Cell Cycle Synchrony in Giardia intestinalis Cultures Achieved by Using Nocodazole and Aphidicolin

Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G₁/S and G₂/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.Giardia intestinalis is a ubiquitous intestinal protozoan parasite causing disease in humans and animals worldwide (1, 11). In developing countries, diarrheal disease is responsible for 80% of the deaths of children under 2 years of age (21), and Giardia is one of the major causes of this condition. As a diplomonad, Giardia has been proposed to represent the earliest diverging lineage of extant eukaryotes, based on single rRNA and single and/or concatenated protein phylogenies developed by considering an archaeal out-group (2, 3, 5, 15, 23), making it a valuable organism for studying the evolution of biological processes in all eukaryotes. Characteristic of the order Diplomonadida, Giardia trophozoites contain two nuclei that remain separate during mitosis, with each daughter cell inheriting one copy of each parental nucleus (19). The trophozoite form, which attaches to the small intestine of the host, has a tetraploid (4N) DNA content in G₁ since each nucleus is 2N (4). Following a round of DNA synthesis, each G₂ nucleus is 4N, making the cell 8N. According to previous flow cytometry results, actively growing Giardia cultures spend the majority of the cell cycle in the G₂/M phase and significantly less time in the G₁ and S phases (4); in contrast, many tissue culture cells display a lengthy G₁ phase. Until recently, an inability to synchronize in vitro Giardia cultures to any degree has severely hampered the ability of researchers to study cell cycle-dependent processes (16, 20).This work demonstrates successful cell cycle arrest by using nocodazole, a microtubule-destabilizing drug that leads to the depolymerization of spindle microtubules in Giardia (6, 20). A brief nocodazole treatment resulted in cells arrested early in mitosis or at the end of G₂, presumably by the activation of a mitotic spindle checkpoint (22). G₂ arrest using nocodazole was combined with G₁ arrest using aphidicolin, a drug that presumably acts through the inhibition of polymerase-dependent DNA synthesis (8, 12, 14, 25). By combining these two treatments, we were able to effectively synchronize Giardia cultures while maintaining cell viability. These synchronization methods were used to enrich cultures with mitotic spindles at the M phase. Moreover, these methods will be a valuable tool for studying other aspects of Giardia biology such as encystation, the time in the life cycle when the trophozoite transforms into an infectious cyst.     

Computational Methods for Estimation of Cell Cycle Phase Distributions of Yeast Cells

Two computational methods for estimating the cell cycle phase distribution of a budding yeast (Saccharomyces cerevisiae) cell population are presented. The first one is a nonparametric method that is based on the analysis of DNA content in the individual cells of the population. The DNA content is measured with a fluorescence-activated cell sorter (FACS). The second method is based on budding index analysis. An automated image analysis method is presented for the task of detecting the cells and buds. The proposed methods can be used to obtain quantitative information on the cell cycle phase distribution of a budding yeast S. cerevisiae population. They therefore provide a solid basis for obtaining the complementary information needed in deconvolution of gene expression data. As a case study, both methods are tested with data that were obtained in a time series experiment with S. cerevisiae. The details of the time series experiment as well as the image and FACS data obtained in the experiment can be found in the online additional material at http://www.cs.tut.fi/sgn/csb/yeastdistrib/.     

Induction of Cell Division Synchrony and Variation of Cytokinin Contents through the Cell Cycle in Tobacco Cultured Cells

