Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures

As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G(1) phase of the cell cycle. The steady-state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady-state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. (c) 1992 John Wiley & Sons, Inc.     

The role of p21cip1 in adaptation of CHO cells to suspension and protein-free culture

The up-regulation of cyclin-dependent kinase inhibitor p21 has been shown to enhance productivity of monoclonal antibodies and has been linked to various regulatory processes. To identify the potential role of p21 in adaptation to suspension and protein-free cultures, we studied the survival and growth of anchorage- and serum-dependent CHO cell lines that differed only in the period of p21-induced arrest. p21 overexpression led to rapid adaptation of cells to suspension and protein-free cultures. The period taken to achieve adaptation was correlated with the time the cells were arrested after transfer from the monolayer and serum-fed culture. Interestingly, cell aggregation associated with protein-free suspension culture was reduced in p21 culture in response to the loss of cellular adherence. The processes of adaptation to suspension and arrest did not decrease monoclonal antibody productivity. In contrast, following adaptation to protein-free growth media, an overall increase in specific productivity was observed. The ability of cells to survive in protein-free suspension cultures was due to the requirement of G1 cells to growth factors and to their relatively high resistance to the hydrodynamic forces. This improved process has the advantage of reducing the duration of critical path activity for developing CHO commercial cell lines from 72 to 36 days.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.     

Light and dark control of the cell cycle in two marine phytoplankton species

The effect of light and dark on growth, DNA replication and cell division of two marine phytoplankters Thalassiosira weissflogii (a diatom) and Hymenomonas carterae (a coccolithophorid) was investigated using flow cytometry. The two species displayed very differing behavior. When transferred from light to prolonged darkness, all coccolithophorid cells were arrested at the beginning of the G1 stage of the cell cycle. When shifted back into light, they resumed cycling at a rate slightly slower than prior to arrest. In contrast, diatom cells were arrested either in the G1 or G2 stage of the cell cycle in the dark. Upon re-exposure to light, cells which had been dark-arrested in G1 resumed cycling at the same rate as prior to arrest, while cells arrested in G2 cycled much more slowly. These results suggest that in both species, light control of cell cycle progression is effective only over a restricted part of the cell cycle, as has been hypothesized by Spudich & Sager (J cell biol 83 (1980) 136) [38] for Chlamydomonas. In the coccolithophorid there is a single light-dependent segment located at the beginning of G1, whereas the diatom appears to have two such segments, one in G1 and the other in G2, corresponding to two different light requiring processes.     

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.     

Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: the role of nutrient deprivation

Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle. However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays.     

Relationship between hybridoma growth and monoclonal antibody production.

Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during G1 and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.     

Additive effects of radiation and docetaxel on murine SCCVII tumors in vivo: special reference to changes in the cell cycle

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G(2)/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with (60)Co gamma rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G(2)/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G(2)/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G(2)/M-phase fraction. More than half of the cells arrested in G(2)/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G(1)-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G(2)/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G(2)/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.     

Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture

In this study, a continuous culture system was applied to mammalian cells on large scale, and polyethyleneimine (PEI) mediated transient gene expression (TGE). PEI MAX 40,000 was chosen as a superior reagent from three types of PEI. The cell cycle distribution of cells in batch and continuous cultures was determined, in which the effects of cell cycle distribution on transfection efficiency, post-transfection proliferation and recombinant prothrombin expression were evaluated. Compared with cells from end-log and plateau phase in batch culture, cells from mid-log phase possessed a larger fraction of S and G2/M phase cells and a smaller fraction of G1 phase cells. In the continuous culture, the fraction of cells in the S and G2/M phases increased and the fraction of cells in the G1/G0 phase decreased with increasing dilution rates. Cells from the continuous culture run at highest dilution rate had excellent proliferation, transfection efficiency and protein expression. These results were confirmed by transfecting cells synchronized to different phases. The G2/M arrested cells exhibited a nearly 10-fold increase in recombinant human prothrombin production relative to that of non-dividing cells. The use of continuous culture for large scale transfection demonstrated a better cell physiological state for TGE process.     

Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.     

Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.     

Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair

Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Effect of temperature on hybridoma cell cycle and MAb production

The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34 degrees C to 39 degrees C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37 degrees C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G(1)-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G(1)-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. (c) 1992 John Wiley & Sons, Inc.     

