Mathematical modelling and computer simulation of cell synchrony

A population of cells is said to be perfectly synchronised if all cells pass through the same point of their reproductive cycle at the same time, asynchronous if the percentage of cells in each phase of the cycle remains constant with time. These theoretically precise definitions, however, can seldom be applied in biological experiments, where researchers usually have to deal with partially synchronised or not completely asynchronous cell populations. As a consequence biologists need to calculate the degree of synchronisation of a cell population starting from data obtained with common laboratory measures and they need to know how to control the synchronisation process. We investigated the behaviour of partially synchronised populations using computer simulations. Different kinetic scenarios, characterised by the mean duration of G0, G1, S and G2M phases, by their variability, by cell cycle blocks, etc., were tried out to show the differences in the growth curve and in the percentages of cells in G0, G1, S and G2M phases, as measured by flow cytometry. The effects of intercell variability of transit times on cell population synchrony were investigated and the transit of cells through G0 phase was seen to induce an efficient loss of synchrony.Abbreviation CV coefficient of variation - N Number of cells - R normalised rate of cell division - ps percentage synchronisation - SI synchronisation index - T_c cell cycle time - T_d doubling time - T_G1, T_S, T_G2M G1, S and G2M transit times     

Cell cycle synchronisation by 3-hydroxypyridin-4-one iron chelators

We describe a protocol for the synchronisation of normal and tumour cells grown in suspension cultures using 3-hydroxypyridin-4-one iron chelators. These compounds inhibit ribonucleotide reductase, one of the rate limiting enzymes in DNA synthesis, and so block the cell cycle in late G₁ phase. After removal of the chelator or repletion of cellular iron, cells progress through the cycle and remain synchronised for at least one full cell cycle. Cell viability is unaffected for at least 72 hours post-incubation and chelator treatment has no effect on RNA and protein synthesis. This method of synchronisation has been successful with all cell lines tested including normal and leukaemic human cell lines.     

rhHGF/cHGF体外抑制SMMC-7721人肝细胞癌细胞生长

目的:观察重组人肝细胞生长因子(rhHGF)和天然小牛肝细胞生长因子(cHGF)对SMMC-7721人肝细胞癌细胞株的生长效应。方法:传代后处于指数生长期的SMMC-7721细胞经过(HGF处理组)或不经(对照组)rhHGF/cHGF处理培养1-4d,采用四甲基偶氮唑盐(MTT)法检测细胞数。并通过流式细胞仪对rhHGF处理细胞作细胞周期分析。结果:rhHGF(5-20μg/L)和cHGF(25-100mg/L)对SMMC-7721细胞增殖具有类似的剂量依赖性抑制作用。在实验浓度内,使用最高浓度时(rhHGF为20μg/L、cHGF为100mg/L)对细胞增殖抑制作用最强。使用不同浓度(5μg/L、10μg/L、20μg/L)rhHGF处理3d的培养细胞均有一半以上停留在G₀/G₁期。结论:rhHGF和cHGF对SMMC-7721人肝细胞癌细胞的体外生长都有强烈的抑制效应。这是由于受HGF的作用细胞生长易于终止在G₀/G₁期。     

抑制氯通道阻抑鼻咽癌细胞周期和细胞增殖

目的:研究Cl^-通道在鼻咽癌细胞调节性容积回缩(RVD)、增殖及细胞周期分布中的作用。方法:活细胞图像分析低分化鼻咽癌细胞(CNE－2Z)RVD,用台盼蓝拒染法检测细胞存活率,MTT法检测细胞增殖能力。用流式细胞仪测定细胞周期不同时相细胞百分率。结果:Cl^-通道抑制剂硝基苯丙胺基苯甲酸(NPPB)剂量依赖性抑制RVD和细胞增殖,100μmol/LNPPB明显阻抑细胞周期进程,使细胞停滞于G₁期,G₁期细胞百分率从54%提高到71%,但对细胞存活率没有显著性影响。结论:阻抑Cl^-通道可阻滞细胞于G₁期而抑制细胞增殖。提示Cl^-通道和RVD的激活是促进细胞从G₁期进入S期和维持增殖所必需的因素。     

