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Results for “Contact Inhibition”

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At least 19 recordsLinked to original sources
PubMed1999

Opposite regulation of the expression of cyclin-dependent kinase inhibitors during contact inhibition.

Contact inhibition is a well-known phenomenon, but the details of its mechanism are poorly understood. Recently, cyclin-dependent kinase inhibitors have been studied intensively with respect to their regulatory role in the cell cycle, and of them, p27(Kip1) is particularly involved in contact inhibition. p27(Kip1) is believed to be regulated primarily through posttranscriptional mechanisms. We now report that cyclin-dependent kinase inhibitors, including p27, are regulated differently at the mRNA level during contact inhibition in murine BALB/c-3T3 fibroblasts. The mRNA expression of p15, p16, and p27 was up-regulated as the cell density increased, but, on the contrary, the mRNA level of p21(Cip1/WAF1/Sdi1) markedly decreased when the cells became confluent. The protein levels of these genes were regulated in the same way as their mRNA levels, and cyclin-dependent kinase-2 activity was markedly inhibited on density-mediated growth arrest of the cells. These results indicate that the regulation of mRNA expression of cyclin-dependent kinase inhibitors appears to contribute to their protein levels and to the arrest of cell growth through contact inhibition.

3T3 Cells↗
PubMed1969

Cellular deoxyribonucleic acid synthesis and loss of contact inhibition in irradiated and contact-inhibited cell cultures infected with fibroma virus.

Cultural changes that follow infection of rabbit kidney cells with fibroma virus were studied. Characteristic alterations of cell morphology and development of multilayered piles and cords of cells were found to occur in infected cultures in which cell division was blocked by gamma radiation or by cell crowding and serum deprivation, thus indicating no dependence upon cell division. Fibroma virus infection did not remove blocks to cell division, but it did exert distinct effects upon nuclear deoxyribonucleic acid synthesis in cells blocked by radiation or cell crowding. Use of tritium-labeled thymidine and autoradiography demonstrated that after infection initial inhibition of nuclear incorporation was followed by sharply increased nuclear labeling at a time that coincided with beginning alterations of cell morphology and development of cell piling.

Animals↗
PubMed2002

Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2.

PURPOSE: Contact inhibition has been implicated as an important antiproliferative mechanism in developing and mature corneal endothelium. Although exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry in cultured rat corneal endothelial cells, it is not known whether TGF-beta2 contributes to the mitotic inhibition that occurs during in vivo endothelial development. TGF-beta receptors I, II, and III must be coexpressed for a TGF-beta2-induced intracellular signal to be transmitted. The current study was conducted to determine whether TGF-beta2 contributes to mitotic inhibition during endothelial development, by investigating when these receptors become coexpressed in the endothelium of neonatal rats. Cyclin-dependent kinase inhibitors (CKIs), such as p27kip1 and p15INK4b, help mediate mitotic inhibition in other cell types. The role of CKIs in inhibiting proliferation in corneal endothelium was examined by first determining the kinetics of p27kip1 expression in neonatal rat corneal endothelium. Studies were then extended to cultured cells to more directly compare the effects of TGF-beta2 and cell-cell contact on the relative protein and mRNA expression of the CKIs, p27kip1, and p15INK4b. METHODS: Immunocytochemistry (ICC) detected TGF-beta receptors I, II, and III (RI, RII, RIII, respectively) in the endothelium of rat corneas on postnatal days 1, 10, and 21, and in adult (3-month-old) rats. ICC for p27kip1 was conducted on postnatal days 1, 7, 14, and 21. Samples were taken for p27kip1 RT-PCR on postnatal days 7, 14, and 21 and from adult rats. The effect of TGF-beta2 on p27kip1 and p15INK4b expression was determined in G(0)-phase synchronized subconfluent rat corneal endothelial cells incubated for 24 hours in 10% serum +/- 5 ng/mL TGF-beta2. CKI expression was also examined in fully confluent cultures. RT-PCR and Western blot analysis detected p27kip1 and p15INK4b mRNA and protein expression, respectively. The effect of releasing cells from cell-cell contact on proliferation and p27kip1 protein expression was studied in confluent cultures treated for 1 hour with and without 2.0 mg/mL di-sodium EDTA and then maintained for 24 hours in 10% serum. Cultures were then either fixed for ICC of Ki67, a marker of actively cycling cells, or extracted for Western blot determination of p27kip1 protein. RESULTS: Positive staining for TGF-beta RIII was detected on postnatal day 10, and staining for RI and RII was detected on postnatal day 21. The endothelium stained positively for p27kip1 on postnatal day 1 and thereafter, and p27kip1 PCR product was detectable at the earliest time point tested (postnatal day 7). In cultured cells, TGF-beta2 and cell-cell contact had relatively little effect on expression of p27kip1 or p15INK4b mRNA. TGF-beta2 lowered the levels of both proteins, but p27kip1 remained at a higher level than p15INK4b. In confluent cultures, p15INK4b protein was reduced; however, p27kip1 protein levels increased 20-fold. Positive staining for Ki67 was detected, and p27kip1 protein levels substantially decreased in EDTA-treated confluent cells compared with the untreated control. CONCLUSIONS: Previous studies from this laboratory showed that corneal endothelial cell proliferation ceases in neonatal rat by postnatal day 13. This timing correlated with the formation of stable cell-cell contacts, implicating contact inhibition as an important mechanism of growth arrest during endothelial development. The current studies showed that coexpression of TGF-beta RI, RII, and RIII occurred too late for TGF-beta2 to have a significant role in inhibiting proliferation during endothelial development. Studies in cultured cells suggest that p27kip1 mediates inhibition of proliferation induced by TGF-beta2, although the response to this cytokine was relatively weak. ICC and RT-PCR of p27kip1 in neonatal endothelium and RT-PCR and Western blot studies in cultured cells indicate that contact inhibition is mediated, in large part, through the activity of p27kip1. These results, together with previous data from this laboratory, strongly suggest that contact inhibition is an important mechanism responsible for inducing cell cycle arrest during corneal endothelial development and for maintaining the mature monolayer in a nonproliferative state. In both cases, contact-induced inhibition is mediated, at least in part, by p27kip1. TGF-beta2 appears not to induce mitotic arrest in the developing endothelium, but may function to maintain the mature endothelium in a nonreplicative state should cell-cell contact be lost in the monolayer.

