Cell size and mutual cell adhesion. II. Evidence for a relation between cell size, long-range electrostatic repulsion and intercellular adhesiveness during density-regulated growth in suspension.

The strength of the long-range electrostatic repulsion forces on HeLa cells is measured by agglutinative titration using low molecular weight polylysine (M.W. 11,000). Repulsion forces, found to be present on the smaller HeLa cells from density-inhibited suspension cultures, are weakened by incubation of the cells in hypotonic NaCl solutions. Repulsion forces, found to be absent on the larger cells from fast growing cultures, can be induced on these cells by incubation in hypertonic NaCl solutions. Both effects of anisotonicity are reversible, and disappear on restoration of the medium to normal tonicity. Induction of repulsion forces on fast growing cells is prevented by previous treatment of the cells with neuraminidase. Neuraminidase also abolishes repulsion on density-inhibited cells. It is proposed that alterations of the cell size, produced by anisotonicity or occurring during growth in isotonic suspension medium, affect mutual cell adhesiveness by modifying the strength of the repulsion forces generated by cell surface sialic acids.     

Intercellular adhesiveness and neuraminidase effect following release from density inhibition of cell growth

Mutual cell adhesion increases when HeLa cells growing in suspension culture are released from density inhibition of growth. Neuraminidase treatment considerably enhances the adhesiveness of density inhibited cells but produces only a small effect on cells at low density.     

Cell size and mutual cell adhesion. I. Increase in mutual adhesivenes of HeLa cells from density-inhibited suspension cultures by hypotonic treatment.

HeLa cells harvested from density-inhibited or fast growing suspension cultures, were incubated in NaCl solutions of different tonicity. Cell size enlargement produced by hypotonicity is accompanied by an increased sedimentation rate of the density-inhibited cells, whereas no appreciable change is observed in the sedimentation rate of fast growing cells. Hypotonicity also has no effect on the sedimentation rate of density-inhibited cells which previously had been treated with neuraminidase or trypsin. It is shown that the effect of hypotonicity on density-inhibited cells cannot be ascribed to release of cell surface sialic acids during hypotonic incubation. Several arguments are presented which indicate that the changes in sedimentation rate, as measured in the rotating suspension system, are not the direct consequence of the alterations in cell size, but rather must be attributed to differences in intercellular adhesiveness resulting from the size alterations. Analogous changes in intercellular adhesiveness and cell size are shown to occur during growth in isotonic suspension culture. The results can be explained by assuming that changes in cell size affect the intercellular adhesiveness by modifying the extent to which cell surface sialic acids counteract adhesion.     

Cell size and mutual cell adhesion

Summary HeLa cells harvested from density-inhibited or fast growing suspension cultures, were incubated in NaCl solutions of different tonicity. Cell size enlargement produced by hypotonicity is accompanied by an increased sedimentation rate of the density-inhibited cells, whereas no appreciable change is observed in the sedimentation rate of fast growing cells. Hypotonicity also has no effect on the sedimentation rate of density-inhibited cells which previously had been treated with neuraminidase or trypsin. It is shown that the effect of hypotonicity on density-inhibited cells cannot be ascribed to release of cell surface sialic acids during hypotonic incubation. Several arguments are presented which indicate that the changes in sedimentation rate, as measured in the rotating suspension system, are not the direct consequence of the alterations in cell size, but rather must be attributed to differences in intercellular adhesiveness resulting from the size alterations. Analogous changes in intercellular adhesiveness and cell size are shown to occur during growth in isotonic suspension culture. The results can be explained by assuming that changes in cell size affect the intercellular adhesiveness by modifying the extent to which cell surface sialic acids counteract adhesion.     

Evidence for long-range electrostatic repulsion between HeLa cells

Agglutination curves obtained on addition of low molecular weight poly-l-lysines (mol. wt 4 000–23 000) to HeLa cells, show a deviation from linearity at low polymer concentration. This probably indicates the existence of a ζ-potential which has to be lowered before agglutination can take place. Experiments with dilysine support the assumption that cell surface charge is lowered on addition of low concentrations of short chain poly-l-lysines.Long poly-l-lysine molecules (mol. wts 70 000; 100 000) yield linear agglutination curves already at the lowest polymer concentrations. This might indicate that these polymers are able to bridge the original repulsion gap between HeLa cells.After removal of peripheral sialic acid by neuraminidase, linear agglutination curves are obtained with all poly-l-lysines irrespective of their chain lengths. This is interpreted as evidence for involvement of sialic acid residues in the charge organization responsible for electrostatic repulsion.The magnitude of the presumed repulsion effect is shown to vary with the cell density at the time the HeLa cells were harvested from the culture. The largest repulsion effect is obtained with cells from density inhibited cultures which also have the lowest tendency for mutual adhesion. With fast growing cells from low density cultures linear agglutination curves are obtained with short chain poly-l-lysines. This is interpreted as evidence for a strong diminishment or absence of long-range electrostatic repulsion between such cells.     