Cytotoxicity by Tumor Necrosis Factor is Linked with the Cell Cycle But Does Not Require DNA Synthesis

Abstract

The relationship between the kinetics of cell death induced by TNF and the cell cycle in L929.10 target cells was investigated by comparison of growing, asynchronous cells with target cells synchronized at G₁/S using a double thymidine block. The induction phase of lysis, the time following TNF addition but before loss of cell viability, was shortened in asynchronous cells by increasing the level of saturation of the TNF receptor. However, in synchronized target cells, the length of the induction phase showed no dependence on receptor occupancy. Almost all cell death occurred within a 3 hr period 4-7 hr after the addition of TNF regardless of the concentration of TNF. Target cell lysis in Synchronized cells was concomitant with mitosis as verified by flow cytometry and DNA staining with propidium iodide. The narrow window of cytotoxicity was not due to cell cycle-related changes in the expression of the TNF receptor as measured by [¹²⁵I]TNF binding. Treatment with TNF did not accelerate or retard the progression of cells through S and G₂/M nor did target cells accumulate at G₂/M. When the kinetic experiments were repeated in the presence of 2 MM thymidine, TNF-treated cells died with identical dose and kinetic responses as those in which the thymidine block had been removed. Under these conditions, flow cytometric analysis revealed that DNA synthesis remained inhibited. These results suggest that TNF-induced cytotoxicity is linked to cell cycle-associated processes and that TNF is capable of overriding the normal cellular controls that coordinately link the DNA replicative cycle with the mitotic cycle. In the L929.10 target cell, TNF may induce a fatal mitosis-linked event.     

Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

Cytogenetic study of cell sub-populations in human leukemias (AML, CML) sorted by flow cytometry

Multivariate analysis, flow cytometry and sorting were used to distinguish and enrich subpopulations of bone marrow cells in cases of acute myeloblastic leukemia and blast crisis of chronic myelocytic leukemia: blast cells with a high rate of S/G2-M phase of the cell cycle; non-blast cells or blasts with a low rate of S/G2-M. These populations were separated on the basis of their forward angle and large angle light scatter of the laser beam, and of their Hoechst 33342 fluorescence intensity. About one million cells of each population were sorted and sorting purity was controlled by cytometry and microscope examination. Cell viability was good. Karyotypes of sorted cell populations were carried out using a cell synchronisation technique and showed different chromosomal markers.     

积雪草提取物对脾淋巴细胞细胞周期及细胞内游离钙的影响

目的: 观察积雪草提取物对小鼠脾淋巴细胞细胞周期及细胞内游离Ca²⁺的影响,并对其免疫调节作用机制进行初步探讨。方法: 采用乙醇浸提、极性萃取分离得到积雪草粗提物,利用Sephadex-LH20凝胶柱层析进一步分离纯化获得积雪草各组份提取物,通过观察各组份抑制1,1-二苯基-2-苦基苯肼(DPPH)自由基和羟自由基的抗氧化作用及对脾淋巴细胞增殖的影响,筛选出其活性成份。采用流式细胞术观察此活性成份对脾淋巴细胞细胞周期影响,并用Fluo-3/AM荧光探针结合流式细胞术分析积雪草提取物对脾淋巴细胞内游离Ca²⁺影响。结果: (1)获得进一步纯化并具有抗氧化活性的积雪草提取物D;(2)积雪草提取物D对脾淋巴细胞增殖抑制作用显著;(3)积雪草提取物D可降低脾淋巴细胞内游离Ca²⁺浓度,且呈量效关系;(4)积雪草提取物D能阻滞脾淋巴细胞进入S期和G₂/M期。结论: 积雪草提取物抑制小鼠脾淋巴细胞增殖,其作用机制可能是通过降低细胞内游离Ca²⁺浓度从而抑制脾淋巴细胞进入细胞分裂期而发挥作用。     

Xenon-induced inhibition of Ca2+-regulated transitions in the cell cycle of human endothelial cells