Animals↗
PubMed1974

A diffusible factor restoring contact inhibition of growth to malignant melanocytes.

Contact inhibition of growth is the property in vitro whose loss is most closely correlated with tumorigenicity in vivo. A contact-inhibited melanocytic cell line produces a diffusible protein-containing factor capable of restoring contact inhibition of cell division to highly malignant hamster melanocytes. Its addition to subconfluent cultures of the malignant cells is followed by the obligatory acquisition, at confluence, of the contact-inhibited state. Cultures are flat, oriented, and fibroblast-like, and show a 55% decrease in saturation density with no loss of viability. The effect is reversible. Present in conditioned media of cultures of the contact-inhibited melanocytic cell line, isolated by column chromatography on Sephadex G-200, the factor appears to be of high molecular weight. Activity is preserved in aqueous solutions at 4 degrees for at least 8 weeks, but is destroyed by repeated freezethawing or by treatment with Pronase. This newly recognized contact-inhibitory factor may be a prototype for a more general and fundamental mechanism for regulation of normal cell-cell interactions. It is the first cell-elaborated factor to be isolated that is capable of restoring the capacity for contact inhibition of growth to malignant cells.

Animals↗
PubMed2004

Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells.

Increasing cell density arrests epithelial cell proliferation by a process termed contact inhibition. We investigated mechanisms of contact inhibition using a model of contact-inhibited epithelial cells. Hepatocyte growth factor (HGF) treatment of contact-inhibited Madin-Darby canine kidney (MDCK) cells stimulated cell proliferation and increased levels of phosphorylated ERK1/2 (phospho-ERK1/2) and cyclin D1. MEK inhibitors PD-98059 and U0126 inhibited these HGF-dependent changes, indicating the dependence on phosphorylation of ERK1/2 during HGF-induced loss of contact inhibition. In relation to contact-inhibited high-density cells, low-density MDCK cells proliferated and had higher levels of phospho-ERK1/2 and cyclin D1. PD-98059 and U0126 inhibited low-density MDCK cell proliferation. Trypsinization of high-density MDCK cells immediately increased phospho-ERK1/2 and was followed by a transient increase in cyclin D1 levels. Reformation of cell junctions after trypsinization led to decreases in phospho-ERK1/2 and cyclin D1 levels. High-density MDCK cells express low levels of both cyclin D1 and phospho-ERK1/2, and treatment of these cells with fresh medium containing HGF but not fresh medium alone for 6 h increased phospho-ERK1/2 and cyclin D1 levels compared with cells without medium change. These data provide evidence that HGF abrogates MDCK cell contact inhibition by increasing ERK1/2 phosphorylation and levels of cyclin D1. These results suggest that in MDCK cells, contact inhibition of cell proliferation in the presence of serum occurs by cell density-dependent regulation of ERK1/2 phosphorylation.

