The effect of cationic electrolytes on the adhesion of cells

The adhesion rate of cells under charge regulation onto a spherical collector with constant potential is investigated in this paper. Particularly, the effect of the presence of cationic electrolytes in the suspension medium on the adhesion rate is examined. The result reveals that the presence of cationic electrolytes in the suspension medium raises the electrostatic repulsion force between cell and collector surface, when the separation distance between them is small than a critical value. This has the effect of decreasing the adhesion rate of cells. The adhesion rate of cells is quite sensitive to the value of Hamaker constant, especially at a high ionic strength value.     

Influence of carboxymethyl dextran and ferric citrate on the adhesion of CHO cells on microcarriers

Due to the inherent risks of animal-derived raw materials, the biopharmaceutical industry has an increasing demand for serum-free and protein-free media for industrial cell culture bioprocesses. The absence of serum often changes the characteristics of mammalian cells, especially growth, productivity, and adherence properties. This study is mainly focused on the influence of media additives on cell adherence characteristics. An array of different carboxymethyl dextrans and different ferric citrate concentrations was tested with a number of CHO clones, using standard cell culture Roux-flasks and Cytoline 1 macroporous microcarriers. A prototype mixing system with controlled shear force input was developed as a screening system for adherence characteristics. The results of this evaluation revealed a negatively correlated dose-dependent influence on adhesion for ferric citrate. It was also found that certain carboxymethyl dextrans are capable of increasing the adherence on Roux-flasks and microcarriers.     

Rapid detection of DNA-damaging agents using repair-deficient CHO cells

A screening method is introduced to detect and claissify DNA-damaging agents using DNA repair-deficient strains of Chinese hamster ovary cells. Differential cytotoxicity (relative growth) of the mutant cells compared to the wild-type cells was interpreted as a measure of lethal, potentially repairable damage to DNA. The assay consists of exposing the wild-type cells and three mutant strains to the test compound in a 24-well tray and using staining intensity to estimate growth after 72 h. The battery of mutants consists of two UV-sensitive strains (UV4 and UV5) that are deficient in different aspects of nucleotide excision repair, and strain EM9, which is defective in DNA-strand-break rejoining. The assay was highly reproducible, and the magnitude of the differential cytotoxicity response compared favorably with the amount of differential killing measured by colony-formation survival curves for several chemicals. 15 direct-acting and 7 metabolism-dependent agents that were expected to produce bulky, covalent DNA adducts were tested in the assay, and all produced a differential cytotoxicity response in at leastwo of the test UV5 showed a response to all of the test compounds whereas EM9 showed a response to 7 of the test compounds. Thus, the pattern of mutant responses presumably reflects the types of DNA damage produced by a compound. Although this aspect is still under development, these results indicate the potential of a larger battery of mutants to classify a wide spectrum of chemicals according to the lesions they produce. 13 non-DNA damaging agents were also tested and none produced a differential cytotoxicity response, suggesting that this endpoint is specific for DNA damage. We conclude that this assay may be a cost-effective alternative or adjunct to the existing short-term tests.     

Influence of reducing agents on the secretion rate of recombinant erythropoietin from CHO cells

The specific secretion rate of recombinant erythropoietin (EPO) from Chinese Hamster Ovary (CHO) cells was increased by reducing agents. Addition of 5 mM cysteamine to serum free medium led to almost 8-fold increase of specific EPO secretion rate although it was not effective in thiol free medium. Each reducing agent has its own optimal concentration for maximizing the EPO secretion from CHO cells.     

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125- kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.     