 Xenon is an anesthetic with very few side-effects, yet its targets at the cellular level are still unclear. It interferes with many aspects of intracellular Ca²⁺ homeostasis, but so far no specific event or defined regulatory complex of the Ca²⁺-signaling system has been identified. Specific effects of xenon were found by investigating its effects on the cell cycle in human endothelial cells: there is a relationship between two cell cycle transition points, their regulation by Ca²⁺, and specific blocks induced by xenon. Within the group of substances studied (xenon, isoflurane, desflurane, helium, and N₂), only xenon blocks the cells almost completely at the G₂–M transition after a 2-h treatment; those cells that slip through this block are then arrested at metaphase. If xenon is removed, cells that have been accumulating at the G₂–M boundary move into mitosis, and cells blocked at metaphase complete their mitosis normally. No such specific block of the cell cycle was found with the other substances studied. An artificial increase of intracellular Ca²⁺ in the submicromolar range, using a very low dose of the Ca²⁺ ionophore ionomycin, or a threefold increase of the external Ca²⁺ concentration suffices to lift the xenon-induced metaphase block; the cells enter anaphase despite the presence of xenon and complete cell division. Thus, the specific but completely reversible inhibition by xenon of the G₂–M transition and the block at metaphase suggest an interaction with a Ca²⁺-dependent event involved in the control of these processes. The results are consistent with the hypothesis that suppression of Ca²⁺ signals can be considered as a common denominator of the effects of xenon on the cell cycle and on the neuronal system during anesthesia. Received: 11 November 1998 / Accepted: 14 December 1998     

Antigen‐specific CD8 T cells in cell cycle circulate in the blood after vaccination

Although clonal expansion is a hallmark of adaptive immunity, the location(s) where antigen‐responding T cells enter cell cycle and complete it have been poorly explored. This lack of knowledge stems partially from the limited experimental approaches available. By using Ki67 plus DNA staining and a novel strategy for flow cytometry analysis, we distinguished antigen‐specific CD8 T cells in G₀, in G₁ and in S‐G₂/M phases of cell cycle after intramuscular vaccination of BALB/c mice with antigen‐expressing viral vectors. Antigen‐specific cells in S‐G₂/M were present at early times after vaccination in lymph nodes (LNs), spleen and, surprisingly, also in the blood, which is an unexpected site for cycling of normal non‐leukaemic cells. Most proliferating cells had high scatter profile and were undetected by current criteria of analysis, which under‐estimated up to 6 times antigen‐specific cell frequency in LNs. Our discovery of cycling antigen‐specific CD8 T cells in the blood opens promising translational perspectives.     

Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2

Summary The cell cycle of Schizosaccharomyces pombe in continuous culture is controlled at two steps, one which limits the transition from G₁ to S phase and the other which determines the timing of cell division. We have investigated, by means of flow-cytofluorometry, the cell cycle characteristics of nutritionally starved cells in stationary phase. Cells were shown to become arrested in either G₁ or G₂, in ratios which depended on the composition of the growth medium. G₁ and G₂ stationary phase cells share certain properties. (1) They become relatively resistant to heat shock. (2) They can reenter the cell cycle after subculture into fresh medium. (3) The G₁ and G₂ arrested populations have equal long-term viability in stationary phase. (4) Both populations require the activity of the cdc2 ⁺ gene for reentry into the cell cycle. We suggest that cell cycle arrest in stationary phase is regulated by the activity of the same G₁ and G₂ controls which limit the rate of cell cycle progression in continuous culture. The data demonstrate that in fission yeast the transition from G₁ to S phase does not mark a point of commitment to the completion of the cell cycle.     

The DNA-binding subunit p140 of replication factor C is upregulated in cycling cells and associates with G1 phase cell cycle regulatory proteins

