Cell-cycle-phase

S (11 h), G2 (1 h), and M (1 h). As explained in the text and in Fig. 10.3a, entrainment by the circadian clock occurs in the model via a semi-sinusoidal rise in Weel (from 4 p.m. to 4 a.m.) and a similar, subsequent rise in Cdkl (from 10 p.m. to 10 a.m.). The variations in Weel and Cdkl from 0 acu to 100 acu (arbitrary concentration units) are represented schematically in panels (c) and (d) below the curves showing the fractions of cells in the various phases. The probability of premature G2/M transition in G2 depends on Cdkl 

4925 for (b), and 0.5125 for (d) these values ensure homeostasis of the cell population, 

the number of cells in the population oscillates around and eventually reaches a stable steady state value. Panels (a) and (b) start initially with 15 000 cells in Gl. Panel (c) and (d) start initially with 10 000 cells in steady state. The data in panels (a) and (b) are normalized by 15 000 cells, in panel (c) by 16 000 and in panel (d) by 14 000 (maximal cell number). 

The top panels in Fig. 10.2 show the oscillations in the fraction of cells in the different cell cycle phases, as a function of time, in the absence of entrainment by the circadian clock. In the case considered, the duration of the cell cycle is 22 h, and the variability V is equal to 0% (Fig. 10.2a) or 15% (Fig. 10.2b). When variability is set to zero, no desynchronization occurs and the oscillations in the successive phases of the cell cycle are manifested as square waves that keep a constant amplitude in a given phase. Conversely, when variability increases up to 15% in the absence of entrainment (Fig. 10.2b), the amplitude of the oscillations decreases, reflecting enhanced desynchronization. 

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Cell Cycle Control. Figure 1 Cell cycle regulation by Cyclin dependent kinases (CDKs). Different cyclins bound to different CDKs promote the transition from one cell cycle phase into another. CDK-dependent phosphorylation of Rb is required to release active E2F transcription factors, which promotes entry into S phase. 

Bryant When you study those later phases do you see a change in the cell cycle phase distribution ... 

Mailer There are some stories coming out that Weel comes up in meiosis II. There might be low HI kinase activity, but there is still cyclin B present in a tyrosine-phosphorylated Cdc2 complex, and this might or might not be able to signal something about cell cycle phase. 

Frahm SO, Rudolph P, Dworeck C, et al. Immunoenzymatic detection of the new proliferation associated protein plOO by means of a cellular ELISA specific detection of cells in cell cycle phases S, G2 and M. J. Immunol. Methods 1999 223 147-153. 

It is hypothesized that cells proliferate uniformly and that the distribution of cell cycle phases is synchronized in the G0/G1 phase in the PMBV/PVA hydrogel. It has also been reported that cells synchronized at G0/G1 phase express a high level of cell-specific functions [59]. Thus, it is expected that the PMBV/PVA hydrogel can avoid a reduction in activity of entrapped cells and preserve cells with high functionality. 

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

Unfortunately monoparametric DNA content analysis by PI is not able to discriminate from different cell cycle phases, and as exemplified in Figure 4, monoparametric analysis by PI show its limitations, since this flow cytometric assay does not display any details S-phase activities after drug treatments. At this point, cells can be arrested in a specific cell phase (eg. Gi or G2/M) and obviously a decrease of S-phase is observed. 

Cell cycle analysis by dedicate software (e g. Modfit or Flowjo ) [26] usually underestimates percentage of cells in S-phase, since Gi and G2/M peaks are fitted by a gaussian model with modelling of cell cycle phases, and early (ES) and late S-phase (LS) are included inside fitted peaks (Figure 4). 

BrdU Pulse Labeling and Chase of for Separating Cell Cycle Phases and Their Fates... 

Data analysis for pulse-labelling and-chase experiment of BrdU labelled and unlabelled fractions normalized for cell count permit to analyze cytostatic and cytotoxic effects during recovery time after lh- treatment. In different cell cycle phases in white area we have shown drug effects for both fractions, while in grey what was expected for untreated samples. 

SN-38 preferentially blocked replication in cells that were synthesizing DNA during treatment. Edotecarin, however, blocked replication in all cells regardless of the cell-cycle phase they occupied during treatment (Figures 8 and 9). 

