On the measurement of cytokinetics by continuous labeling with bromodeoxyuridine with applications to chick wing buds.

The cytokinetic properties, specifically the phase-transit times, TG1, TS, and TG2+M, of chick wing bud cells were estimated using data obtained from continuous labeling of stage 20 embryos with bromodeoxyuridine (BrdUrd). The presence of BrdUrd was detected with monoclonal antibodies, and the amount of DNA in the cells was determined with propidium iodide. The fraction of cells in each cell cycle phase, the fraction of labeled cells, and the relative movement, a measure of the mean DNA content, of all labeled cells were evaluated using bivariate flow cytometry at successive times following introduction of the label. Equations are presented to describe the fraction of unlabeled cells in G2 + M, which gives a direct estimate of TG2+M; the fraction of all labeled cells, which can then be used to estimate TG1; and, finally, the relative movement, which provides an estimate of TS. Thus, the data measured in these experiments together provide estimates of the progression through the cell cycle of limb mesoderm cells.     

Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells

The mechanism of CD4(+) T-cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected individuals remains unknown, although mounting evidence suggests that direct viral cytopathicity contributes to this loss. The HIV-1 Vpr accessory protein causes cell death and arrests cells in the G(2)/M phase; however, the molecular mechanism underlying these properties is not clear. Mutation of hydrophobic residues on the surface of its third alpha-helix disrupted Vpr toxicity, G(2)/M arrest induction, nuclear localization, and self-association, implicating this region in multiple Vpr functions. Cytopathicity by virion-delivered mutant Vpr protein correlated with G(2)/M arrest induction but not nuclear localization or self-association. However, infection with whole virus encoding these Vpr mutants did not abrogate HIV-1-induced cell killing. Rather, mutant Vpr proteins that are impaired for G(2)/M block still prevented infected cell proliferation, and this property correlated with the death of infected cells. Chemical agents that inhibit infected cells from entering G(2)/M also did not reduce HIV-1 cytopathicity. Combined, these data implicate Vpr in HIV-1 killing through a mechanism involving inhibiting cell division but not necessarily in G(2)/M. Thus, the hydrophobic region of the third alpha-helix of Vpr is crucial for mediating G(2)/M arrest, nuclear localization, and self-association but dispensable for HIV-1 cytopathicity due to residual cell proliferation blockade mediated by a separate region of the protein.     

双标记RC对KHT肉瘤的细胞周期分析(英文)

本文利用测定G_1期和中S期细胞内放射性变化的方法(RC)测出小鼠KHT肉瘤的细胞周期时相的时间及其变异系数(CV)。腹腔注射~3H-UdR后,8小时再注射~(125)I-UdR,按2小时间隔取肿瘤制成单个细胞悬液,DNA特异性染料色霉素A_3染色,根据细胞DNA含量用FACS荧光激活细胞分类器分离出纯的G_1期和中S期细胞,分别测定细胞中~(125)I和~3H的放射性,用多室数学模型根据每个细胞内~(125)I和~3H的放射性变化,计算出TG_1为6.7小时,Ts为9.0小时,TG2M为3小时,生长指数为1。     

PROLIFERATION KINETICS OF SHAY CHLOROLEUKAEMIA CELLS GROWN IN DIFFUSION CHAMBERS IN VIVO

The proliferation kinetics of Shay chloroleukaemia cells was studied by the labelled mitoses technique and some other methods. Detailed estimates of the phase durations of the cell cycle for proliferating cells were obtained using young, exponentially growing diffusion chamber cultures; estimates of kinetic parameters were also obtained for 'old' cultures and for local subcutaneous chloroma tumours. The results enabled determination of the growth fraction and the cell loss factor.
Cell loss was found to be the dominant factor determining the growth rate of chloroleukaemia cell populations. The mechanism of cell loss was cell death. Some implications of these findings are discussed.     

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.     

Radiosensitivity of early and late M-phase HeLa cells isolated by a combination of fluorescent ubiquitination-based cell cycle indicator (Fucci) and mitotic shake-off

The mitotic shake-off method revealed the remarkable variation of radiosensitivity of HeLa cells during the cell cycle: M phase shows the greatest radiosensitivity and late S phase the greatest radioresistance. This method harvests all M-phase cells with a round shape, making it impossible to further subdivide M-phase cells. Recently, the fluorescent ubiquitination-based cell cycle indicator (Fucci) was developed; this system basically causes cells in G(1) to emit red fluorescence and other cells to emit green fluorescence. Because the green fluorescence rapidly disappears at late M phase, two-dimensional flow cytometry analysis can usually detect a green(high)/red(low) fraction including S-, G(2)- and early M-phase cells but not a transitional fraction between green(high)/red(low) and green(low)/red(low) including late M-phase cells. However, combining the shake-off method concentrated the transitional fraction, which enabled us to separate early and late M-phase cells without using any drugs. Here we demonstrate for the first time that cells in early M phase are more radiosensitive than those in late M phase, implying that early M phase is the most radiosensitive sub-phase during the cell cycle.     