Animals↗
PubMed1975

[Restoration of growth contact inhibition in malignant melanocytes from man, mice, and hamsters].

Contact inhibition of growth is the in vitro property whose loss is most closely correlated with in vivo tumorigenicity. A contact inhibited line of hamster melanocytes produces a diffusible high molecular weight protein (melanocyte contact inhibitory factor, MCIF) which restores contact inhibition of growth to hamster malignant melanocytes. MCIF, extracted from culture medium of confluent contact-inhibited cultures, is now shown to restore the same capacity for in vitro growth regulation to malignant melanocytes of human and murine origins. In all 3 species MCIF produces characteristic changes in growth and morphology of cultures, with significant reductions of saturation densities. MCIF, the first protein identified which restores contact inhibition of growth to malignant cells, may be the prototype for a closely related class of surface-associated proteins concerned with regulation of normal cell-cell interactions leading to feedback inhibition of growth.

Animals↗
PubMed1978

Cell movements in a confluent monolayer are not caused by gaps: evidence for direct contact inhibition of overlapping.

According to the hypothesis of contact inhibition of movement, cells in a confluent monolayer are restrained from major overlapping by a directional inhibition of locomotion. This explanation of monolayering proposes that contact between 2 cells locally paralyses the locomotory function, preventing movement in the direction that would lead to overlapping. Consequently, a cell in contact on all sides with neighbouring cells should be immobilized. Yet in strictly monolayered cultures of confluent chick liver or mouse 3T3 cells, we have previously observed both translational cell movements and re-shufflings of relative cell positions. The 'confluence' was not perfect, however, and it seemed possible that the movements observed were due to release from contact inhibition by occasional transitory gaps seen to open up between cells. In the present study, detailed gap experiences and cell movements were recorded for 31 cells over a total of 1637 cell-hours. There was no significant correlation between frequency of gaps experienced and the extent of cell movement measured as neighbour-exchanges. We conclude that gaps are not a major cause of the movements observed. The hypothesis based on contact inhibition of motion, which attempts to explain monolayering indirectly by imposing a restraint on cell locomotion, cannot explain the substantial cell movements seen in the confluent cell monolayer studied here. To explain contact inhibition of overlapping, the evidence favours a more direct hypothesis which places no restriction on cell movement other than that overlapping be avoided. Such direct avoidance of overlapping could result from differences in the strengths with which cells adhere to one another and to the substratum.

Cell Movement↗
PubMed1995

The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.

Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.

Activating Transcription Factor 1↗
PubMed1974

Differences in membrane fluidity and structure in contact-inhibited and transformed cells.

Studies on contact-inhibited mouse embryo fibroblast 3T3 cells and 3T3 cells transformed by oncogenic RNA and DNA viruses and by a chemical carcinogen have demonstrated differences in plasma membrane architecture. Spin-label and freeze-fracture ultrastructural studies have shown that contact-inhibited cells have ordered membrane lipids and aggregated intramembranous particles, whereas transformed cells have fluid membrane lipids and randomly distributed intramembranous particles. These findings suggest a model for how changes in the cell membrane may account for some of the characteristic differences observed between contact-inhibited and transformed cells.

Animals↗
PubMed1996

Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells.

We postulated that contact inhibition and transforming growth factor (TGF)-beta 1 may target the same molecules to negatively regulate the Mv1Lu cell cycle in G0/G1. Both contact inhibition and TGF-beta 1 suppressed the expression of a 45-kDa protein (p45); cyclins D2 and B1; cyclin-dependent protein kinase (Cdk)-4, Cdc-2, and Cdc-2-associated activity; and the phosphorylation of retinoblastoma tumor-suppressor protein (pRb) but did not affect the expression of cyclins D1, E, and A or the expression of Cdk-2 and Cdk-5. Expression of p45 reappeared 12 h after release from contact inhibition and 6-8 h after release from TGF-beta 1, while TGF-beta 1 prevented release from contact inhibition and maintained suppression of both p45 and cyclin D2. Additionally, cyclin D2 phosphorylation and its associated kinase activity were strongly inhibited by contact inhibition and TGF-beta 1. Thus suppression of p45, cyclin D2/Cdk-4, and cyclin B1/Cdc-2 expression and/or activities is targeted both by contact inhibition and by TGF-beta 1 and may define common mechanisms through which these negative growth signals are integrated.

