Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

Interstitial stem cell proliferation in hydra: evidence for strain-specific regulatory signals.

We have examined the growth behavior of small numbers of interstitial stem cells transplanted into tissue of genetically unrelated strains of Hydra magnipapillata. We show that such stem cells, which are at low density following transplantation, proliferate more rapidly than the stem cells of the host, which are at normal density. The rapid proliferation is similar to the proliferation rate of stem cells transplanted into interstitial cell free tissue. The results suggest that stem cells transplanted into heterotypic tissue are unable to "sense" the presence of host stem cells and to adopt their growth rate to that of the surrounding cells. Thus, the feedback signal which negatively regulates stem cell growth as a function of stem cell density must be strain specific.     

Noninvasive estimation of cell cycle phase and proliferation rate of human mesenchymal stem cells by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent mesenchymal stem cells (MSCs) using a fluorescence microscope after staining with 4′,6-diamidine-2′-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by phase-shifting laser microscopy (PLM) revealed that the laser phase shift of cells in the G₂/M phase was markedly higher than that of cells in the G₀/G₁ phase. Even in the synchronous cultures to G₀/G₁ and G₂/M cell cycle phases, the laser phase shift of the cells in the G₂/M phase was markedly higher than that of the cells in the G₀/G₁ phase. The analysis of the cultures of MSCs from different donors with the addition of FGF2 at different concentrations revealed that there was a marked negative correlation between the average phase shift and mean generation time. In conclusion, it is possible to estimate noninvasively the proliferation activity of MSCs population by measuring the phase shift using PLM.     

Cell cycle regulation in hematopoietic stem cells

Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells. Because HSCs must persist for a lifetime, the balance between their proliferation and quiescence is carefully regulated to ensure blood homeostasis while limiting cellular damage. Cell cycle regulation therefore plays a critical role in controlling HSC function during both fetal life and in the adult. The cell cycle activity of HSCs is carefully modulated by a complex interplay between cell-intrinsic mechanisms and cell-extrinsic factors produced by the microenvironment. This fine-tuned regulatory network may become altered with age, leading to aberrant HSC cell cycle regulation, degraded HSC function, and hematological malignancy.     

Geometrical constraints in the scaling relationships between genome size, cell size and cell cycle length in herbaceous plants

Plant nuclear genome size (GS) varies over three orders of magnitude and is correlated with cell size and growth rate. We explore whether these relationships can be owing to geometrical scaling constraints. These would produce an isometric GS-cell volume relationship, with the GS-cell diameter relationship with the exponent of 1/3. In the GS-cell division relationship, duration of processes limited by membrane transport would scale at the 1/3 exponent, whereas those limited by metabolism would show no relationship. We tested these predictions by estimating scaling exponents from 11 published datasets on differentiated and meristematic cells in diploid herbaceous plants. We found scaling of GS-cell size to almost perfectly match the prediction. The scaling exponent of the relationship between GS and cell cycle duration did not match the prediction. However, this relationship consists of two components: (i) S phase duration, which depends on GS, and has the predicted 1/3 exponent, and (ii) a GS-independent threshold reflecting the duration of the G1 and G2 phases. The matches we found for the relationships between GS and both cell size and S phase duration are signatures of geometrical scaling. We propose that a similar approach can be used to examine GS effects at tissue and whole plant levels.     

Cell division cycle 20 promotes cell proliferation and invasion and inhibits apoptosis in osteosarcoma cells

Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in human tumorigenesis. However, the function of Cdc20 in osteosarcoma (OS) has not been investigated. In the current study, we aim to explore the role of Cdc20 in human OS cells. Multiple approaches were used to measure cell growth, apoptosis, cell cycle, migration and invasion in OS cells after depletion of Cdc20 or overexpression of Cdc20. We found that down-regulation of Cdc20 inhibited cell growth, induced apoptosis and triggered cell cycle arrest in OS cells. Moreover, Cdc20 down-regulation let to inhibition of cell migration and invasion in OS cells. Consistently, overexpression of Cdc20 in OS cells promoted cell growth, inhibited apoptosis, enhanced cell migration and invasion. Mechanistically, our Western blotting results showed that overexpression of Cdc20 reduced the expression of Bim and p21, whereas depletion of Cdc20 upregulated Bim and p21 levels in OS cells. Altogether, our findings demonstrated that Cdc20 exerts its oncogenic role partly due to regulation of Bim and p21 in OS cells, suggesting that targeting Cdc20 could be useful for the treatment of OS.     

