Postconfluence Inhibition Of Cell Division Research Articles

Mitogenic response of near‐diploid mouse cell line m5S/1M induced by epidermal growth factor

A nonmalignant near-diploid cell line m5s/1M, established by Sasaki and Kodama (J. Cell. Res., 131:114-122, 1987), was shown to respond to the epidermal growth factor (EGF). The m5s/1M cells showed high sensitivity to post-confluence inhibition of cell division and formed a uniform monolayer after the cells had become confluent. The addition of EGF resulted in loss of contact-dependent inhibition of growth and caused a massive piling up of a multilayered array of cells after they had become confluent. When EGF was removed from the medium, the cell number decreased rapidly, and the cells formed a uniform monolayer at the density observed in the absence of EGF. m5S/1M cells have high- and low-affinity receptors for EGF (approximately 40,000 receptors per cell), and the apparent dissociation constants of the EGF-binding reactions were 3.3 nM and 0.15 nM, respectively. The effect of EGF on the intracellular mobilization of Ca2+ and the formation of inositol phosphates was studied by using the calcium-sensitive fluorescent indicator fura 2 and [3H]inositol. EGF had no effect either on the mobilization of cytosolic free calcium [( Ca2+]i) or on the formation of inositol phosphates in m5s/1M cells, whereas bradykinin induced a rapid increase in both [Ca2+]i and inositol phosphates. Analysis of the glycosphingolipid (GSL) composition of m5S/1M cells showed that globotriaosylceramide (Gb3Cer), which is known to be a Burkitt lymphoma-associated antigen, is specifically expressed in the EGF-treated cells. The expression of Gb3Cer is dependent on the presence of EGF, with a reversible shift in GSL composition being observed in the presence or absence of EGF.

Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method

The present studies intend to heighten the sensitivity of BALB/3T3 cells to chemical carcinogens in a transformation assay, by including exposure of carcinogen-treated cells to a tumor promoter, 12- O-tetradecanoylphorbol 13-acetate (TPA). In the assay, cells were first treated with a known or suspected carcinogen for 72h, cultured in normal medium for 3 days, exposed to media with and without TPA for 2 weeks, and cultured in normal medium for an additional 3 weeks. Benzo[ a]pyrene, a potent carcinogen with a polycyclic aromatic hydrocarbon structure, caused transformation in the presence and absence of TPA. N-Methyl- N′-nitro- N-nitrosoguanidine (MNNG), a carcinogen with direct-acting alkylating ability, did not induce significant transformation without TPA, while treatment with MNNG followed by TPA produced numerous transformed foci, classifying MNNG as an initiating agent of transformation under the condition presented in this report. 3-Amino-1,4-dimethyl-5 H-pyrido[4,3- b]indole (Trp-P-1), 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), sodium nitrite and butylated hydroxyanisole (BHA), which are carcinogenic and/or mutagenic, produced transformed foci in significant numbers of treated dishes in the presence but not in the absence of TPA. Butylated hydroxytoluene (BHT) and sodium saccharin, which are considered to be a modifier and a promoter of carcinogenesis, did not cause significant transformation with or without TPA treatment. These studies suggest that this 2-stage transformation system is capable of detecting a wider range of chemical carcinogens as initiating agents than the standard assay. Studies on the transformation assay schedule revealed that the proportion of dishes with foci, the number of foci per dish and sizes of foci all increased in the normal medium after the termination of TPA treatment. Therefore, transformed cells appear to proliferate independenty of TPA after those cells are released by TPA from postconfluence inhibition of cell division.

Establishment and some mutational characteristics of 3T3-like near-diploid mouse cell line.

