Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de-adaptation of EUE cells to a hypertonic medium

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.     

Cell cycle effects of hypertonic stress on various human cells in culture

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE. cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immuno-labelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protien band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G_0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however, differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.     

New insights into the cell cycle profile of Paracoccidioides brasiliensis

The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus' multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus' unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature.     

A 33 kDa protein band is enhanced during long-term adaptation of EUE cells to a hypertonic medium

A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

Adaptation to hypertonicity and selection of cell hybrids.

The ability to survive in hypertonic media varies markedly among different cell strains. This property was exploited to set up a selection procedure to isolate cell hybrids, in cell fusion experiments. The results of a cross between two EUE sublines, with a differential sensitivity to hypertonicity, are discussed.     

Flow cytometric measurements of cell volumes and DNA contents during culture of indigenous soil bacteria

Indigenous soil bacteria were released from a clay loam soil by repeated washing and centrifugation followed by density gradient centrifugation to remove enough soil particles to allow a flow cytometric (FC) study of cell numbers, cell sizes, and DNA content in single cells. The bacteria were suspended in liquid soil extract medium and incubated at 15°C for 60 h, during which direct fluorescence microscopic counts (acridine orange direct counts, AODC) were done along with the FC measurements. Cells of Escherichia coli with a known number of whole genomes per cell (rifampicin treated) were used as a calibration standard both for the DNA measurements (mitramycin-ethidium bromide stain) and cell volumes (light scatter). In response to the nutrients in the soil extract medium, the indigenous soil bacteria increased in numbers and respiration rate after a lag period of about 17 h. The onset of growth was seen first as an increase in respiration rate, numbers of large cells, and the amounts of DNA per cell in the large cells. Respiration and direct microscopical determination of biovolume was used to calculate the average growth yield on the basis of cell carbon, which was found to be 20–30% during the period of active growth. For separate volume groups of the indigenous cells, the DNA content ranged from 1.5 to 15 fg DNA per cell, the majority being below 4 fg DNA. During growth in soil extract medium, the numbers of large cells (volume > 0.18 m³) increased, and the frequency of cells with high DNA contents increased as well for this group. For the smallest sized cells (volumes < 0.065 m³) it was not possible to detect any increase in numbers during the 60-h incubation, and the DNA contents of these cells remained virtually unchanged. Compared with cell volumes based on microscopy (AODC), the FC-light scatter data grossly overestimated the volume for indigenous cells but apparently not for the newly formed cells during growth in the suspension. This probably reflects differences in light scatter properties due to adsorbed materials on the indigenous cells. The FC-DNA measurements confirmed earlier findings in that the average DNA content per cell was low (around 2 fg DNA per cell), but demonstrated a positive relationship between cell size and DNA content for indigenous cells.     

Rapid assay for cell age response to radiation by electronic volume flow cell sorting

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. The sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [3H]thymidine autoradiography of the sorted cell populations, we demonstrate that the resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. The variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. This new method has several advantages, including: no treatment of the cells is required, thus, this method is noncytotoxic; no cell cycle progression is needed to obtain different cell age compartments; the cell population can be held in complete growth medium at any desired temperature during sorting; and a complete radiation age-response assay can be plated in 2 h. The application of this method to problems in radiobiology and chemotherapy is discussed, along with some of the technical limitations.     

Rearrangement of nuclear ribonucleoproteins and extrusion of nucleolus-like bodies during apoptosis induced by hypertonic stress

Short-term hypertonic (HT) stress induces apoptotic cell death in human EUE cells in culture, as observed by electron microscopy, agarose-gel electrophoresis of low-molecular-weight DNA, DNA flow cytometry and annexin-V-propidium iodide double-staining. During HT-induced apoptosis, nuclear ribonucleoprotein (RNP)-containing structures undergo rearrangement, with the formation of Heterogeneous Ectopic RNP-Derived Structures (HERDS) which pass into the cytoplasm, as already reported for other examples of spontaneous and drug-induced apoptosis. Of special interest was the observation that nucleolus-like bodies (NLBs) which resemble morphologically nuclear functional nucleoli may be extruded into the cytoplasm of apoptotic cells and are observed inside the cytoplasmic fragments blebbing-out at the cell surface; these NLBs still contain immunodetectable nucleolar proteins (such as fibrillarin). This is an additional example of RNP-containing structures of nuclear origin which are extruded from the nucleus, in an almost "native" form, during apoptosis.     

