Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells

The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, is markedly modulated in cells during growth activation, differentiation, motility and cell transformation. The stimulation of quiescent cells by serum factors and the culturing of cells on highly adhesive matrices induce vinculin gene expression, whereas the transformation of fibroblast and epithelial cells often results in decreased vinculin expression (reviewed in Rodriguez Fernandez, J. L., B. Geiger, D. Salomon, I. Sabanay, M. Zoller, and A. Ben-Ze'ev. 1992. J. Cell Biol. 119:427). To study the effect of reduced vinculin expression on cell behavior, 3T3 cells were transfected with an antisense vinculin cDNA construct, and clones displaying decreased vinculin levels down to 10-30% of control levels were isolated. These cells showed a round phenotype with smaller and fewer vinculin-positive plaques localized mostly at the cell periphery. In addition, they displayed an increased motility compared to controls, manifested by a faster closure of "wounds" introduced into the monolayer, and by the formation of longer phagokinetic tracks. Moreover, the antisense transfectants acquired a higher cloning efficiency and produced larger colonies in soft agar than the parental counterparts. The results demonstrate that the regulation of vinculin expression in cells can affect, in a major way, cell shape and motility, and that decreased vinculin expression can induce cellular changes reminiscent of those found in transformed cells.     

Redox signalling in anchorage-dependent cell growth

Current data have provided new perspectives concerning the regulation of non-transformed cell proliferation in response to both soluble growth factors and to adhesive cues. Non-transformed cells are anchorage dependent for the execution of the mitotic program and cannot avoid the concomitant signals starting from mitogenic molecules, as growth factors, and adhesive agents belonging to extracellular matrix. Reactive oxygen species play a key role during both growth factor and integrin receptor signalling and these second messengers are recognised to have a synergistic function for anchorage-dependent growth signalling. Redox regulated proteins include protein tyrosine phosphatases and protein tyrosine kinases, although with opposite regulation of their enzymatic activity, and cytoskeletal proteins as beta-actin. In this review we support a role of ROS as key second messengers granting a proper executed mitosis for anchorage-dependent cells, through redox regulation of several downstream targets. Deregulation of these redox pathways may help to guide transformed cells to elude the native apoptotic response to abolishment of signals started by cell/ECM contact, sustaining ectopic anchorage-independent cancer cell growth.     

Modulation of the cell cycle during reversible growth inhibition of 3T3 and SV40-3T3 cells with succinylated concanavalin A

Succinylated concanavalin A (ConA), a non-toxic, non-agglutinating derivative of the jack bean lectin ConA inhibits the growth of both normal and SV40-transformed 3T3 cells. The quantitative but not the qualitative growth inhibitory effect of succinyl-ConA can be modulated by the composition of the growth medium. Succinyl-ConA inhibited cells show a low rate of DNA synthesis and accumulate in the G0/G1 phase of the cell cycle. Upon removal of the succinyl-ConA, inhibited cells re-enter the cell cycle synchronously.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Monitoring and modeling density-dependent growth of anchorage-dependent cells

The density-dependent growth of Chinese hamster ovary (CHO) cells was monitored on-line by using an inverted microscope. A flow system was employed for cell cultivation so that nutrient concentration could be maintained and metabolic wastes were removed. With the help of video image analysis, local cells density could be accurately calculated and cell motility and exposed cell surface area could be estimated. A computer program which accounted for change of sell size and translocation of cells was developed to stimulate cell growth. The stimulated results of the population dynamics and the variations in cell size showed good agreement with our experimental observations, Cell motility and initial cell distribution on the substratum were found to have strong effect on cell growth.     

Segregated mathematical model for growth of anchorage-dependent MDCK cells in microcarrier culture

To describe the growth behavior of anchorage-dependent mammalian cells in microcarrier systems, various approaches comprising deterministic and stochastic single cell models as well as automaton-based models have been presented in the past. The growth restriction of these often contact-inhibited cells by spatial effects is described at levels with different complexity but for the most part not taking into account their metabolic background. Compared to suspension cell lines these cells have a comparatively long lag phase required for attachment and start of proliferation on the microcarrier. After an initial phase of exponential growth only a moderate specific growth rate is achieved due to restrictions in space available for cell growth, limiting medium components, and accumulation of growth inhibitors. Here, a basic deterministic unstructured segregated cell model for growth of Madin Darby Canine Kidney (MDCK) cells used in influenza vaccine production is described. Four classes of cells are considered: cells on microcarriers, cells in suspension, dead cells, and lysed cells. Based on experimental data, cell attachment and detachment is taken explicitly into account. The model allows simulation of the overall growth behavior in microcarrier culture, including the lag phase. In addition, it describes the time course of uptake and release of key metabolites and the identification of parameters relevant for the design and optimization of vaccine manufacturing processes.     

New perfluorocarbon system for multilayer growth of anchorage-dependent mammalian cells.

A novel tissue culture system has been developed that supports the multilayer growth of Hep G2 cells. The system consists of growing cells on collagen-coated perfluorocarbon substrata in the wells of a multi-well plate designed so that, even at very high densities, the oxygen in the cultures is replenished as rapidly as it is consumed. Hep G2 cells, which are typically contact inhibited, grow to form more than 10 layers of cells that continue to secrete albumin. Both multilayer growth and high rates of albumin depend on using a very enriched nutrient medium, compared to media usually used for monolayer culture of Hep G2 cells. The role played by increased oxygenation, enriched media, and the unique properties of the perfluorocarbon substrata for the 3-D growth of anchorage-dependent cells is discussed.     