A stable and nonmalignant near-diploid cell line, designated m5S, has been established by serial in vitro transfers of embryonic skin cells of ICR mouse. The cells are stable in karyotype and growth, display high sensitivity to contact inhibition of cell division, are nontumorigenic in nude mice, and the transformation and mutation may be reproducibly tested. Unlike most of the mouse cell lines, the m5S underwent a unique karyotypic change during establishment and showed high stability in a diploid range, making amenable to the study of mutagenesis. The m5S is nearly unique; it represents the second near-diploid cell line sensitive to postconfluence inhibition of cell division derived from mouse embryo cells to be reported and characterized. These cells are being successfully used for qualitative and quantitative studies of oncogenic transformation as well as mutation to drug resistance by radiation and chemicals.

Cellular differentiation and neoplasia: characterization of subpopulations of cells that have neoplasia-related growth properties in Syrian hamster embryo cell cultures.

Cellular subpopulations having two of the growth properties of neoplastically transformed cells--lack of postconfluence inhibition of cell division (CI-) and anchorage independence of growth (AD-)--were found in cell cultures established from 10- to 13-day-old Syrian hamster embryos. The subpopulations having these properties decrease with increasing gestation period of the embryo as well as with continuing passage in vitro. The decrease in these subpopulations was also observed when they were cultured on a lethally irradiated confluent monolayer of contact-inhibited cells (cell mat), a selection condition for CI- cells. Therefore, negative selection cannot be the explanation for the loss of CI- cells in the population, leaving two other possibilities: either the loss of proliferative capacity of the CI- cells or the acquisition of sensitivity to postconfluence inhibition of cell division (CI+) of this subpopulation on in vitro culture or in vivo growth. The CI- subpopulations were isolated clonally from cell mats and were cultured continuously on both cell mats or plastic dishes. The results indicate that these cells did not lose proliferative capacity but acquired the contact-inhibited phenotype. This result, together with the fact that embryonic development in vivo also decreases CI/AD-subpopulations, suggests that the disappearance of these subpopulations is due to cellular differentiation of the CI-/AD- cells to become CI+/AD+ cells.

Existence of an upper-limit to elongation of the prereplicative period in confluent cultures of [formula omitted] cells

Cultures of C3H 10T 1 2 CL8 mouse embryo fibroblasts show a high degree of postconfluence inhibition of cell division in the absence of serum depletion. Confluent cultures had their serum concentrations increased from 3 to 10% at 6 1 4 , 6 1 2 , 8, 15, 22, and 28 days postplating, stimulating synchronized DNA synthesis as measured by the uptake of [ 3H]thymidine into acid-insoluble material. The prereplicative period, from serum increase to the beginning, or median, of synchronized thymidine uptake, increased with culture age. This movement into longer prereplicative times occurs in exponentially declining increments, indicating that an upper limit exists.

Toxicity of firemaster FF-1 and 2,2',4,4',5,5'-hexabromobiphenyl in cultures of C3H/10T 1/2 mammalian fibroblasts.

A procedure for purifying 2,2', 4,4',5,5'-hexabromobiphenyl (HBB) from FireMaster FF-1 in gram amounts by crystallization is presented. Following purification, the structural assignment of HBB was made by using proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy, elemental analysis, and mass spectroscopy (MS). The growth of C3H/10T 1/2 cells treated with 5, 37, 75, and 150 microgram of HBB and FF-1 per milliliter of medium was measured at 4, 8, and 13 days following treatment. FF-1 was more toxic at 37 and 75 microgram/ml at both 4 and 8 days, but the same at 13 days. At 150 microgram/ml cell growth was completely inhibited by both compounds. Growth of cells was stimulated at 5 microgram/ml, by HBB at 4 and 8 days, and FF-1 at 8 and 12 days. HCB was compared with HBB and FF-1 for cell growth toxicity at 37 and 75 microgram/ml. At 75 microgram/ml, HCB was more toxic than HBB and FF-1 during the entire time period. At 37 microgram/ml, HCB was more toxic than HBB and FF-1 at 4 and 8 days. Cells seeded at high densities and treated with HBB for three days lost the high degree of postconfluence inhibition of cell division observed in control cultures. Cells treated with FF-1 for three days did not adhere well to the plastic growth surface. Ultrastructural features of the HBB- and FF-1-treated cells included decreased surface villi and increased lysosomes relative to the control cells.

Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment.

5-AZACYTIDINE (aza-CR)—a s-triazine nucleoside analogue of cytidine—has strong inhibitory effects in various biological systems including neoplasms: it is a powerful bacteriostatic, antitumour and mutagenic agent1. Aza-CR also exhibits immunosuppressive, antimitotic, radioprotective and virostatic effects1. The cytostatic effect of aza-CR has primarily been directed against leukaemia2 and it has been used to treat human solid tumours3. During a study on the morphological transformation of C3H 10T½ C18 cells by cancer chemotherapeutic agents, it was noted that elongated multinucleated cells arose in cultures exposed to aza-CR4. The C3H 10T½ C18 line is a clonal line of mouse embryo cells developed at the McArdle Laboratory for Cancer Research for use in studies on chemical oncogenesis in vitro5–10. The cells are highly sensitive to post-confluence inhibition of cell division, show a remarkably low rate of spontaneous transformation and grow with a fibroblast-like morphology with long cytoplasmic processes in sparse cultures. When confluent, they form flat even monolayers and appear epithelioid. Scanning electron microscopy has shown the confluent cells to be extremely thin and polygonal with smooth surfaces resembling epithelial cells11. Here we describe experiments showing that the multinucleated tubular cells which arise in C3H 10T½ C18 cells treated with aza-CR are in fact functional myotubes.

Lack of correlation between agglutinability, the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.

Malignant hemangioendotheliomas produced by subcutaneous inoculation of Balb/3T3 cells attached to glass beads.

The Balb/3T3 mouse embryo cell line has been frequently used in cancer research as representative of nontumorigenic cells with the characteristic in vitro properties of postconfluence inhibition of cell division, low saturation density, and anchorage dependence. On the reasoning that anchorage dependence might also apply in vivo, each of nine mice were subcutaneously inoculated with an average of 15,400 Balb/3T3 cells attached to two glass beads 3 millimeters in diameter. After 8 weeks, all the mice had developed large bloody tumors that microscopically proved to be hemangioendotheliomas. Ther inoculation of Balb/3T3 cells alone or beads alone produced no tumors. Transplants of each tumor into normal mice grew to kill the animal within 6 weeks. Tumor cells from collagenase-disaggregated tumor tissue had a plating efficiency of 21.2 percent compared to that of normal adult subcutaneous fibroblasts of less than 0.1 percent. The tumor cells in vitro closely resembled Balb/3T3 cells in appearance and were tumorigenic at a dose of 10-4 cells. A second, repeat experiment produced the same type of tumors grossly and microscopically in 17 of 25 mice between 99 and 211 days after inoculation of the Balb/3T3 cells attached to glass beads. These findings require a reassessment of the postulate that low saturation density, postconfluence of cell division, and anchorage dependence are characteristic in vitro properties only of nonneoplastic cells.

Movement in a confluent 3T3 monolayer and the causes of contact inhibition of overlapping.

ABSTRACT The existence of contact inhibition of nuclear overlapping (monolayering) has previously been interpreted to mean that intercellular contact locally inhibits the cell’s locomotory apparatus and hence that cells in a confluent monolayer should be immobilized. Garrod & Steinberg, however, observed gross movements and exchanging of nuclear nearest neighbour relationships in a confluent monolayer of chick epithelial-like cells. Data presented in the present report reveal that similar movements occur in confluent monolayers of mouse 3T3 fibroblasts, previously shown to display contact inhibition of speed, contact inhibition of overlapping, and postconfluence inhibition of cell division. Cytoplasmic boundaries frequently could not be resolved in the present study. However, during a 22-day period, more than one third of originally adjacent pairs of nuclei became separated by 1·5 intervening nuclei, and their average distance of separation increased more than 2-fold. Also, each nucleus was displaced, on average, more than one mean cell diameter in a random direction. These indications of gross cellular movements were seen during the stationary density phase of perfused culture, as well as during the doubling of cell density which occurs after confluence but before the stationary phase. If the intensity of cell-substratum adherence exceeds that of cell-cell adherence, this differential in adhesive strength could, by itself, produce the observed avoidance of overlapping, without invoking local inhibition of the cell’s internal ‘motor’ by contact. This differential adhesion hypothesis for contact inhibition of overlapping is consistent with the extensive movements very likely occurring in at least certain confluent monolayers. On the other hand, the probable existence of these movements seems less easily reconciled with the hypothesis that contact inhibition of overlapping results from contact-induced local paralysis of an intracellular locomotory apparatus, although this latter hypothesis is not excluded.