Cell proliferation and cell cycle dependent changes in the methylthioadenosine phosphorylase activity in mammalian cells

The objective of this study is to investigate the activity of methylthioadenosine phosphorylase (MTA-Pase) in mammalian cells stimulated by serum to proliferate and during their cell cycle. A direct correlation between growth rate and MTA-Pase activity in chinese hamster ovary (CHO) cells was observed. High MTA-Pase activity was observed during the exponential growth phase followed by a low enzyme activity during plateau phase of growth. To understand whether the fluctuations in the enzyme activity was cell cycle dependent, initially the activity of MTA-Pase was studied in plateau phase (G0) CHO cells as they synchronously go into S phase upon plating in fresh medium. The MTA-Pase activity in G0 cells before initiation of growth was 10.3 n.mol/mg protein/30'. A peak activity of 16.0 n.mol/mg/30 min was found at 12 hr after stimulation of proliferation by serum. These results indicate a peak MTA-Pase activity between 10-12 hr after stimulation of proliferation coinciding with the initiation of DNA synthesis. The activity of the enzyme slowly decreased as the cells completed their DNA synthesis. To understand whether these fluctuations are cell cycle specific, HeLa cells were synchronized in different phases and MTA-Pase activity was studied. The specific activities of the enzyme were 2.76, 2.99, 3.97, 3.28 and 3.65 n.moles/mg/30 min. in mitosis, early G1, late G1, S and G2 phases of the cell cycle respectively. These results indicate that MTA-Pase activity peaks in late G1 phase before the initiation of DNA synthesis, similar to the polyamine biosynthetic enzymes and might play a role in the initiation of DNA synthesis by salvage of adenine into nucleotide pools.     

The relationship between size,budding rate,and growth efficiency in three species of hydra

Three species of the fresh water carnivore hydra, H. littoralis, H. pseudoligactis, and C. viridissima present a graduation in size with the first species the largest and albino Chlorohydra the smallest. When presented with a daily overabundance of food (artemia), considerable variation in food intake and gross efficiency of growth (proportion of food energy consumed that is turned into new protoplasm or buds) existed among the species. The degree of association between size of species and food intake was highly significant. However, budding efficiency among the species was found to be independent of food intake (when the effects of species size were eliminated) and of species size (when the effects of food intake were removed). However, species with high (low) efficiencies have significantly higher (lower) reproductive rates. A lowering of the temperature from 25° to 15° C. increased the size of the species, increased food intake, but decreased reproductive rate. In all species except H. pseudoligactis a corresponding increase in the production of bud energy with no change in efficiency also occurred. On the other hand, lowering of the temperature for H. pseudoligactis significantly lowered reproductive efficiency but had no effect on the total calorific output of buds. This species, in constrast to the others, appears to have a compensatory ability to adjust its efficiency to maintain a high calorific output when temperature increases. It was also found that albino Chlorohydra have budding efficiencies of around 35 percent which are not influenced by changes in food intake or light. Normal green hydras, however, have efficiencies which range from 40 to 62 percent above their albino counterparts when fed once a day and once every two days in light respectively. It it concluded first, that the symbiotic algae in the gastrodermals cells of green hydra contribute quantitatively in the order of the above amounts to the growth process in this species, and second, that green hydras have the ablity to increase their growth efficiency when food intake is reduced thus reducing the drop in calorific but output that normally occurs in the albino (control) form.     

Telomere length, stem cells and aging

Telomere shortening occurs concomitant with organismal aging, and it is accelerated in the context of human diseases associated with mutations in telomerase, such as some cases of dyskeratosis congenita, idiopathic pulmonary fibrosis and aplastic anemia. People with these diseases, as well as Terc-deficient mice, show decreased lifespan coincidental with a premature loss of tissue renewal, which suggests that telomerase is rate-limiting for tissue homeostasis and organismal survival. These findings have gained special relevance as they suggest that telomerase activity and telomere length can directly affect the ability of stem cells to regenerate tissues. If this is true, stem cell dysfunction provoked by telomere shortening may be one of the mechanisms responsible for organismal aging in both humans and mice. Here, we will review the current evidence linking telomere shortening to aging and stem cell dysfunction.     