Influence of radiofrequency radiation on chromosome aberrations in CHO cells and its interaction with DNA-damaging agents

A limited number of contradictory reports have appeared in the literature about the ability of radiofrequency (rf) radiation to induce chromosome aberrations in different biological systems. The technical documentation associated with such reports is often absent or deficient. In addition, no information is available as to whether any additional genotoxic hazard would result from a simultaneous exposure of mammalian cells to rf radiation and a chemical which (by itself) induces chromosome aberrations. In the work described, we have therefore tested two hypotheses. The first is that rf radiation by itself, at power densities and exposure conditions which are higher than is consistent with accepted safety guidelines, can induce chromosome aberrations in mammalian cells. The second is that, during a simultaneous exposure to a chemical known to be genotoxic, rf radiation can affect molecules, biochemical processes, or cellular organelles, and thus result in an increase or decrease in chromosome aberrations. Mitomycin C (MMC) and Adriamycin (ADR) were selected because they act by different mechanisms, and because they might put normal cells at risk during combined-modality rf radiation (hyperthermia)-chemotherapy treatment of cancer. The studies were performed with suitable 37 degrees C and equivalent convection heating-temperature controls in a manner designed to discriminate between any thermal and possible nonthermal action. Radiofrequency exposures were conducted for 2 h under conditions resulting in measurable heating (a maximum increase of 3.2 degrees C), with pulsed-wave rf radiation at a frequency of 2450 MHz and an average net forward power of 600 W, resulting in an SAR of 33.8 W/kg. Treatments with MMC or ADR were for a total of 2.5 h and encompassed the 2-h rf radiation exposure period. The CHO cells from each of the conditions were subsequently analyzed for chromosome aberrations. In cells exposed to rf radiation alone, and where a maximum temperature of approximately 40 degrees C was achieved in the tissue culture medium, no alteration in the frequency from 37 degrees C control levels was observed. Relative to the chemical treatment with MMC alone at 37 degrees C, for two different concentrations, no alteration was observed in the extent of chromosome aberrations induced by either simultaneous rf radiation exposure or convection heating to equivalent temperatures. At the ADR concentration that was used, most of the indices of chromosome aberrations which were scored indicated a similar result.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.     

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.     

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.     

The differential effect of normal cells and their neoplastic transformants on embryonic cell adhesion

The adhesion of 7 day old embryonic chick neural retina cells was reduced by adult calf serum whereas foetal calf serum diminished adhesion to a lesser extent. Conditioned media from normal cells either increased the adhesion of neural retina cells or had no effect. Media from malignant cells reduced the adhesion of the retina cells. The results are discussed in relation to malignant invasion.     

The effect of insulinomimetic agents on protein degradation in H35 hepatoma cells

A wide variety of agents are shown to mimic insulin action and inhibit rates of intracellular protein degradation in H35 hepatoma cells. For oxidizing agents such as NaNO₂, H₂O₂ and oxidized glutathione, inhibition of protein breakdown is reversed by adding catalase. Phenylhydrazine behaves like an oxidant and mimics insulin action in a manner potentiated by superoxide dismutase and reversed by catalase. Similarly the effect of insulin itself is increased by superoxide dismutase and reduced by catalase. Sulfhydryl reagents also mimic insulin action: inhibition of protein breakdown is seen following addition of 2-mercaptoethanol or a brief pre-treatment with N-ethylmaleimide or iodoacetate. Mild pre-treatment with trypsin also inhibits subsequent rates of protein breakdown. A model is proposed suggesting that these insulinomimetic actions involve a common mechanism which links the generation of active oxygen species through the redox potential of the cell to the activation of a proteinase.     

Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively.     

SCE induction in Chinese hamster ovary cells (CHO) exposed to G agents

Cultured Chinese hamster ovary (CHO) cells were exposed to two neurotoxic organophosphates, either satin (GBI, GBII) at 1.4 x 10⁻³ M or soman (GD) at 1.1 and 2.2 x 10^t-3 M for 1 h, grown and their metaphase chromosomes scored for sister-chromatid exchanges (SCE). No cytotoxicity was seen with either agent at any dose level tested. Since histograms of SCE per cell showed that they were non-symmetrically arrayed around the mean, the number of SCEs were analyzed by using the nonparametric tests, Mann-Whitney and Kruskall-Wallis. Agents GBI and GBII did not show any significant increase in SCE over baseline. On the other hand, GD demonstrated a statistically significant increase in SCE with and without metabolic activation. Ethyl methanesulfonate (EMS) alone at 5 x 10⁻³ M and cyclophosphamide (CP) at 10⁻⁴ M in the presence of rat microsomes (S9) induced a 3- and 8-fold increase in SCE per cell, respectively.     

Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (μPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNF in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNF-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.