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.

Contact inhibition of what? An analytical review.

AbstractQuite a number of phenomena having to do with cells' influences upon one another's movements have come to be regarded as expressions of “contact inhibition.” However, no single, central mechanism has been shown to underlie them all. Consequently, the term “contact inhibition” should not be used without operational modifiers. Inhibitions of individual cell movements imputed to be mediated by cell‐cell contacts include inhibition of overlapping (which results in monolayering), of colony expansion, of cell speed (nuclear translocation), of ruffling, of orthogonal movement (proposed to explain spontaneous parallel alignment of cells), and of neighbor exchanges.The six inhibitions listed above are operationally distinct, and only two (overlapping and colony expansion) are known to result from a common mechanism. A seventh phenomenon, so‐called “contact inhibition of cell division” (more operationally termed postconfluence inhibition of cell division) is in a separate category and is not considered here.Evidence eliminating action‐at‐a‐distance is available only for the first three, and hence only these should at present be termed contact inhibitions. Inhibition of neighbor exchanges is yet hypothetical; at its extreme, it would immobilize cells in a confluent monolayer, but such immobilization has been found not to occur.Contact inhibition of overlapping, the most studied of the six, is not displayed by invasive cells with respect to normal cells; invasive tumor cells overlap freely upon normal cells, although not necessarily upon one another. Contact inhibition of overlapping, and its loss by invasive cells, can readily be interpreted, by means of the differential adhesion hypothesis, as consequences of cell‐type‐specific differences in cell‐cell and cell‐substratum “strengths of adhesion.” These strengths of adhesion are formulated as specific interfacial free energies, which are the only parameters of cellular adhesiveness that have been rigorously shown to determine equilibrium configurations of cell populations.

The role of cell-cell contact in "contact" inhibition of cell division: a review and new evidence.

AbstractThe term “contact inhibition of cell division” was borrowed from “contact inhibition of cell movement.” We prefer the term “postconfluence inhibition of cell division” as being more operational and less mechanistically biased; it is operationally defined as a pronounced depression of the mitotic rate in a postconfluent culture which displays a stationary density despite periodic nutrient renewal, the inhibition being locally reversibly by removal of the adjacent cells.The mechanism of postconfluence inhibition is of considerable interest because of the inverse correlation between postconfluence inhibition and the tumorigenicity of a number of cell lines. Several hypotheses, involving direct cell‐to‐cell contacts or locally restricted diffusion gradiens, could explain postconfluence inhibition.With the goal of discriminating among these hypotheses, time‐lapse films were taken of carefully regulated, perfused cultures of 3T3 mouse cells, in which the transition from rapid growth to the stationary phase was recorded. Measurements of cell‐to‐cell contact, local cell density, and generation times were made on an individual cell level and analyzed with the aid of a computer.We observed that all‐around cell‐cell contact or a high local cell density present throughout G1 often did not produce immediate inhibition of cell division. We conclude that either (i) simple visible cell‐cell contacts or a high local cell density are not the direct cause of postconfluence inhibition of cell division, or (ii) their effects often do not inhibit cell division until after a delay of about one cell generation time. Such a delay may be partly responsible for the 50% overshoot past the stationary density that we observed in 3T3 cultures.