Cell cycle and cell size dependence of susceptibility to hydrodynamic forces

Exposure of animal cells to intense hydrodynamic forces exerted in turbulent capillary flow, and by controiled agitation and aeration, resulted in preferential destruction of S and G(2) cells and the extent of destruction of these cells was dependent upon the intensity of the action. The loss of these cells was possibly due to their larger size. However, the appearance of large numbers of membrane-bound vesicular structures similar to apoptotic bodies as well as cells with low DNA stainability (in a sub-G(1) peak) suggested that the action of adverse hydrodynamic forces on these large cells may at least in part be to induce an apoptotic response. (c) 1995 John Wiley & Sons, Inc.     

Cell cycle-dependent expression of potassium channels and cell proliferation in rat mesenchymal stem cells from bone marrow

OBJECTIVE: Recently, our team has demonstrated that voltage-gated delayed rectifier K(+) current (IK(DR)) and Ca(2+)-activated K(+) current (I(KCa)) are present in rat bone marrow-derived mesenchymal stem cells; however, little is known of their physiological roles. The present study was designed to investigate whether functional expression of IK(DR) and I(KCa) would change with cell cycle progression, and whether they could regulate proliferation in undifferentiated rat mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Membrane potentials and ionic currents were recorded using whole-cell patch clamp technique, cell cycling was analysed by flow cytometry, cell proliferation was assayed with DNA incorporation method and the related genes were down-regulated by RNA interference (RNAi) and examined using RT-PCR. RESULTS: It was found that membrane potential hyperpolarized, and cell size increased during the cell cycle. In addition, IK(DR) decreased, while I(KCa) increased during progress from G(1) to S phase. RT-PCR revealed that the mRNA levels of Kv1.2 and Kv2.1 (likely responsible for IK(DR)) reduced, whereas the mRNA level of KCa3.1 (responsible for intermediate-conductance I(KCa)) increased with the cell cycle progression. Down-regulation of Kv1.2, Kv2.1 or KCa3.1 with the specific RNAi, targeted to corresponding gene inhibited proliferation of rat MSCs. CONCLUSION: These results demonstrate that membrane potential, IK(DR) and I(KCa) channels change with cell cycle progression and corresponding alteration of gene expression. IK(DR) and intermediate-conductance I(KCa) play an important role in maintaining membrane potential and they participate in modulation of proliferation in rat MSCs.     

Characterization of interstitial stem cells in hydra by cloning.

A procedure has been developed for cloning interstitial stem cells from hydra. Clones are prepared by introducing small numbers of viable cells into aggregates of nitrogen mustard-inactivated host tissue. Clones derived from added stem cells are identified after 1–2 weeks of growth by staining with toluidine blue. The incidence of clones increases with increasing input of viable cells according to one-hit Poisson statistics, indicating that clones arise from single cells. After correction for cell losses in the procedure, about 1.2% of the input cells are found to form clones. This compares with estimates from in vivo experiments of about 4% stem cells in whole hydra [David, C. N., and Gierer, A. (1974). Cell cycle kinetics and development of Hydra attenuata. III. Nerve and nematocyte differentiation. J. Cell Sci.16, 359–375.]Differentiation of nematocytes and nerve cells in clones was analyzed by labeling precursors with [³H]thymidine and scoring labeled nerves and nematocytes 2 days later. Nine clones examined in this way contained both differentiated nerve cells and nematocytes, demonstrating that the interstitial stem cell is multipotent. This result suggests that the observed localization of nerve and nematocyte differentiation in whole hydra probably occurs at the level of stemcell determination. The observation that differentiated cells occur very early in clone development suggests that a stem cell's decision to proliferate or differentiate is regulated by shortrange feedback signals which are already saturated in young clones.     

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.     

Cloned interstitial stem cells grow as contiguous patches in hydra

The migration of interstitial cells was analyzed during the growth of stem cell clones in vivo. The spatial distribution of cloned cells was analyzed at a time by which extensive migration of interstitial cells could have occurred. All interstitial cell clones were found to form large contiguous patches of cells. The results indicate that there is little migration of large interstitial cells in undisturbed tissue during normal growth. This finding is surprising since numerous grafting experiments have shown extensive migration of these cells. The implications of finding nonrandomly distributed stem cells are discussed.