The DNA-binding subunit of replication factor C (RFCp140) plays an important role in both DNA replication and DNA repair. The mechanisms regulating activation of RFCp140 thereby controlling replication and cellular proliferation are largely unknown. We analyzed protein expression of RFCp140 during cell cycle progression and investigated the association of RFCp140 with cell cycle regulatory proteins in cell lines of various tissue origin and in primary hematopoietic cells. Western and Northern blot analyses of RFCp140 from synchronized cells showed downregulation of RFCp140 when cells enter a G₀-like quiescent state and upregulation of RFCp140 in cycling cells. Translocation from the cytoplasmic compartment to the nucleus did not account for the significant increase in RFCp140 protein levels observed in cycling cells. To investigate a potential association of RFCp140 with cell cycle regulatory proteins coimmunoprecipitation assays were performed. These studies demonstrated specific binding of RFCp140 to cdk4-kinase in hematopoietic and fibroblast cell lines. Additional coimmunoprecipitation studies revealed specific association of RFCp140 with cyclin D1, p21, proliferating cell nuclear antigen, and retinoblastoma protein. These findings link DNA replication and repair factor RFCp140 to G₁ phase cell cycle regulatory elements critically involved in cell cycle control. Received: 13 May 1998 / Accepted: 1 February 1999     

Enhancement of 1,25-Dihydroxyvitamin D3– and all-Trans Retinoic Acid-Induced Differentiation of Human Leukemia HL-60 Cells by Phyllostachys nigra var. henonis

Human myeloid leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid (RA), respectively. In this study, the effect of acetone fraction prepared from bamboo leaf on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 50–400 μg/ml acetone fraction of bamboo leaf for 72 hr inhibited cell proliferation and induced a little increase in cell differentiation, as demonstrated by the MTT and nitroblue tetrazolium reduction assay. Interestingly, synergistic induction of HL-60 cell differentiation was observed when the acetone fraction of bamboo leaf was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans RA. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and the acetone fraction of bamboo leaf stimulated differentiation predominantly to monocytes, whereas combinations of all-trans RA and the acetone fraction of bamboo leaf stimulated differentiation predominantly to granulocytes. These results suggest that the acetone fraction of bamboo leaf enhanced leukemia cell differentiation and suggest a possibility of bamboo in the treatment of leukemia.     

An intermediate-conductance Ca2+-activated K+ channel mediates B lymphoma cell cycle progression induced by serum

We have previously reported that Kv1.3 channel is expressed in Daudi cells. However, the present study demonstrates that Daudi cell cycle progression is not affected by margatoxin, a Kv1.3 channel blocker, but can be suppressed by tetraethylammonium (TEA) and 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), a selective blocker of intermediate-conductance Ca²⁺-activated K⁺ (IK) channels. Our patch-clamp data indicate that Daudi cells express an IK channel because it has a unit conductance of about 30 pS, is voltage-independent, and can be activated by submicromolar Ca²⁺ and blocked by TRAM-34. Fetal bovine serum (FBS) elevated intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activated this IK channel. Conversely, Rituximab, a human–mouse chimeric monoclonal antibody of CD20, significantly decreased [Ca²⁺]_i and inhibited the channel. Furthermore, both FBS-induced IK channel expression and cell cycle progression were attenuated by the treatment with LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. These data together suggest that a growth factor(s) in FBS triggers cell cycle progression by elevating both IK channel activity via CD20 and IK channel expression on the cell surface via PI3K. Thus, elevated IK channel activity and expression may account, in part, for Daudi cell malignant growth and proliferation.     

Comparative cytotoxicity of five current dentin bonding agents: Role of cell cycle deregulation

To compare the cytotoxicity of three nano-dentin bonding agents (nano-DBAs) and two non-nano-DBAs using Chinese hamster ovary (CHO-K1) cells. We found that nano fillers were not the major contributing factor in DBA cytotoxicity, as analyzed by colony forming assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Exposure of CHO-K1 cells to all three tested total-etching DBAs led to G₀/G₁ cell cycle arrest, whereas exposure to higher concentrations of two tested nano-DBAs induced G₂/M arrest. All five DBAs further induced apoptosis at the highest concentration, as analyzed by propidium iodide staining flow cytometry. The toxicity of all DBAs (1:4000 v/v or higher) is related to increased reactive oxygen species (ROS) production, as analyzed by single cell DCF fluorescence flow cytometry. These results indicate that clinical application of DBAs may be potentially toxic to dental pulp tissues. Cytotoxicity of DBAs is associated with ROS production, cell cycle deregulation and apoptosis. Presence of methacrylate monomers such as PENTA and UDMA is possibly the major cytotoxic factor for DBAs. Further studies on other toxicological endpoints of nano-DBAs are necessary to highlight their safe use.     