Cell division synchrony was induced in tobacco {Nicotiana tabacum)cultured cells by several treatments. Very high synchrony throughouttwo cell cycles was induced by aphidicolin treatment (inhibitorof DNA polymerase , 10 µg/ml) and by treatment with lowtemperature (4°C) and hydroxyurea (50 µg/ml). Themitotic index reached its maximum (52% and 40% in aphidicolinand hydroxyurea treatments, respectively) at 11 h after removalof the added chemical. During the treatments, the cells werearrested in the G₁/S phase of the cell cycle. In the aphidicolin-inducedsystem, incorporation of ¹⁴C-thymidine confirmed that DNA synthesiswas started immediately after removal of the chemical. The aphidicolin-induced synchronous cells were used to studythe contents of butanol-soluble cytokinins during the cell cycle.Cytokinin contents increased conspicuously at the G₂/M boundary. ¹Present address: Department of Biology, Otsuma Women's University,Chiyodaku, Tokyo 102, Japan. (Received May 14, 1985; Accepted November 8, 1985)     

A Mathematical Model of Cell Cycle Effects in Gastric Cancer Chemotherapy

A mathematical model is presented to investigate the relationship between drug order and treatment response in gastric cancer chemotherapy involving a taxane (either paclitaxel or docetaxel) coupled with flavopiridol. To model treatment effects, we simulate treatment by bolus injection and employ a pulsing condition to indicate cell kill as well as instantaneous changes to the cell’s transition rates. Cell population growth is described using an ordinary differential equation model whereby we examine the treatment effects upon cells in various stages of the cell cycle. Ultimately, the results generated support prior clinical investigations which indicate that for an enhanced synergistic effect, flavopiridol must be administered following taxane therapy.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

A Transgenic Mouse Model for High Content,Cell Cycle Phenotype Screening in Live Primary Cells

High content cell-based genetic and small molecule library screens are powerful strategies in drug discovery and investigations of disease mechanisms. We report that primary cells derived from a transgenic mouse model expressing a fluorescence mitosis biosensor provide unambiguous phenotype readouts without the need for transfection or immunocytochemistry. Phenotype profiles of cell cycle disruption and of apoptosis are easily detectable at a single time point selected from time-lapse live fluorescence microscopy. Most importantly, this transgenic mouse model may be crossed with cancer mouse models to derive biosensor-expressing primary cancer cells for use in high content screening strategies targeting discovery of tumor-specific chemotherapeutic compounds.     

Quasi-Exponential Generation Time Distributions From A Limit Cycle Oscillator

In spite of the apparently random behaviour and the often exponential distribution of generation times expressed in cell populations, there is evidence for rather precise timekeeping in the cell cycle. In experiments using time-lapse video-tape microscopy, we have noted that cell generation times are often not distributed smoothly but in many cases seem to cluster at roughly 4 hr intervals. Phase shift responses following application of heat shock, ionizing radiation or serum pulses in each case show a pattern which is repeated twice in cells with an 8–9 hr modal generation time. We describe here a cell cycle model with an independent cellular clock controlling cell cycle events which accounts for the phase response data, while also reconciling the stochastic and periodic behaviour characteristic of animal cells.     

A Probabilistic Model of Perception

The development of electronic computers has made it possible to transfer to them a number of the "intellectual" functions of man. At present such machines are being employed to model broader and broader regions of man's psychological activity. One of the most important problems in this connection is the building of "perceptive machines," such as reading machines, "the printing phonograph" and so forth. Perceptive machines are a necessary element in the development of automatic equipment to operate in complicated environments.     

The Cell Cycle Time In Intestinal Crypts By Simulation of Flm Experiments

Abstract. A computer program is developed that permits simulation of the dynamic behaviour of cells in intestinal crypts (Meinzer & Sandblad, 1985). Here we present the simulation of FLM data which is compared with the experimental findings of Al-Dewachi et al. (1974). the phase durations and total cycle times of cells in the jejunal crypts of rats were calculated. Additionally, the influence of various control parameters on the simulation output is discussed, e.g. the standard errors of phase times and the grain dilution at mitosis.     