A STUDY ON THE MECHANISM OF INTERCELLULAR ADHESION : Effects of Neuraminidase, Calcium, and Trypsin on the Aggregation of Suspended HeLa Cells

Aggregation of suspended HeLa cells is increased on removal of cell surface sialic acid. Calcium ions promote aggregation whereas magnesium ions have no effect. The calcium effect is abolished by previous treatment of the cells with neuraminidase. Trypsinization of the HeLa cells followed by thorough washing diminishes the rate of mutual cell aggregation. Subsequent incubation with neuraminidase restores the aggregation rate to the original value before trypsin treatment. Cells which had acquired a greater tendency for aggregation after removal of peripheral sialic acid lose this property when subsequently treated with trypsin. Calcium ions have no aggregative effect on trypsinized cells. In contrast to HeLa cells, aggregation of human erythrocytes was not increased after treatment with neuraminidase or on addition of calcium. The results with HeLa cells are interpreted as follows: (a) Trypsin-releasable material confers adhesiveness upon the cells. (b) The adhesive property of this material is counteracted by the presence of cell surface sialic acids. (c) Calcium ions exert their effect by attenuating the adverse effect of sialic acid.     

Inositol trisphosphate stimulates the release of calcium from intact vacuoles isolated from Acer cells

On addition of inositol trisphosphate, intact vacuoles isolated from Acer pseudoplatanus cell suspension cultures release part of their calcium content. The process was specific, dose-dependent (IC₅₀ = 0.2μM) and was inhibited by an intracellular calcium antagonist. The calcium efflux elicited by inositol trisphosphate increased with the age of the cell suspension cultures, the maximum effect being obtained when the cultures reached the stationary phase. It is suggested that vacuoles play a role as an endocellular calcium store that is responsive to inositol trisphosphate in plants.     

Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration of spatial distribution of viability

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc.     

Effect of cell density on energy-dependent calcium uptake by Balb/c 3T3 membranes is independent of protein synthesis and attachment to substratum.

Membranes isolated from subconfluent cultures of Balb/c 3T3 cells have low energy-dependent calcium uptake activity. Replating confluent cells at low density results in a prompt fall of energy-dependent calcium uptake by membrane fractions. The level to which uptake activity falls is a function of the density at which the cells are plated (Moore and Pastan, '77b). To determine if regulation of energy-dependent uptake of calcium by membrane fractions is dependent upon attachment to a substrate and to further characterize conditions that regulate the process, we examined calcium uptake activity of membranes isolated from cells in suspension. With cells in suspension energy-dependent calcium uptake activity of isolated membranes falls promptly if cells are diluted to a low density (less than 10(5) cells/ml) and is a function of cell density. When cells in suspension at low cell densities are concentrated to high cell densities (greater than 2 x 10(6) cells/ml), calcium uptake activity of the isolated membrane fraction is increased as a function of cell density. These changes of membrane calcium uptake activity occur promptly and do not require protein synthesis.     

Adhesion of Drosophila imaginal disc cells in vitro

Summary We have used our imaginal disc cell lines to carry out in vitro studies on the cell-cell and cell-substrate adhesion of Drosophila leg and wing disc cells. Single cells were allowed to reaggregate in roller culture, and this process was found to be partially dependent on the presence of magnesium and calcium ions in the suspension medium. Varying rates of reaggregation were observed in cells from different stages of a passage, correlating with the pattern of morphogenesis which occurs during the passage. We have demonstrated that cloned cell lines can be produced showing certain selected characteristics, such as reduced cell adhesiveness.     

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures ("confluent-log" cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that "confluent-log" cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors

Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.     

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.     

