Morphometric characteristics such as cell area, dispersion, elongation and orientation were studied in normal and transformed fibroblasts, and in epitheliocytes cultured on flat or cylindrical substrata. Cylindrical surfaces with a high degree of curvature (12-13 or 25 microns radii) were shown to affect cell size, shape and alignment. The reaction of the cells to the curvature of cylindrical substrata was different in various cell types studied and depended on the pattern of actin microfilament bundles. The cells containing pronounced straight actin bundles (mouse embryo fibroblasts at the polarization stage of spreading, single spread cells of the ‘normal’ epithelial FBT line or the fully transformed epithelial IAR 6–1 line) were relatively resistant to bending around a cylindrical substratum, and became elongated and oriented along the cylinder. Cells with circular actin bundles as the predominant pattern (mouse embryo fibroblasts at the radial stage of spreading, single spread cells of ‘normal’ epithelial IAR 20 line) and cells with insufficient or no actin bundles (transformed fibroblastic L line) were prone to bending around a cylinder with much less pronounced elongation and orientation along its axis. The data obtained indicate that the reaction of cultured cells to the geometry of the substratum surface and, in particular, to a cylindrical surface is determined not only by the presence or absence of actin microfilament bundles but by their pattern in the cell.
ABSTRACTto as normal fibroblasts, and (b) fibroblasts of the L line (2). The subline of L cells kept in this Institute is weakly tumorigenic (all previously irradiated C3H mice injected with 106 cells developed tumors). Possibly the characteristics of this subline (e.g., sensitivity to topoinhibition) are somewhat different from those of L cells maintained in other laboratories (3); however, comparison is difficult as the detailed characteristics of other sublines are not published. Absence of pleuropneumonia-like organisms was checked by autoradiography with [3H]thymidine. Methods of cell culture, of wounding the confluent cultures, of autoradiography, of time-lapse microcinematography, and of transmission electron microscopy were described (4, 5). For scanning electron microscopy, cells cultured on coverslip were fixed in 5% glutaraldehyde, dehydrated in alcohols and in amylacetate, shadowed with silver, and examined with a Stereoscan microscope (Cambridge Instruments Ltd.).The aim of the experiments described in this paper was to find out why neoplastic fibroblasts of the L line grown together with their parent cells (mouse embryo fibroblasts) are sorted out from these cells and form the colonies above the normal monolayer. This ability to grow at the top of a sheet of normal cells is a characteristic property of many types of tumor cells, although there are few types of transformed cells that do not grow under these conditions (1).Data presented in the first part of this paper suggest that the behavior of L cells cannot be adequately described as due to loss of contact inhibition of movements or of topoinhibition of growth. Decreased attachment of L cells to the glass appears to be the main factor responsible for their abnormal interactions with embryo fibroblasts.The second part of the paper deals with the possible structural basis of this deficient attachment. The attachment of normal cells to the substratum is accompanied by formation at the cell periphery of a cytoplasmatic zone with certain special structural characteristics (lamellar cytoplasm); the lamellar cytoplasm of L cells is structurally abnormal and is smaller. Thus, decreased attachment of L cells to the substratum may result from a deficiency of reactions involved in the formation of lamellar cytoplasm. (a) Their rate of proliferation is density-dependent. The proportion of nuclei in the cultures pulse-labeled with [3H]dT that were fixed at 40 hr after change of medium gradually decreased as cell density increased. At 20-24 hr after wounding, the proportion of labeled nuclei in the wounded area of a confluent culture was much higher than in the surrounding cell sheet. The index of topoinhibition (6) for cultures in 0.2% serum-medium was 0.8-0.9. Saturation density was 6 X 105-10 X 105 cells/cm2, as compared with 4 X 105-5 X 105 cells/ cm2 for normal fibroblasts. Labeled L cells seeded upon the monolayer of the same cells were incorporated into this monolayer and did not increase in number (Fig. 1).(b) Contact inhibition of movement is obvious...
Cells were cultivated on transparent conductive substrates. glass slides coated with indium oxide: individual cells were marked with a diamond indentor. Cell cultures were frozen ( -15"C), thawed, and then stained with fluorescent dyes to determine cell damage. The marked cells were examined by phase contrast. fluorescence, and Nomarski DIC microscopy. After aldehyde and osmium tetroxide fixation, the cell preparations were sequentially treated with tannic acid, uranyl acetate, and lead citrate. The same marked cell could be sequentially studied by light microscopy (LM; in water immersion conditions), scanning electron microscopy (SEM; after dehydration and critical point drying), and transmission electron microscopy (TEM: after embedding of cell samples in epoxy resin and laser marking of the cell previously marked with a diamond indentor). The method used ensures good preservation of cell morphology, cell surface relief, and intracellular structures. The treatment used renders the cells conductive and permitted SEM of uncoated culture cells on conductive substrates.
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