Edotecarin was more efficient to block Gi and G2/M cells during shorter treatment than SN-38 also at lower concentration. Results have show that SN-38 acted mainly against cells that were actively synthesizing DNA during treatment with Topi Inhibitor, whereas edotecarin affected all cell cycle phases [17, 18]. 

Kurose A, Tanaka T, Huang X, Halicka HD, Traganos F, Dai W, Darzynkiewicz Z (2005) Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, apoptosis. Cytometry A 68(1) 1—9... 

Determination of Cell-Cycle Phase Distributions by Staining with PI. 318... 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

This protocol describes one of several useful procedures for constructing DNA content frequency histograms that yield information about cell-cycle phase distributions. The method uses ethanol-fixation to prepare cells for staining with PI. 

Cell-cycle phase distribution in an asynchronous population of CEM cells that were fixed in ethanol and stained with PI. The data, acquired with a BD FACScan, are presented as a histogram of PI fluorescence (a measure of DNA content), recorded in units of channel numbers. Analysis of the DNA frequency content histogram was performed with Modfit LT software... 

The cell cycle is a key process that recurs in a periodic manner. Early cell cycles in amphibian embryos are driven by a mitotic oscillator. This oscillator produces the repetitive activation of the cyclin-dependent kinase cdkl, also known as cdc2 [131]. Cyclin synthesis is sufficient to drive repetitive cell division cycles in amphibian embryonic cells [132]. The period of these relatively simple cell cycles is of the order of 30 min. In somatic cells the cell cycle becomes longer, with durations of up to 24 h or more, owing to the presence of checkpoints that ensure that a cell cycle phase is properly completed before the cell progresses to the next phase. The cell cycle goes successively through the phases Gl, S (DNA replication), G2, and M (mitosis) before a new cycle starts in Gl. After mitosis cells can also enter a quiescent phase GO, from which they enter Gl under mitogenic stimulation. 

Plitidepsin (Aplidin) (203) Cyclic depsipeptide Plitidepsin (Aplidin ) (203) Oncology Inhibitor to VEGF, VEGFRLandGl/ G2 phase cell cycle Phase II PharmaMar 934-936... 

For anti-tumour drugs, Ozawa et cd. [27] proposed the following models. For cell cycle phase non-specific drugs (type I drug), the cytotoxic activity depends on the drug exposure, as reflected in the area under the intracellular concentration-time profile (AUC), and can be modelled using the following formula [2,28] ... 

The cytotoxic activity of cell cycle phase specific drugs (type II drugs) is time-dependent, and is different for cells in the sensitive phase (As) and in the resistant phase (Ar), as described as follows [2,27,28] ... 

The progression of the cell cycle is regulated by interconversion processes, in each phase, special Ser/Thr-specific protein kinases are formed, which are known as cyclin-depen-dent kinases (CDKs). This term is used because they have to bind an activator protein (cyclin) in order to become active. At each control point in the cycle, specific CDKs associate with equally phase-specific cyclins. if there are no problems (e.g., DNA damage), the CDK-cyclin complex is activated by phosphorylation and/or dephosphorylation. The activated complex in turn phosphorylates transcription factors, which finally lead to the formation of the proteins that are required in the cell cycle phase concerned (enzymes, cytoskeleton components, other CDKs, and cyclins). The activity of the CDK-cyclin complex is then terminated again by proteolytic cyclin degradation. 

It has a cytocidal effect on both proliferating and nonproliferating cultured human cells, suggesting lack of cell cycle phase specificity. 

The mechanisms of interaction between fluorouracil and radiation are not clearly understood. Different hypotheses have been postulated to explain the synergistic or potentiated effect of 5-FU with radiation including redistribution of cells to a more radiosensitive cell cycle phase, deranged pyrimidine pools with reduced capacity for repair of DNA damage, and activation of apoptosis. The effect of 5-FU on radiation damage also appears to vary in different cell lines, thus complicating the extrapolation of laboratory results into clinical practice. 

The degree of sensitization achieved also appears to be profoundly affected by the cell-cycle phase. It is well established that cells in S phase are much more radioresistant than cells in other phases of the cell cycle (28). Interesting results from Latz et al. show quite clearly that cells that are pretreated with gemcitabine no longer show a progressive increase in radioresistance as they move toward DNA replication and therefore sensitization appears to be greatest in S phase (21). 

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