Distributions Selected for Use in Probabilistic Human Health Risk Assessments in Oregon

In 1995, Oregon enacted amendments to its state Cleanup Law that emphasize risk-based remedial action decisions and allow a responsible party to conduct probabilistic human health risk assessments. This change required selection and/or development of probability density functions for exposure factors frequently used in human health risk assessments. Methods used to obtain distributions for body weight, soil, water, vegetable/fruit, fish, and animal product ingestion, soil adherence, daily inhalation rate, various event and exposure frequencies, and exposure duration are described. Primary data sources were U.S. Environmental Protection Agency guidance and peer-reviewed scientific literature. These distributions of exposure factors may be used, in conjunction with a probabilistic age- and gender-based model, to calculate prospective exposures and risks. A brief overview of this model, which handles temporal parameters (age, exposure frequency, exposure duration) in a manner substantially different from that typically used in deterministic assessments, is also provided. Oregon's development of an age/ gender-based exposure model, and its selection of exposure factor value distributions for that model, represents one of the first attempts to develop practical approaches to using probabilistic techniques in a hazardous waste regulatory program.     

Cyclin and DNA Distributed Cell Cycle Model for GS-NS0 Cells

Mammalian cell cultures are intrinsically heterogeneous at different scales (molecular to bioreactor). The cell cycle is at the centre of capturing heterogeneity since it plays a critical role in the growth, death, and productivity of mammalian cell cultures. Current cell cycle models use biological variables (mass/volume/age) that are non-mechanistic, and difficult to experimentally determine, to describe cell cycle transition and capture culture heterogeneity. To address this problem, cyclins—key molecules that regulate cell cycle transition—have been utilized. Herein, a novel integrated experimental-modelling platform is presented whereby experimental quantification of key cell cycle metrics (cell cycle timings, cell cycle fractions, and cyclin expression determined by flow cytometry) is used to develop a cyclin and DNA distributed model for the industrially relevant cell line, GS-NS0. Cyclins/DNA synthesis rates were linked to stimulatory/inhibitory factors in the culture medium, which ultimately affect cell growth. Cell antibody productivity was characterized using cell cycle-specific production rates. The solution method delivered fast computational time that renders the model’s use suitable for model-based applications. Model structure was studied by global sensitivity analysis (GSA), which identified parameters with a significant effect on the model output, followed by re-estimation of its significant parameters from a control set of batch experiments. A good model fit to the experimental data, both at the cell cycle and viable cell density levels, was observed. The cell population heterogeneity of disturbed (after cell arrest) and undisturbed cell growth was captured proving the versatility of the modelling approach. Cell cycle models able to capture population heterogeneity facilitate in depth understanding of these complex systems and enable systematic formulation of culture strategies to improve growth and productivity. It is envisaged that this modelling approach will pave the model-based development of industrial cell lines and clinical studies.     

Synchrony in Human,Mouse and Bacterial Cell Cultures: A Comparison

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.     

A Cell Cycle Timer for Asymmetric Spindle Positioning

The displacement of the mitotic spindle to one side of a cell is important for many cells to divide unequally. While recent progress has begun to unveil some of the molecular mechanisms of mitotic spindle displacement, far less is known about how spindle displacement is precisely timed. A conserved mitotic progression mechanism is known to time events in dividing cells, although this has never been linked to spindle displacement. This mechanism involves the anaphase-promoting complex (APC), its activator Cdc20/Fizzy, its degradation target cyclin, and cyclin-dependent kinase (CDK). Here we show that these components comprise a previously unrecognized timer for spindle displacement. In the Caenorhabditis elegans zygote, mitotic spindle displacement begins at a precise time, soon after chromosomes congress to the metaphase plate. We found that reducing the function of the proteasome, the APC, or Cdc20/Fizzy delayed spindle displacement. Conversely, inactivating CDK in prometaphase caused the spindle to displace early. The consequence of experimentally unlinking spindle displacement from this timing mechanism was the premature displacement of incompletely assembled components of the mitotic spindle. We conclude that in this system, asymmetric positioning of the mitotic spindle is normally delayed for a short time until the APC inactivates CDK, and that this delay ensures that the spindle does not begin to move until it is fully assembled. To our knowledge, this is the first demonstration that mitotic progression times spindle displacement in the asymmetric division of an animal cell. We speculate that this link between the cell cycle and asymmetric cell division might be evolutionarily conserved, because the mitotic spindle is displaced at a similar stage of mitosis during asymmetric cell divisions in diverse systems.     

