Cell Contact Inhibition Research Articles

Activation of nuclear chromatin and the release from contact-inhibition of 3T3 cells

A rapid cytophotometric method was developed to measure binding of acridine orange (AO) to the nuclear chromatin of mouse fibroblasts. Contact-inhibited 3T3 cells bound 50–70% less dye than did growing cells in G1 phase of the cell cycle. Release from contact inhibition by fresh serum resulted in stimulated binding of AO within 30 min. No such changes were observed in SV40 virus transformed cells. Differences in AO-binding disappeared at high dye concentration, suggesting that activated cells have a greater affinity to the dye rather than an increased number of binding sites.

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10 −4 M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10 4/cm 2 irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10 5/cm 2. Inhibition of growth did not occur in KB cells when the density was below 1 × 10 4 cells/cm 2. When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.

Contact inhibition of speed in 3T3 and its independence from postconfluence inhibition of cell division.

AbstractThe average speed of nuclear translocation of 3T3 cells, recorded in a time‐lapse film of a perfused culture, was negatively correlated with the number of contacting cells, and, to a lesser degree, with the amount of a cell's perimeter in contact with other cells. When a cell was in contact with five or more other cells, its speed was reduced by 50%, on the average, although the variation in individual cell speed was considerable at each level of contact. A partial correlation analysis showed that any extracellular soluble factors governed by the local cell density had little or no effect on speed, relative to the prominent effect of the number of cell‐cell contacts, and hence that 3T3 cells display true contact inhibition of speed. This confirms the original demonstration by Abercrombie and Heaysman (1952), who studied chick embryo heart fibrpolasts. In our study, the relationships between average speed and age of the culture was such that a possible independent contribution of a time‐associated factor other than contact to the diminution in average speed, although not necessary to account for the data, could not be excluded.The same intercellular contacts found to inhibit speed in this study were previously reported to cause no immediate prolongation of individual cell generation times, despite the fact that the filmed culture was undergoing so‐called “contact” inhibition of cell division. In the present study, moreover, no correlation was observed between the average speeds of individual cells and their generation times. Hence, postconfluence inhibition of cell division and contact inhibition of speed of cell movement seem to be independent phenomena.

Cell locomotion and contact inhibition of normal and neoplastic rat cells.

AbstractThe morphology, locomotion and contact inhibition of locomotion of normal and neoplastic rat cells were studied in relation to oncogenicity. Embryonic fibroblasts (LWF) and a spontaneously neoplastic variant (LW13) were both fibroblastic in appearance, with a monolayered arrangement of cells at moderate densities. A variant transformed by Rous sarcoma virus (RsK4) was epithelioid and was also well monolayered. Scanning electron microscopy revealed a smooth dorsal surface on LWF cells, microvilli on the dorsal surface of LW13 cells and numerous microvilli, blebs and ruffles over the entire surface of RsK4 cells. LWF cells and LW13 cells moved with broad fan‐shaped lamellae on which lamellipodia (ruffles) arose at the leading edge. These cells showed polarized locomotion and migrated rapidly into wounds made in confluent cultures. RsK4 cells neither possessed large, fan‐shaped lamellae nor showed polarized movement. They did not invade wounds efficiently. After treatment with colcemid LWF and LW13 cells resembled RsK4 cells more closely and lost polarity of movement. On analysis of cell collisions by time‐lapse cinemicrography, the three cell types showed pronounced homotypic contact inhibition of cell locomotion, as judged by cessation of forward displacement. LWF and LW13 cells exhibited paralysis of ruffling, contraction of the leading edge, cell to cell adhesion and inhibition of pinocytosis typical of contact inhibition between fibroblasts. Paralysis and contraction were not evident in RsK4 cell collisions. Colcemid treatment did not prevent the initiation of contact inhibition but reduced the lasting paralysis of LWF and LW13 cells. In collisions between tumour cells and normal cells, cell locomotion and membrane ruffling were inhibited in the normal cells but not in the tumour cells. Such non‐reciprocal contact inhibition resulted in apparent monolayering in mixed cultures. The loss of contact inhibitory responses of neoplastic cells to normal cells appears to be more closely related to oncogenicity than are homotypic contact inhibition and cellular morphology. The contact inhibitory stimuli of certain neoplastic cells differ from those of normal fibroblasts, and may be preserved by fixation with glutaraldehyde.

