggesting that control of length is cell-type-specific.

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Behaviour of epitheliocytes and fibroblasts on special discontinuous substrata (metallic grids with square openings of 45x45 microm2) was examined in order to compare the ability of these cells to spread in two mutually perpendicular directions and to stretch over the void spaces. Two cell types with typical fibroblastic morphology, the AGO 1523 line of human foreskin fibroblasts and secondary cultures of mouse embryo fibroblasts, and three cell types with typical epithelial morphology, primary mouse hepatocytes, the IAR-2 line of rat liver cells and the MDCK line of canine kidney epithelial cells (clone 20) were used. We also examined the epitheliocytes (MDCK cells, clone 20) transformed to fibroblast-like morphology by treatment with hepatocyte growth factor/scatter factor (HGF/SF). Time-lapse video microscopy, scanning electron microscopy and immunofluorescence microscopy were used to examine cell reorganizations at various stages of spreading. It was found that early stages of spreading of fibroblasts and epitheliocytes were similar: the cell spread along two bars, perpendicular to each other (bar and crossbar), with the formation of a small triangular lamellar cytoplasm stretched over the opening. Later central parts of the bodies of the fibroblasts retracted from the bars so that the cells remained attached only by their polar lamellae. Successive expansions and partial retractions of these lamellae led to elongation of the cell body crossing several openings of the grid. Epitheliocytes, in contrast to fibroblasts, at the late stages of spreading did not retract their bodies and did not contract polar lamellae. As a result, their central lamellae stretched progressively over the openings. As a result of the treatment of MDCK epitheliocytes with HGF/SF the behaviour of the cells on the grids became similar to that of fibroblasts. It is suggested that these distinct spreading patterns of epitheliocytes and fibroblasts are due to the type-specific differences in the actin-myosin cortex. Experiments with microtubule-specific drugs, colcemid and taxol, indicate that the organization of this cortex is under microtubular control.

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This review summarizes data on cellular and molecular mechanisms underlying phenotypical characteristics of tumor cells that determine their ability for invasion. These mechanisms include dysregulation of adhesive interactions of tumor cells with each other and with extracellular matrix, protease production, locomotion reactions of tumor cells, and induction of angiogenesis in tumor. Data on structure and functions of transmembrane adhesion molecules and their ligands, molecular composition of adhesion structures (intercellular and focal contacts), and role of adhesion molecules as transducers of intracellular signals are considered. Alterations of expression of adhesion molecules and cytoplasmic proteins in adhesion structures and hyperphosphorylation of these molecules by oncogene products are described as a precondition of invasion activity of tumor cells. The contact interaction between circulating tumor cells and vascular endothelium is considered as the important stage of the metastatic process. Secretion of proteases by tumor cells and regulation of their activity by specific stromal inhibitors are described. Function of motogens in the acquisition by a tumor cell of locomotor phenotype facilitating invasion and impairments of topographic reactions of cells playing an important role in the invasion are considered. Attention is given to mechanisms of neoangiogenesis in the tumor providing additional ways for dissemination of tumor cells.

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Fragments of parietal and visceral pleura were studied by total films preparation, light microscopy and SEM at different times after intrapleural injection of asbestos in Wistar rats. Pleural rat mesothelium in histological slices consists normally of one layer of oblong cells. By SEM the cells are flat and coated with microvilli of different lengths. In total films the parietal mesothelium was composed of large polygonal cells covering intercostal spaces and small cells covering spaces over the ribs. Inflammatory reaction and permanent pathological regenerative processes were observed in the mesothelium during 24 months after inoculation of asbestos fibres. Different lesions which we regarded as preneoplastic or premesotheliomatous were observed against the background of or without these processes. They were diffuse irregular hyperplasia and proliferation of epithelium-like or fibroblast-like cells and focal nodous proliferates composed of such cells with various morphological structures. The number of thymidine-labelled cells was significantly more inside the proliferates than in the surrounding tissue. They were confirmed by SEM and histological slices of the same fields. Chronic pathological regeneration of pleural mesothelium could be the background against which preneoplastic lesions and mesotheliomas develop easily.