Animals↗
PubMed1979

Contact inhibition and malignancy.

The role of contact inhibition in influencing the behaviour of malignant cells is discussed in a review. Although tissue culture cannot simulate the immense complexity of the conditions in vivo, some of the distinctive features of malignant invasion can be conveniently observed with this technique. The evidence derived from this technique indicates that defective contact inhibition of movement of malignant cells does contribute to their invasiveness.

Animals↗
PubMed1999

Growth arrest of melanoma cells is differentially regulated by contact inhibition and serum deprivation.

Both growth-factor deprivation and contact inhibition suppress cell growth; however, the mechanisms by which they inhibit cell proliferation may not be identical. The function of antiproliferative genes and the induction of programmed cell death are among the potential differences between these growth-arrest mechanisms. Specifically, an inverse relation between the expression of cyclin-dependent kinase inhibitors (CDKIs) and the susceptibility to apoptosis has been reported. To test this relation, we examined the features of growth arrest in a canine melanoma cell line, TLM1. Both contact inhibition and serum deprivation halted cell-cycle progression of TLM1 cells in the G1 phase. Prolonged growth arrest of the cells without restimulation resulted in apoptosis; conversely, the cells reentered the cell cycle after release from contact inhibition or on restimulation with serum. Cell-to-cell contact, but not serum deprivation, led to the expression of p53 and p21/Waf-1. The expression of p21/Waf-1 did not prevent apoptosis. Moreover, the ectopic overexpression of CDKIs increased apoptosis. These results support the premise that growth arrest induced by contact inhibition and serum deprivation are mediated through distinct mechanisms. Furthermore, CDKIs are not universal inhibitors of apoptosis, and in some cases, they may initiate or enhance the apoptotic program.

Animals↗
PubMed1967

Contact inhibition in colony formation.

Contact inhibition of replication of the established mammalian cell line 3T3 was examined during growth of the colony and compared with that of the Chinese-hamster cell line CHL-1. The growth curves of cells in the colonies conformed to the predicted exponential and linear rates for CHL-1 and 3T3 respectively. Autoradiographs of colonies in which DNA was labeled with tritiated thymidine showed that in 3T3 colonies, only peripheral cells were labeled, while CHL-1 colonies were labeled throughout.

Animals↗
PubMed1975

Contact inhibition of phagocytosis in epithelial sheets: alterations of cell surface properties induced by cell-cell contacts.

Contact inhibition of phagocytosis was found to be characteristic for epithelial sheets formed in cultures by several cell types: normal and transformed mouse kidney cells, and differentiated mouse hepatoma cells. In these sheets most central cells surrounded by other cells had very low phagocytic activity. In contrast, marginal cells having a free edge were able to perform an active phagocytosis. Contact inhibition of phagocytosis was absent in dense cultures of mouse embryo fibroblasts and in cultures of anaplastic mouse hepatoma 22a. The upper surface of epithelial sheets was nonadhesive for prelabeled epithelial cells and fibroblasts. In contrast, the upper surface of dense cultures of mouse fibroblasts was adhesive for these cells. These and other data strengthen the suggestion that contact inhibition of phagocytosis is a result of different adhesiveness of the upper cell surface and of the surfaces near the free edge. Agents inhibiting cell surface movements at the free edges of marginal epithelial cells (cytochalasin, azide, sorbitol, low temperature) prevented adhesion of particles to these edges. Possibly, the surface of actively moving cytoplasmic processes is the only cell part that has adhesive properties necessary for the formation of attachments with other cellular and noncellular surfaces. In epithelial sheets, in contrast to fibroblast cultures, Colcemid did not activate movements of immobile contacting cell edges. These results indicate that mechanisms of contact immobilization of cell surface may be different in epithelium and fibroblasts. Firm contacts formed between epithelial cells are sufficient for stable immobilization of the surface; additional stabilization of the surface by microtubules is not essential. Fibroblasts do not form firm contacts and the Colcemid-sensitive stabilization process is essential for maintenance of the immobile state of their surfaces. Differences in the stability of cell surface immobilization produced by cell-cell contacts may also explain different adhesiveness of the upper surfaces of dense fibroblastic and epithelial cultures.

Animals↗