Histochemical study of the distribution of sialic acid on the surface of HeLa cells in culture

Summary The distribution of sialic acid on the surface of HeLa cells is studied using Hale's staining technique. Treatment of the cells with neuraminidase before staining, indicates that the staining technique is specific for the demonstration of sialic acid.HeLa cell monolayers, grown in Leighton tubes, are treated with a solution of E.D.T.A. During separation and rounding up, cells are fixed in calcium-formalin and stained. We found a gradual increase of the Hale's positivity during treatment with E.D.T.A.HeLa cells from suspension cultures are grown in Rose-chambers. They are fixed and stained after various periods of incubation. We found a decrease of Hale's positivy during spreading out of cells and monolayer formation.These findings are discussed in terms of surface charge density and formation of stable cell contacts.The authors thank Mr. C. Dragonetti for technical assistance.     

DIFFERENTIATION OF THE GREEN ALGA CODIUM FRAGILE

Clonal cultures of Codium fragile were established from both swimming cells and vegetative filaments. In the laboratory axis primordia differentiate from heterotrichous juveniles only when cultures are agitated on a reciprocating shaker. The shear forces created by mechanical agitation are essential both for initiation and maintenance of primordia. Contact guidance of growing coenocytic filaments indicates mutual adhesion of filaments as the basis for the differentiation process.     

Methanogenesis is Ca2+ dependent in Methanothermobacter thermautotrophicus strain DeltaH

The effect of Ca2+ ions on methanogenesis and growth of Methanothermobacter thermautotrophicus was investigated. The calcium chelator ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetra-acetic acid, calcium ionophore A23187 and ruthenium red all inhibited growth of this strain. Methane formation was strongly dependent on the external Ca2+ concentration in a resting cell suspension. In addition, methanogenesis of Ca2+ preloaded cells was stimulated by 400%. Inhibitor studies revealed that Co2+ and Ni2+, inorganic antagonists of Ca2+ transport, strongly inhibited methanogenesis in these cells. Interestingly, our findings imply that one of the enzymes of methanogenesis might catalyse a Ca2+ -dependent step and allow a direct activation of methanogenesis by Ca2+ ions.     

Use of Hydrocyclones for Mammalian Cell Retention: Separation Efficiency and Cell Viability (Part 1)

A hydrocyclone with a volume of 2.56 cm³ was studied as a potential cell retention device for mammalian cell cultures (6 L volume). For the feasible operation range (0.9 to 1.6 L/min flow corresponding to pressure drops of 0.4 to 1.3 bar) the hydrocyclone was characterized with regard to flow split (underflow‐to‐overflow ratio) and flow ratio (underflow to supply). Cultures of BHK and HeLa cells (with low cell concentrations) were applied to measure separation efficiency and cell viability for a hydrocyclone operation period of 3 min corresponding to a cell suspension throughput of 2.7 to 4.8 L. Cell separation efficiencies ranged from 0.77 to 0.97 and cell viability was not affected except for BHK cells in the overflow at the highest pressure drop (1.3 bar). As the overflow is commonly used for product harvest and cells are discarded, the application of the hydrocyclone has no detrimental effect on the reactor perfusion system. The results indicate that only cells passing from the primary vortex downwards into the inner secondary vortex and from there upwards could be damaged. Evidence for this hypothesis is obtained from operating the hydrocyclone with closed overflow (only centrifugal forces acting) for a period of 3 h. In these studies no significant effect on cell viability could be detected for HeLa and CHO cells. Hence, the results indicate that the hydrocyclone can be appropriately used for cell retention and separation in perfusion cultures. Application at higher pressures is recommended whereby separation efficiencies of 0.97 without any loss in viability can be achieved.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

Inhibition of HeLa-S3 cell proliferation and biosynthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)

Treatment of HeLa-S3 cells in suspension cultures with 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) for 18-30 hr stops the growth of the cell population when treatment is carried out at 37 degrees C in Eagle's spinner culture medium supplemented with 5% fetal bovine serum. The length of the period of no growth after termination of treatment is directly related to the duration of DRB treatment. Upon resumption of growth, the rate becomes exponential and is not distinguishably different from the control rate (doubling time: 19 hr). The growth of the progeny population of the previously DRB-treated cells is as sensitive to inhibition by DRB as the growth of control populations not treated with DRB. After treatment of cells with DRB for 30 hr at 39.5-40 degrees C, the population which grows out has a prolonged doubling time. DRB treatment at 37 degrees C for 5 hr markedly inhibits uridine uptake and cellular RNA synthesis in the presence either of 5 or 15% serum. After treatment for 48 hr in 15% serum, inhibition of RNA synthesis by DRB is significantly decreased. DRB treatment does not inhibit leucine uptake in HeLa cells growing in suspension cultures. Protein synthesis is moderately inhibited in 5% serum and only slightly inhibited in 15% serum after either 5- or 48-hr period of treatment.