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The influence of the cylindrical substratum surface on the actin microfilament bundle and the extracellular matrix (fibronectin and laminin) patterns in nontransformed epithelial cells of the IAR-2 rat line and in their N-ras-transformed descendants, IAR-ras-c4 cells, was studied. The cells were cultured on substrata with cylindrical surfaces-fused quartz glass fibers with a diameter of 32 microm. Quantitative analysis of actin microfilament bundle alignment and immunomorphological study of fibronectin and laminin were used. IAR-2 epitheliocytes on the cylindrical substrata formed straight actin microfilament bundles and fibronectin- or laminin-positive fibrils aligned predominantly transversely to the cylinder axis. In contrast, in the majority of IAR-ras-c4 cells on the cylindrical substrata, the revealed straight microfilament bundles and the fibrils of the extracellular matrix were oriented approximately longitudinally to the cylinder axis; a small part of the transformed cells formed microfilament bundles and extracellular matrix patterns similar to those in the normal epitheliocytes on the cylindrical substrata. These results show that transformed epitheliocytes that acquired polarized morphology react to the curvature of the cylindrical substratum surface by actin cytoskeleton and extracellular matrix reorganization changes essentially different from those characteristic of normal discoid epitheliocytes on the cylindrical substrata, but similar to those observed in normal polarized cells, e.g., fibroblasts.

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Cylindrical culture substrata are known to induced longitudinal orientation of polarized fibroblasts and corresponding alignment of actin microfilament bundles in these cells. We studied microfilament bundle distribution in two cell types, fibroblasts and epitheliocytes, spread on two kinds of anisotropic substrata, quartz glass cylinders with a diameter 32 microns and narrow (25-40 microns wide) flat glass adhesive strips with non-adhesive borders. Rat embryo and human diploid fibroblasts, as expected, formed predominantly longitudinally aligned bundles on both substrata. In contrast, transverse bundles on cylinders and randomly oriented bundles on flat strips were formed in IAR-2 and MDCK epithelial cells. We interpret these data as showing that the epitheliocyte attempts to override the guiding influence of anisotropic substrata. The microfilament bundle pattern on cylinders depends on the integrity of the microtubules. Colcemid-induced microtubule depolymerization caused formation of longitudinal as well as transverse bundles both in fibroblasts and epitheliocytes, thus diminishing the differences in microfilament bundle patterns in two cell types. These results show that microtubules control the cell-type-specific distribution of microfilament bundles both in polarized fibroblasts and in discoid epitheliocytes. However, the results of this control are opposite: microtubules enhance cell polarization in fibroblasts, but prevent it in epithelial cells.

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The standard preparation of cell suspensions (e.g., blood cells, cell suspensions derived from monolayer cultures or from various tissues, etc.) for scanning electron microscopy (SEM) includes fixation of the cells in suspension and subsequent dehydration and critical point drying (CPD) of the cells after their preliminary attachment to the special substrata. In the course of the SEM examination of cell suspensions of various origins, unusual morphological cell surface structures, filopodia-like protrusions (FLP), were consistently detected in 3-20% of the cells in the populations. FLP can be effectively observed only by using a stage tilt angle of no less than 30 degrees. FLP were single or multiple thin threads extending from basal parts of a spherical cell and attaching to the substratum surface used. FLP strikingly resembled substratum-attached filopodia formed by a viable cell at its earliest stages of spreading. The percentages of the cells with FLP were not significantly affected by the character of cell fixation (primary aldehyde fixation alone or primary aldehyde with subsequent OsO4 post-fixation) or the raising of the temperature and pressure inside the CPD bomb. It seems that the protrusions imitating the natural cell surface structures can probably be formed at later stages of the preparation of cell suspensions for SEM, namely during dehydration and (or) CPD when the cells undergo substantial shrinkage. One of the possible mechanisms by which the cell shrinkage could induce FLP formation follows. A prefixed spherical cell which settles down to the substratum sticks to it at some discrete points on the cell surface. As a result of the subsequent cell shrinkage, FLP could be formed and then stretched, connecting the same points on the cell surface with the points of the initial cell-substratum adhesion.