A Mathematical Model of Cell Cycle Dysregulation Due to Human Papillomavirus Infection

Human papillomaviruses (HPVs) that infect mucosal epithelium can be classified as high risk or low risk based on their propensity to cause lesions that can undergo malignant progression. HPVs produce the E7 protein that binds to cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein (RB) to modulate cell cycle control. Generally, high-risk HPV E7 proteins bind to RB with a higher affinity than low-risk HPV E7s, but both are able to deactivate RB and trigger S phase progression. In uninfected cells, RB inactivation is a tightly controlled process that must coincide with growth factor stimulation to commit cells to division. High-risk HPV E7 proteins short-circuit this control by decreasing growth factor requirement for cell division. We develop a mathematical model to examine the role that RB binding affinity, growth factor concentration, and E7 concentration have on cell cycle progression. Our model predicts that high RB binding affinity and E7 concentration accelerate the \(\mathrm {G_{1}}\) to S phase transition and weaken the dependence on growth factor. This model thus captures a key step in high-risk HPV oncogenesis.     

A Mathematical Model of Cell Cycle Progression Applied to the MCF-7 Breast Cancer Cell Line

In this paper, we present a model of cell cycle progression and apply it to cells of the MCF-7 breast cancer cell line. We consider cells existing in the three typical cell cycle phases determined using flow cytometry: the G1, S, and G2/M phases. We further break each phase up into model phases in order to capture certain features such as cells remaining in phases for a minimum amount of time. The model is also able to capture the environmentally responsive part of the G1 phase, allowing for quantification of the number of environmentally responsive cells at each point in time. The model parameters are carefully chosen using data from various sources in the biological literature. The model is then validated against a variety of experiments, and the excellent fit with experimental results allows for insight into the mechanisms that influence observed biological phenomena. In particular, the model is used to question the common assumption that a ‘slow cycling population’ is necessary to explain some results. Finally, an extension is proposed, where cell death is included in order to accurately model the effects of tamoxifen, a common first line anticancer drug in breast cancer patients. We conclude that the model has strong potential to be used as an aid in future experiments to gain further insight into cell cycle progression and cell death.     

Cell Cycle Markers for Live Cell Analyses

Many cellular processes are regulated by cell cycle dependent changes in protein dynamics and localization. Studying these changes in vivo requires methods to distinguish the different cell cycle stages. Here we demonstrate the use of DNA Ligase I fused to DsRed1 as an in situ marker to identify S phase and the subsequent transition to G2 in live cells. Using this marker, we observed changes in the nuclear distribution of Dnmt1 during cell cycle progression. Based on the different nuclear distribution of DNA Ligase I and Dnmt1 in G2 and G1, we demonstrate that the combination of both proteins allows the direct discrimination of all cell cycle phases using either immunostainings or fusions with fluorescent proteins. These markers are new tools to directly study cell cycle dependent processes in both, fixed and living cells.     

A Mathematical Model for the Effects of HER2 Over-Expression on Cell Cycle Progression in Breast Cancer

In this paper, we present a mathematical model predicting the fraction of proliferating cells in G1, S, and G2/M phases of the cell cycle as a function of EGFR and HER2. We show that it is possible to find parameters for the mathematical model so that its predictions agree with the experimental observations that HER2 over-expression results in: (1) a shorter G1-phase and early S-phase entry; (2) and that with a 1-to-1 ration between EGFR and HER2, the growth advantage in HER2 over-expressing cells is indeed associated with the increase of the HER2 expression level.