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.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

Reovirus-induced G(2)/M cell cycle arrest requires sigma1s and occurs in the absence of apoptosis

Serotype-specific differences in the capacity of reovirus strains to inhibit proliferation of murine L929 cells correlate with the capacity to induce apoptosis. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation and induce apoptosis to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G(2)/M cell cycle arrest. Using T1L x T3D reassortant viruses, we found that strain-specific differences in the capacity to induce G(2)/M arrest, like the differences in the capacity to induce apoptosis, are determined by the viral S1 gene. The S1 gene is bicistronic, encoding the viral attachment protein sigma1 and the nonstructural protein sigma1s. A sigma1s-deficient reovirus strain, T3C84-MA, fails to induce G(2)/M arrest, yet retains the capacity to induce apoptosis, indicating that sigma1s is required for reovirus-induced G(2)/M arrest. Expression of sigma1s in C127 cells increases the percentage of cells in the G(2)/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G(2)/M arrest. Inhibition of reovirus-induced apoptosis failed to prevent virus-induced G(2)/M arrest, indicating that G(2)/M arrest is not the result of apoptosis related DNA damage and suggests that these two processes occur through distinct pathways.     

Oxaline, a fungal alkaloid, arrests the cell cycle in M phase by inhibition of tubulin polymerization

Oxaline and neoxaline, fungal alkaloids, were found to inhibit cell proliferation and to induce cell cycle arrest at the G(2)/M phase in Jurkat cells. CBP501 (a peptide corresponding to amino acids 211-221 of Cdc25C phosphatase), which inhibits the G(2) checkpoint, did not affect the G(2)/M arrest caused by oxaline, suggesting that oxaline causes M phase arrest but not G(2) phase arrest. The Cdc2 phosphorylation level of oxaline-treated cell lysate was lower than that of the control cells, indicating that oxaline arrests the M phase. Oxaline disrupted cytoplasmic microtubule assembly in 3T3 cells. Furthermore, oxaline inhibited polymerization of microtubule protein and purified tubulin dose-dependently in vitro. In a binding competition assay, oxaline inhibited the binding of [(3)H]colchicine to tubulin, but not that of [(3)H]vinblastine. These results indicate that oxaline inhibits tubulin polymerization, resulting in cell cycle arrest at the M phase.     

The analysis and interpretation of DNA distributions measured by flow cytometry

A principal use of flow cytometers is for the measurement of fluorescence distributions of cells stained with DNA specific dyes. A large amount of effort has been and is being expended currently in the analysis of these distributions for the fractions of cells in the G1, S, and G2 + M phases. Several methods of analysis have been proposed and are being used; new methods continue to be introduced. Many, if not most, of these methods differ only in the mathematical function used to represent the phases of the cell cycle and represent attempts to fit exactly distributions with known phase fractions or unusual shapes. In this paper we show that these refinements probably are not necessary because of cell staining and sampling variability. This hypothesis was tested by measuring fluorescence distributions for Chinese hamster ovary and KHT mouse sarcoma cells stained with Hoechst-33258, chromomycin A3, propidium iodide, and acriflavine. Our results show that: a) single measurements can result in phase fraction estimates that are in error by as much as 40% for G2 + M phase and 15-20% for G1 and S phases; b) different dyes can yield phase fraction estimates that differ by as much as 40% due to differences in DNA specificity; c) the shapes of fluorescence distributions and their interpretation are very dependent on the dye being used and on its binding mechanism.     

Apoptosis and cell cycle progression in an acidic environment after irradiation

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.     

BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M

Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G(2)/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G(2)/M phase of the cell cycle. G(2)/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G(2)/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G(2)/M.     

Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells.

BrdU-Hoechst flow cytometry was employed to study the proliferation kinetics of blood lymphocytes from patients with Fanconi anemia (FA). Compared to controls, untreated FA lymphocytes show normal response to PHA stimulation, normal G0/G1 exit rates, and normal first S-phase durations. The G2 phase of the first cell cycle, however, is severely prolonged, and 24% of the recruited population become arrested during the first chromosome cycle (S, G2/M phases). The delay suffered during G2 appears to be compensated in part by a subsequent G1 phase duration that is unusually short for postnatal human cells (3.7 +/- 0.5 hrs). In analogy to what has been observed in other cell systems after experimental delays of the chromosome cycle, we therefore postulate that at least some FA cells enter their second growth phase without prior completion of the delayed chromosome cycle. Renewed replication would ensue in such cells without prior passing through mitosis and cytokinesis, leading to endoreduplication, which is a frequent finding in the FA syndrome.     

Cell cycle progression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors and implications for proliferation and productivity

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (micro(N, max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining micro(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.     

Cell cycle perturbation of cultured C6 glioma cells following short-term contact with a low dose of ACNU

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1-(4-amino-2-methyl-5-pyrimidyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU-labeled cells with mid-S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively. The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.     

Flow cytometric determination of cell cycle parameters of V79 cells by continuous labeling with bromodeoxyuridine

A simple method with which to determine the cell cycle parameters, TG1, TS and TG2M (the durations of the G1, S and G2 + M phases) is described. V79 Chinese hamster lung cells were used to evaluate the method. After continuous labeling with bromodeoxyuridine (BrdU), V79 cells were stained with anti BrdU-monoclonal antibody with FITC (fluorescein isothiocyanate) and with PI (propidium iodide). The individual cells were checked by flow cytometry for green and red fluorescences whose signal intensities corresponded to the BrdU and cellular DNA contents. The durations of G1, S and G2 + M phases of V79 cells were determined by measuring the cell fractions containing the nonlabeled G1, labeled S and nonlabeled G2 + M phases. The reliability of this method is discussed.