An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery

The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease.     

Phosphoinositide 3-kinase signaling overrides a G2 phase arrest checkpoint and promotes aberrant cell cycling and death of hematopoietic cells after DNA damage

DNA damage activates arrest checkpoints to halt cell cycle progression in G1 and G2 phases. These checkpoints can be overridden in hematopoietic cells by cytokines, such as erythropoietin, through the activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. Here, we show that PI3K activity specifically overrides delayed mechanisms effecting permanent G1 and G2 phase arrests, but does not affect transient checkpoints arresting cells up to 10 hours after gamma-irradiation. Assessing the status of cell cycle regulators in hematopoietic cells arrested after gamma-irradiation, we show that Cdk2 activity is completely inhibited in both G1 and G2 arrested cells. Despite the absence of Cdk2 activity, cells arrested in G2 phase did retain detectable levels of Cdk1 activity in the absence of PI3K signaling. However, reactivation of PI3K promoted robust increases in both Cdk1 and Cdk2 activity in G2-arrested cells. Reactivation of Cdks was accompanied by a resumption of cell cycling, but with strikingly different effectiveness in G1 and G2 phase arrested cells. Specifically, G1-arrested cells resumed normal cell cycle progression with little loss in viability when PI3K was activated after gamma-irradiation. Conversely, PI3K activation in G2-arrested cells promoted endoreduplication and death of the entire population. These observations show that cytokine-induced PI3K signaling pathways promote Cdk activation and override permanent cell cycle arrest checkpoints in hematopoietic cells. While this activity can rescue irradiated cells from permanent G1 phase arrest, it results in aberrant cell cycling and death when activated in hematopoietic cells arrested at the G2 phase DNA damage checkpoint.     

Digital Holographic Microscopy for Non-Invasive Monitoring of Cell Cycle Arrest in L929 Cells

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.     

MyoD can induce cell cycle arrest but not muscle differentiation in the presence of dominant negative SWI/SNF chromatin remodeling enzymes

Cell cycle arrest is critical for muscle differentiation, and the two processes are closely coordinated but temporally separable. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that have been shown to be required for muscle differentiation in cell culture and have also been reported to be required for Rb-mediated cell cycle arrest. We therefore looked more closely at how SWI/SNF enzymes affect the events that occur during MyoD-induced myogenesis, namely, cell cycle regulation and muscle-specific gene expression, in cells that inducibly express dominant negative versions of Brahma (BRM) and Brahma-related gene 1 (BRG1), the ATPase subunits of two distinct SWI/SNF complexes. Although dominant negative BRM and BRG1 inhibited expression of every muscle-specific regulator and structural gene assayed, there was no effect on MyoD-induced activation of cell cycle regulatory proteins, and thus, cells arrested normally. In particular, in the presence or absence of dominant negative BRM or BRG1, MyoD was able to activate expression of p21, cyclin D3, and Rb, all of which are critical for cell cycle withdrawal in the G1/G0 phase of the cell cycle. These findings suggest that at least one basis for the distinct mechanisms that regulate cessation of cell proliferation and muscle-specific gene expression during muscle differentiation is that SWI/SNF-mediated chromatin-remodeling enzymes are required only for the latter.     

Rb+ influxes differentiate between growth arrest of cells by different agents

The effect of cell cycle on Rb+ (K+) fluxes was studied in NIH 3T3 mouse fibroblasts. Serum starvation or isoleucine deprivation resulted in cell arrest at an early G1/G0 phase, accompanied by a marked decrease in both ouabain-sensitive and ouabain-resistant Rb+ influx. On the other hand, cells arrested at late G1/G0 phase by hydroxyurea treatment have high ouabain-sensitive and ouabain-resistant Rb+ influx. Butyric acid treatment resulted in cell arrest at an early G1/G0 phase, but in contrast to serum or isoleucine starvation did not decrease Rb+ influxes. It is thus shown that quiescent cells may have Rb+ influx rates as high as that of logarithmically growing cells. The results are consistent with the hypothesis that an increased ion permeability of the cell is initiated at a critical stage in G1/G0 phase, and that butyric acid may arrest the cell beyond that stage.