Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

An in vivo study on the synchronizing effect of hydroxyurea

The effect of hydroxyurea (HU; 0.5 mg/g body wt) on L 1210 ascites tumor cells has been studied using various cell kinetic methods. In contrast to the general assumption that HU blocks cells at the G1/S boundary [J. Brachet (1985) Molecular Cytology, Vol. I, p. 266, Academic Press, New York], the present results show that the cells are not held at G1/S but enter S at about the normal rate and are accumulated in early S phase due to a dose-dependent inhibiting effect of HU on DNA synthesis. Partial synchronization of the cells demonstrated by a distinct mitotic peak 10 h after HU application is not due to a G1/S block of the cells and their subsequent synchronous passage through the cycle after release from the block but is due to rather complex mechanisms of action of HU: a differential cytocidal effect and an effect on the passage of the cells through the cycle, both depending on the position of the cells throughout the cycle. HU kills S-phase cells, mainly cells in early S phase; i.e., a great portion of the cells "accumulated" in early S phase is killed by the drug, while G1-phase cells are almost not affected by the lethal effect of HU. These G1-phase cells pass through the cycle more rapidly after cessation of the HU effect. The same is true for the surviving cells accumulated in early S phase, while part of the cells in the remaining S phase are delayed in their passage through the cycle. This causes partial synchronization, since a great portion of all cells that survive HU treatment reach mitosis at the same time.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

Cytostatic effect of cyclophosphane on esophageal tumors and epithelium of mice

Investigation of cytostatic activity of cyclophosphamide in sarcoma 37 and esophagus epithelium in albino mice with respect to the diurnal rhythm of mitotic activity and the number of labeled nuclei was performed. Apparently the tumour cells in the G1-phase and at the beginning of the S-phase of the mitotic cycle were the most sensitive to the inhibitory effect of this drug. During the completion of the DNA-synthesis period the cell resistance to the action of the cytostatic increased. Cells at the G1-phase of the mitotic cycle were sensitive to the inhibitory action of cyclophosphane in the esophageal epithelium.     

Heat production of mammalian cells at different cell-cycle phases

1. 1.|Heat production of Reuber H35 rat hepatoma cells and murine C1300 neuroblastoma cells at different stages of the cell cycle were measured microcalorimetrically.

2. 2.|Reuber H35 monolayer cultures of G1-phase cells and cells in S-phase were trypsinized, reincubated in suspension culture and immediately used for microcalorimetric measurements. There was a remrkable difference in the heat evolution of H35-cells in suspension derived from a monolayer culture of G1-phase cells and that of cells in S-phase of the cell cycle. Heat production of G1-cells was relatively continuous during the experiment, in contrast to S-phase cells that showed a decrease in heat production after an initial maximum.

3. 3.|Neuroblastoma cells synchronized by mitotic shake-off and cultured in suspension progressed through their cell cycle. They showed maximal heat production shortly before and durign mitosis.

Cell-cycle phase-dependence of drug-induced cycle progression delay.

The effect of adriamycin on cell cycle phase progression of CHO cells synchronized into the various phases of the cell cycle by elutriation was investigated by high resolution pulse cytophotometry. Cells treated in all phases of the cell cycle showed delay in their subsequent progression. In addition to the wellknown block of cells in the G2-phase, a delay in passage of cells from G1 to S and a decreased rate of transit through the S-phase were observed. A broadening of the DNA distributions of the treated cells was observed after cell division indicating induction of chromosomal abnormalities.     

DNA-synthesis in synchronized L-cells after irradiation during the G1-phase of the cell cycle

Mouse fibroblast L-929 cells synchonized by mitotic selection were irradiated during the G1-phase of the cell cycle with a dose of 1000 rad. The rat of DNA synthesis was measured by 3H-thymidine incorporation, and the progression of the cells through the cell cycle was determined using a pulse-cytophotometer. Irradiation caused a decrease in the rate of DNA synthesis to half the control value, and an extension of the S-phase to twice its normal duration.     

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.     