Cell growth, cell division and cell size homeostasis in Swiss 3T3 cells

By separating large and small 3T3 cells we show here that cell growth (in volume) after stimulation from quiescence is not 'autocatalytic'. Rather, large cells grow significantly more slowly, in relative terms, than small cells. It follows that 3T3 cells do not require a size control mechanism operating at the level of division timing in order to achieve cell size homeostasis.     

An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines

A3 adenosine receptor agonists have been reported to influence cell death and survival. Here we report the effects of an A3 adenosine receptor agonist, IB-MECA, on the cell growth of human breast cancer cell lines, MCF-7 (estrogen receptor positive) and MDA-MB468 (estrogen receptor negative). Therefore, this study was aimed to investigate the expression and possible action of A3 receptor in the human breast cancer cell lines. IB-MECA, at 1-100 microM, resulted in a significant cell growth inhibition (P < 0.05) which reached the maximum at 48 h, in the cell lines. In both cell lines, agonist-induced effects were antagonized by pretreatment with a selective A3 adenosine receptor antagonist, MRS1220. Using RT-PCR method, further confirmation was provided by the presence of mRNA of A3 receptor in the cells. In addition, IB-MECA was able to inhibit forskolin-stimulated cAMP levels, which indicate the functional form of A3 receptor on the cell surface of these breast cancer cell lines. These results suggest that the inhibitory effect of IB-MECA on the growth of human breast cancer cell lines is mediated through activation of A3 adenosine receptor.     

An autocrine function of nerve growth factor for cell cycle regulation of vascular endothelial cells

Nerve growth factor (NGF) regulates maintenance, survival, and function of not only neuronal cells but also various kinds of non-neuronal cells. Here we clearly demonstrated that mouse aortic endothelial cells (AEC) produced bioactive NGF, and the production was enhanced by a proinflammatory cytokine, interleukin (IL)-1beta. AEC expressed both high affinity (TrkA) and low affinity (p75(NGFR)) receptors for NGF. Exogenously added NGF induced rapid phosphorylation of TrkA tyrosine kinase. Addition of anti-NGF neutralizing antibody resulted in an increase in the proportion of AEC in S and G(2)/M phases and in a hypodiploid range. Since the vascular endothelium plays a pivotal role in inflammatory conditions, these results strongly suggest that NGF, whose production is enhanced at the affected site, may contribute to maintenance, survival, and function of vascular endothelial cells by autocrine and/or paracrine mechanisms.     

Regulation of the rate of cell cycle progression in quiescent cytolytic T cells by T cell growth factor: analysis by flow microfluorometry

We have previously shown that greater than 90% of B6.1 cells, a murine cytolytic T lymphocyte (CTL) cloned line which is solely dependent on T cell growth factor (TCGF) for continuous growth in vitro, accumulates in the G1 phase of the cell cycle after transfer into culture medium containing no TCGF. Moreover, when such quiescent cells are exposed again to TCGF, greater than 85% reenter the S phase and subsequently divide in a relatively synchronous fashion. In this study, the regulation of the rate of cell cycle progression of quiescent B6.1 cells after exposure to TCGF was analyzed using two complementary DNA staining techniques, namely, the propodium iodide method (to enumerate cells entering the S phase) and the Hoechst 33342-bromodeoxyuridine substitution technique (to enumerate cells which have gone through mitosis). After TCGF addition, quiescent B6.1 cells resumed DNA synthesis and divided after a lag phase of 10 and 20 h, respectively. The duration of the lag phase was found to be dependent on the length of time during which quiescent B6.1 cells had been deprived of TCGF, but was independent of the concentration of TCGF used for restimulation. In contrast, the proportion of cells responding to TCGF as well as the rate of their first passage through mitosis was dependent on TCGF concentration. The presence of TCGF for at least 6 h was required for a maximal response. Moreover, direct evidence was obtained that TCGF by itself was able to stimulate proliferation of quiescent B6.1 cells in the absence of other growth factors and serum constituents other than bovine serum albumin, transferrin, and lipids.     

Attachment and growth kinetics of anchorage-dependent BHK cells on microcarriers

Anchorage-dependent Baby Hamster Kidney (BHK) cells were cultivated on polyhydroxyethylmethacrylate (PHEMA), polystyrene (PS), and Cytodex microcarriers. Analysis of the experimental data indicated that there were a finite number of sites on the microcarrier surfaces, available for anchorage. The number of these sites was determined by the chemical and physical structure of the surface. A small fraction of these sites were suitable for attachment of the cells before proliferation. A larger fraction of these sites did not support attachment but the cells could proliferate on them by the help of previously attached mother cells. The attachment and proliferation of the BHK cells on these microcarriers were satisfactorily modeled by surface saturation type of mathematical expressions.     

T cell receptor-induced activation and apoptosis in cycling human T cells occur throughout the cell cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0-G1, S, and G2-M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca(2+) flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.     

Expression of c-fos in NIH3T3 cells is very low but inducible throughout the cell cycle.

It has previously been shown that the c-fos proto-oncogene is rapidly and transiently induced following growth factor stimulation of quiescent NIH3T3 mouse fibroblasts. To investigate a possible role of c-fos in growth control mechanisms we have studied its expression and inducibility during the NIH3T3 cell cycle. Two major conclusions can be drawn from this analysis. First, expression of c-fos is not cell cycle-regulated, and is barely detectable in all phases of the cycle. Second, cells at different stages of the cell cycle (except for mitosis) are as sensitive to c-fos induction by growth factors as quiescent cells. These observations suggest that induction of the c-fos gene does not play a role during the continuous cycling of NIH3T3 cells, but they are fully compatible with the hypothesis that a function of c-fos may be associated with the induction of competence in fibroblasts. Through such a function c-fos may contribute to moving cells out of the quiescent state.