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The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.

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Mouse embryo fibroblasts were cultivated on special substrates with discontinuous surfaces. The substrates were silicon plates with multiple vertical (65-90 microns height) spike-like silicon microcrystals evenly distributed on the plate surfaces. It was shown that the cells were successfully spread and flattened on these substrates. The spread cells formed several discrete attachment zones at the tops and side surfaces of the spikes; these zones were separated from one another by distances considerably greater than the diameter of the unspread cell. At early stages of spreading the unspread cells attached to the tops of single spikes and extended long filopodia attached to the distant spikes. At later stages the lamellae were formed between the filopodia: probably these filopodia served as guidelines for extension of lamellae and progressive cell spreading. These experiments demonstrated that continuity of substrate surface is not a necessary condition for advanced cell spreading.

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The distribution and shape of rat cells cultivated on plastic surfaces containing cylindrical areas of various radii and grooves of various depths were studied. Normal embryo cells and two lines of neoplastic cells (LW13K2 and RsK4) were used. The nuclear shape, orientation of the nuclei and migration of cells from the bottom of the grooves were assessed by quantitative methods. All characteristics of the behaviour of both neoplastic cell lines on the grooved substrates were found to be different from those of normal cells: a) the nuclei of neoplastic cells, in contrast to those of the normal ones, did not undergo additional elongation on the cylindrical areas of the substrate; b) the orientation of neoplastic cells with regard to the axis of the cylindrical substrate was decreased or absent; c) the migration of neoplastic cells from certain types of the grooves was decreased. It is suggested that the different reaction to the geometry of the substrate may be a characteristic feature of transformed cells. Possible mechanisms of these alterations are discussed.

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Attachment of the cells of mouse ascitic hepatoma to various substrata was examined with the aid of SEM. Ascitic cells did not attach themselves in vitro to the mesothelium-covered surface of peritoneum. However, these cells were attached to the surface of peritoneum from which mesothelial layer had been removed. In the course of growth of ascitic tumor in vivo attached ascitic cells on the peritoneal surface were seen only in the areas of stomata but not in the mesothelium-covered areas. Ascitic cells taken from the peritoneal fluid adhered poorly to the glass surface in culture. However, adhesiveness of these cells to the glass and their ability to spread on the glass surface increased considerably after cultivation in vitro. This alteration of adhesiveness was completely reversible: the cultured cells transplanted intraperitoneally restored their poor adhesive properties. It is suggested that non-adhesiveness of mesothelial surface is the main factor preserving suspended state of intraperitoneally growing ascitic cells. Depending on the environment, ascitic cells may undergo reversible changes of adhesiveness.

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Isolated cultures of mouse L-cells are similar to those of normal cells in showing contact inhibition of movement and topoinhibition of growth. In mixed cultures with untransformed mouse embryo fibroblasts, their parent strain, however, L cells are able to form colonies above the monolayer of normal fibroblasts, i.e., they have a property characteristic of transformed cells. Analysis of microcinematographic data suggests that the behavior of L cells in mixed cultures is a result of their defective attachment to the substratum. Scanning electron microscopy showed that attachment of a normal fibroblast was accompanied by the formation of a wide ring of flattened cytoplasm spread on the substratum (lamellar cytoplasm). This structure was observed to disintegrate in newly attached L cells. The structure of the lamellar cytoplasm remained abnormal in the fully spread L cells. The mean area of lamellar cytoplasm was 3- to 4-times less in L cells than in normal fibroblasts. It is suggested that deficient formation of lamellar cytoplasm may be the basis of the inability of L cells to interact normally with embryo fibroblasts.

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