Cytotoxic effects of phallolysin on a human heteroploid cell line

The effect of Phallolysin on cellular growth, macromolecular biosyntheses and cellular membrane structure was analysed using cells from the EUE line. Concentrations of the toxin that do not affect cellular growth, as determined by plating efficiency, have no effect on RNA or protein synthesis, but stimulate DNA synthesis. The doses of Phallolysin that inhibit cell survival do not affect macromolecular biosyntheses, but greatly increase the percentage of cells stainable with Trypan blue after 1 hour of incubation. At the same dose of the toxin the cells, analysed by electron microscopy, show increased vacuolization indicating an alteration of the membrane apparatus.     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Establishment of cell suspension cultures of yew (<Emphasis Type="Italic">Taxus × media</Emphasis> Rehd. and assessment of their genomic stability

Summary Yew cell suspension culture is used as an alternative source of paclitaxel, an anti-cancer drug. To optimize the initiation protocol, highly dormant yew seeds were germinated in vitro and the seedlings used to establish callus culture. The best sources of explant for callus initiation and growth were seedling stems and roots, and the most successful medium was modified B5 medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Calluses were friable and suitable for establishing cell suspension cultures, which were maintained for over 3 yr. Flow cytometric analysis of nuclear DNA content revealed that 2-yr-old cell suspension cultures consisted predominantly of putative euploid and aneuploid cells coexisting as sub-populations. Additional measurements performed 3 and 7 mo. later revealed further genomic instability, with a tendency towards a higher proportion of cells with elevated nuclear DNA content. In a selected cell line, which showed significant taxane production, the addition of 100 μM jasmonic acid strongly enhanced total taxane production and slightly inibited growth while no effect on nuclear DNA content was noted.     

Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G₁ and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G₁ with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G₁ nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.     

Hypertonicity induced apoptosis in HL-60 cells in the presence of intracellular potassium

Cell shrinkage is a hallmark of apoptosis. Potassium efflux, which is involved in cell shrinkage, has been previously described as an essential event of apoptosis. This study was designed to address the importance of potassium efflux in hypertonicity (450 mOsm and 600 mOsm) induced apoptosis. We initiated apoptosis in HL-60 cells in hypertonic medium consisting of either high concentrations of NaCl, mannitol or KCl. Apoptotic activity was evaluated based on the DNA content of the cells, annexin-V staining and calcium content. Apoptosis was initiated in hypertonic conditions consisting of high intracellular K⁺. We demonstrate that apoptosis can occur in the presence of high intracellular potassium contrary to previous predictions.     

Relationship between DNA repair and cell recovery: Importance of competing biochemical and metabolic processes

Summary The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were-irradiated with 1500 rad and then immediately treated for 20 min with a 37° C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. Hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation isotonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damageper se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions.     

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

Energy use efficiency of soil microorganisms: Driven by carbon recycling and reduction

Carbon use efficiency (CUE) is being intensively applied to quantify carbon (C) cycling processes from microbial cell to global scales. Energy use efficiency (EUE) is at least as important as the CUE because (i) microorganisms use organic C mainly as an energy source and not as elemental C per se, and (ii) microbial growth and maintenance are limited by energy, but not by C as a structural element. We conceptualize and review the importance of EUE by soil microorganisms and focus on (i) the energy content in organic compounds depending on the nominal oxidation state of carbon (NOSC), (ii) approaches to assess EUE, (iii) similarities and differences between CUE and EUE, and (iv) discuss mechanisms responsible for lower EUE compared to CUE. The energy content per C atom (enthalpy of combustion, the total energy stored in a compound) in organic compounds is very closely (R² = 0.98) positively related to NOSC and increases by 108 kJ mol⁻¹ C per one NOSC unit. For the first time we assessed the NOSC of microbial biomass in soil (−0.52) and calculated the corresponding energy content of −510 kJ mol⁻¹ C. We linked CUE and EUE considering the NOSC of microbial biomass and element compositions of substrates utilized by microorganisms. The mean microbial EUE (0.32–0.35) is 18% lower than CUE (0.41) using glucose as a substrate. This definitely indicates that microbial growth is limited by energy relative to C. Based on the comparison of a broad range of processes of C and energy utilization for cell growth and maintenance, as well as database of experimental CUE from various compounds, we clearly explained five mechanisms and main factors why EUE is lower than CUE. The two main mechanisms behind lower EUE versus CUE are: (i) microbial recycling: C can be microbially recycled, whereas energy is always utilized only once, and (ii) chemical reduction of organic and inorganic compounds: Energy is used for reduction, which is ongoing without C utilization.