G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated activated/memory phenotype CD4+ cells of older lupus mice

A general characteristic of lupus-prone mice (and humans) is the expedited accumulation of large numbers of presumably self-reactive activated/memory phenotype T cells. The mechanism by which these cells escape apoptosis has not been defined. We used activated/memory phenotype CD4⁺ cells from male BXSB mice with early-life severe lupus-like disease to investigate cell cycle status and apoptosis susceptibility, and to determine the role of corresponding genes in survival of these cells. In vitro acridine orange staining indicated that most of the rapidly accumulating memory phenotype CD4⁺ T cells of 4-month-old male BXSB mice are G1 arrested. Long-term bromodeoxyuridine in vivo labeling also showed that with advanced age, there was a shift of the CD4⁺ CD44^hi male cells from predominantly cycling to predominantly noncycling. Moreover, the CD4⁺ CD44^hi cells of older males were refractory to anti-CD3-induced proliferation and apoptosis. Using a multiprobe RNase protection assay encompassing riboprobe panels for cell cycle and apoptosis-related genes, we found that these cells exhibited high expression of certain members of the Ink4 (p18^Ink4C) and Cip/Kip (p21^Cip1) families of cyclin kinase inhibitors as well as of the apoptosis-inhibiting Bcl-x_L gene. Western blot analysis confirmed increased levels of Bcl-x_L and p21^Cip1, and also identified increases in another cyclin kinase inhibitor, p27^Kip1. We propose that in autoimmunity, self-reactive CD4⁺ cells are subjected to successive rounds of activation/division that eventually lead to a build-up in cyclin-dependent kinase inhibitors. Once high levels of such inhibitors are reached, they cause refractoriness to further activation, impaired cell cycle entry and resistance to apoptosis, a situation akin to replicative senescence.     

The Contribution of Cytolethal Distending Toxin to Bacterial Pathogenesis

Cytolethal distending toxin (CDT) is a bacterial toxin that initiates a eukaryotic cell cycle block at the G₂ stage prior to mitosis. CDT is produced by a number of bacterial pathogens including: Campylobacter species, Escherichia coli, Salmonella enterica serovar Typhi, Shigella dystenteriae, enterohepatic Helicobacter species, Actinobacillus actinomycetemcomitans (the cause of aggressive periodontitis), and Haemophilus ducreyi (the cause of chancroid). The functional toxin is composed of three proteins; CdtB potentiates a cascade leading to cell cycle block, and CdtA and CdtC function as dimeric subunits, which bind CdtB and delivers it to the mammalian cell interior. Once inside the cell, CdtB enters the nucleus and exhibits a DNase I-like activity that results in DNA double-strand breaks. The eukaryotic cell responds to the DNA double-strand breaks by initiating a regulatory cascade that results in cell cycle arrest, cellular distension, and cell death. Mutations in CdtABC that cause any of the three subunits to lose function prevent the bacterial cell from inducing cytotoxicity. The result of CDT activity can differ somewhat depending on the eukaryotic cell types affected. Epithelial cells, endothelial cells, and keratinocytes undergo G₂ cell cycle arrest, cellular distension, and death; fibroblasts undergo G₁ and G₂ arrest, cellular distension, and death; and immune cells undergo G₂ arrest followed by apoptosis. CDT contributes to pathogenesis by inhibiting both cellular and humoral immunity via apoptosis of immune response cells, and by generating necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions produced by pathogens resulting in slow healing and production of disease symptoms. Thus, CDT may function as a virulence factor in pathogens that produce the toxin.     

Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia

Frontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G₁/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degeneration.     

Oscillations: a key event in transformed renal epithelial cells

Summary Intracellular pH (pH_i) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pH_i may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form foci in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca²⁺ flux activates plasma membrane K⁺ channels and Na⁺/H⁺ exchange. This leads to periodic changes of membrane voltage and pH_i at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.Abbreviations CICR calcium-induced calcium release - IP₃a inositol triphosphate - MDCK Madin-Darby canine kidney - pH_ia intracellular pH Dedicated to our friend and teacher Prof. Dr. Gerhard Giebisch in gratitude for his long standing support