Differential Effect of Jasmonic Acid and Abscisic Acid on Cell Cycle Progression in Tobacco BY-2 Cells

Environmental stress affects plant growth and development. Several plant hormones, such as salicylic acid, abscisic acid (ABA), jasmonic acid (JA), and ethylene play a crucial role in altering plant morphology in response to stress. Developmental regulation often has the cell cycle machinery among its targets. We analyzed the effect of JA and ABA on cell cycle progression in synchronized tobacco (Nicotiana tabacum) BY-2 cells. Both compounds were found to prevent DNA replication, keeping the cells in the G1 stage, when applied just before the G1/S transition. However, ABA did not have any effect on subsequent phases of the cell cycle when applied at a later stage, whereas JA effectively prevented mitosis on application during DNA synthesis. This demonstrates that JA treatment can freeze synchronized BY-2 cells in both the G1 and G2 stages of the cell cycle. Jasmonate administered after the S-phase was less effective in decreasing the mitotic index, suggesting that cell sensitivity toward JA is dependent on the cell cycle phase. In cultures detained in the G2-phase, we observed a reduced histone H1 kinase activity of kinases associated with the p13(suc1) protein.     

Cell cycle dependent distribution of proliferating cell nuclear antigen/cyclin and cdc2-kinase in mouse T-lymphoma cells

The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34-cdc2-kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T-lymphoma cells (Yac-1 cells), using fluorescein isothiocyanate (FITC)-labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S-phase. PCNA is essential for cells to cycle through S-phase and its synthesis is initiated during late G1-phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2-kinase was detectable in all phases of the cell cycle and the G2-M-phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 μg/ml) treated Yac-1 cells showed an aneuploid or hypodiploid population. Although the G2-M-phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2-kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S-phase and G2-M-phase, but PCNA expression was evident in a portion of the cell population in G2-M-phase. Although cells blocked in M-phase seem to have high levels of cdc2-kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2-kinase could be responsible for the colcemid arrest of cells in M-phase.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage

In the Drosophila bristle lineage, five differentiated cells arise from a precursor cell after a rapid sequence of asymmetric cell divisions (one every 2 hours). We show that, in mitotic cells, this rapid cadence of cell divisions is associated with cell cycles essentially devoid of the G1-phase. This feature is due to the expression of Cyclin E that precedes each cell division, and the differential expression of the S-transition negative regulator, Dacapo. Thus, apart from endocycles (G/S), which occurred in two out of five terminal cells, two other cell cycles coexist in this lineage: (1) an atypical cell cycle (S/G2/M), in which the S-phase is initiated during the preceding telophase; and (2) a canonical cell cycle (G1/S/G2/M) with a brief G1 phase. These two types of cell cycle result from either the absence or very transient expression of Dap, respectively. Finally, we show that the fate determinant factor, Tramtrack, downregulates Cyclin E expression and is probably involved in the exit of the cells from the cell cycle.     

The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.     

Cell cycle-dependent effects of wortmannin on radiation survival and mutation

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.     

Progression through the G1-phase of the on-going cell cycle

Cell cycle progression is dependent upon the action of cyclins and their partners the cyclin dependent kinases (CDKs). Each cell cycle phase has its own characteristic cyclin-CDK combination, cyclin D-CDK4,6 and cyclin E-CDK2 being responsible for progression through G(1)-phase into S-phase. Progression through G(1)-phase is regulated by signal transduction cascades activated by polypeptide growth factors and by extracellular matrix (ECM) components. Studies aiming to unravel the molecular mechanism by which these extracellular components activate the cyclin-CDK complexes in the G(1)-phase, are usually performed using serum-starved cells (G(0) cells). These cells are activated by addition of growth factors, or the cells are detached from the substratum by trypsinization and subsequently allowed to re-attach. An alternative approach, however, is to study the effects of growth factors and attachment in the ongoing cell cycle by synchronization of the cells by the mitotic shake-off method. These cells are not serum starved and not actively detached from the substratum. In this contribution it is shown that both methods yield significant different results. These observations demonstrate that data obtained with model systems should be interpreted with care, especially if the findings are used to explain cell cycle progression in cells in an intact organism.     

Effect of a chalone-containing preparation from Ehrlich ascites tumor on DNA synthesis and cell division in the tumor in relation to the time of day

The mitosis inhibitory action of chalone-containing preparation of the Ehrlich ascite tumour was shown to depend on the time of its administration on round the clock, and on the circadian rhythm phase of the mitotic activity in this tumour. This allowed a conclusion that the chalone system of the tumour may be involved in the formation of the circadian rhythm of cell division. It was found that Ehrlich's ascite tumour chalone system regulated DNA synthesis influencing the cell passage from G1-phase of the mitotic cycle to S-phase, and the processes occurring during S-phase.     

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.