Differentiation, contact inhibition and intercellular communication in retinal pigment cells

Retinal pigment cells of the chick embryo were grown as clone and mass monolayer cultures. Autoradiographs showed that cells were unpigmented as long as the colonies were growing exponentially. Nondividing cells at the center of the clone became pigmented; dividing cells at the edges of the clones remained unpigmented. Unpigmented cells from clone or mass monolayer cultures were able to give rise to pigmented colonies. This suggests that these cells may inherit the ability to express a specific phenotype for many cell generations. We have studied the effect of whole embryo extract on cell morphology and cell pigmentation. In this experimental system, cell pigmentation and cell proliferation appear as two mutually exclusive phenomena. The pigmented and unpigmented cells are in a different state with respect to contact inhibition. Electrical measurement of cell (junctional) communication revealed no difference in communication for small ions between the two types of cells.

Cell surface glycosyl transferases and acceptors in normal and RNA- and DNA-virus transformed fibroblasts

Cell surface glycosyl transferases and acceptors were found to be present in normal and oncogenic virus transformed fibroblasts. In cells from confluent cultures, the reaction was elevated 2- to 4-fold in MSV-3T3, RSV-3T3 and PY-3T3 cells compared to normal 3T3 cells, but in cells from sparse cultures, the reaction was essentially equivalent in the normal and transformed cells. Using exogenous acceptors, elevated surface levels of some glycoprotein:glycosyl transferases were demonstrated in the RNA- and DNA-virus transformed cells. The results may be important in phenomena such as cell:-cell adhesion and contact inhibition.

Contact-inhibited revertant cell lines isolated from SV40-transformed cells. II. Ultrastructural study.

The ultrastructural appearances of normal 3T3, SV40-transformed 3T3 (SV-3T3), and F1A revertant cell lines are compared. Both confluent and subconfluent cultures are described after in situ embedding of the cells for electron microscopy. There is striking nuclear pleomorphism in F1A revertant cells, with many cells having large nuclei compared to the less variable nuclear morphology of both normal 3T3 and SV-3T3 cells. Under the culture conditions used, deep infoldings of the nuclear envelope are prominent in growing cells, e.g., subconfluent normal 3T3 and confluent SV-3T3 cells. Such infoldings are infrequently seen in cultures which display contact inhibition of growth, e.g., normal 3T3 or F1A revertant cells grown just to confluence. In confluent cultures, the cytoplasmic organelles in revertant cells closely resemble those of normal 3T3 cells. In both normal and revertant cells in confluent culture, the peripheral cytoplasm (ectoplasm) has many 70 A filaments (alpha filaments), which are frequently aggregated into bundles. Alpha filaments are also abundant in the ectoplasm near regions of cell-to-cell apposition and in the motile cell processes (filopodia). The abundance and state of aggregation of alpha filaments correlates with contact inhibition of movement and growth in these cell lines since fewer bundles of alpha filaments are seen in growing cells than in contact-inhibited cells. This observation suggests that these filaments may be an important secondary component in the regulation of contact inhibition of movement and, possibly, of growth in normal and revertant cells.

Contact-inhibited revertant cell lines isolated from SV40-transformed cells. I. Biologic, virologic, and chemical properties.

Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.

Acquired tolerance to elevated temperatures in a poikilothermic cell line (terrapene heart, TH-1)

TH-1 cells, derived from the turtle Terrapene carolina and routinely grown at 23 °C, were originally incapable of indefinite growth at 30 °C, or of any growth at 36 °C, designated high temperature sensitive (hts +). Following brief exposures (16 to 24 days) to 30 °C, two sublines eventually became capable of growth at 36 °C (hts −). Cells which where hts − grew at approximately equal rates at 23 °C, 30 °C and 36 °C, although cells grown at the warmer temperatures had larger surface areas. The attachment rates of both hts + and hts − cells to glass were similar and were directly proportional to temperature, over a temperature range of 15 °C to 36 °C. Hts − cells maintained at 23 °C subsequently developed morphological changes characterized by a change to more fusiform cell type and reduced cell contact inhibition. In sublines of hts − cells maintained at 30 °C and at 36 °C, a variety of nuclear aberrations also appeared. It is suggested that exposure to supra-optimal growth temperatures may have been responsible for the appearance of hts − cell sublines.