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PubMed1998

Phase transitions in the growth of C3H 10T1/2 cells.

In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth--e.g. containing cells in all phases of the growth cycle--as the area density increases, cells are progressively lost from the distribution in the order M phase, G2 phase, S phase and G1 phase, leading to the accumulation of viable cells out of cycle in so-called G0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

Animals↗
PubMed1999

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.

Biological Transport↗
PubMed1989

[Dependence of lethality and incidence of chromosome aberrations induced by treatment of synchronized human diploid fibroblasts with sodium fluoride on different periods of the cell cycle].

The cytotoxic and clastogenic effects of sodium fluoride during various phases of cell cycle of human cultured diploid fibroblasts were examined. The cells in confluence were synchronized at G1/G0 phase by a period of growth in medium containing 1% serum (low serum medium). To obtain the cells in S phase and G2 phase, exponentially growing cells were cultured in low serum medium with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. Synchronized cells were treated with sodium fluoride for 3 hr during the G1 phase or G2 phase, and for each of three 3-hr periods during the S phase which lasted 9 hr. The cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of cell cycle during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, whereas scare lethality was observed in G1 phase. Inducibility of chromosome aberrations of the cells following treatment with sodium fluoride was also dependent upon the phase of cell cycle. Significant increase in the incidence of chromosome aberrations was observed only in cultures treated during early and/or middle S phases of the cell cycle. These results indicate that cytotoxicity and clastogenicity of sodium fluoride to cultured human diploid fibroblasts are cell phase dependent, and that the cells in early and middle S phases are more sensitive to these effects.

Cell Cycle↗
PubMed1995

Nuclear proteins involved in cell division in the ciliate, Tetrahymena.

For the purpose of clarifying the presence of nuclear proteins which may induce cell division, we conducted experiments using ciliate Tetrahymena which can be synchronized at the G2 phase in the cell cycle easily by periodic heat shock treatment (HST): We obtained proteins from the nuclei isolated from the cells grown at the early mid log-phase, at the G2 phase (60 min) and at the G1 phase (150 min), after HST. The proteins were studied by comparing the spots separated by isoelectric point, 10-20% gradient SDS two-dimensional electrophoresis. As a result, in comparison with the intranuclear protein at the log-phase, the proteins at the G2 phase showed a marked increase, but with no great change in the electrophoretic pattern. Meanwhile, the proteins at the G1 phase differed greatly from those at the G2 phase not only in the quantitative changes, but in the electrophoretic patterns. It is considered that the level of the accumulation of the nuclear proteins which should be closely involved in cell division must increase markedly at the G2 phase and decrease at the G1 phase. We confirmed the presence of four proteins: pI 5.8 MW 68 kDa, pI 6.1 MW 75 kDa, pI 8.6 MW 48 kDa and pI 6.6 MW 57 kDa, and then prepared monoclonal antibodies using these nuclear proteins as antigens. Among them, the antibody (IgM) against the pI 8.6 MW 48 kDa polypeptide (p48) was recognized the nuclei by indirect immunofluorescence in ancellular system at the S, G2 and the mid-M phases. However, nuclei at the late M and G1 phases were not stained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Adrenalin has differential effects on epidermal cell cycle progression in mice.

The cell kinetic response after intraperitoneal injection of the 10 micrograms adrenalin was investigated in hairless mouse epidermis. Changes in the proportion of cells in S and G2 phase were studied by means of flow cytometry of isolated basal cells. Changes in the proportion of cells in prophase and metaphase, changes in the mitotic rate (Colcemid method) and in cell cycle progression of 3H-TdR labeled cells were studied in histologic sections. The results showed that adrenalin has a differential effect on cell proliferation in mouse epidermis. The cell progression rate from S phase through G2 phase to metaphase is increased in one cohort of cells shortly after adrenalin injection. Simultaneously another cohort of cells is reversibly delayed or blocked in prophase. In agreement with most previous studies a significantly reduced cell division rate was seen 2-3 hr after adrenalin injection. At this time the proportions of cells in prophase and G2 phase were normalized, whereas a significant increase in the proportion of cells in S phase had cycle progression out of S phase might be responsible for the reduced mitotic rate seen after adrenalin administration.

Animals↗
PubMed1993

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride.

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G0/G1 phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G1/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G1 phase or G2 phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in cultures treated with NaF during the first 3 h of S phase (early S phase). Moderate lethality was observed when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G2 phase. Little lethality was observed in cultures in G1 phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and/or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.

Cell Cycle↗
PubMed1997

Preferential incision of interstrand crosslinks induced by 8-methoxypsoralen plus UVA in yeast during the cell cycle.

Interstrand crosslink (ICL) induction by 8-methoxypsoralen plus UVA and the incision step of the repair have been investigated during the mitotic cell cycle of haploid Saccharomyces cerevisiae. Cells were synchronised by elutriation and events were examined at the level of the MAT alpha and the HML alpha loci in a SIR strain. The DNA sequence of these two loci is identical, but the MAT alpha locus may be replicated earlier in S phase and is transcriptionally active while the HML alpha locus may be replicated later in S phase and is transcriptionally inactive because of Sir repression that creates a heterochromatin-like structure at this locus. ICL were induced to similar extents in both loci during the stages of the cell cycle examined, and these levels were identical to those reported for asynchronous cultures. Preferential incisions occurred for ICL in the MAT alpha locus compared to those in the HML alpha locus, independently of the cell cycle phase studied. The levels of incision were comparable for events in the early G1 phase (eG1), late G1 phase (lG1), early S phase (eS), middle S phase (mS), late S phase (lS) or G2 phase (G2). Thus the preferential incision of ICL observed previously in asynchronous cell culture is maintained throughout the cell cycle and, surprisingly, occurs equally well in G1. Here the opportunities for recombination to further process the incised damaged are substantially limited compared to those in the S and G2 phases.

Cell Cycle↗
PubMed1986

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta.

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30-32 h; 75% of these cells multiplied after 30-32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [35S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [35S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied: [35S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [35S]proteoglycans secreted into the medium. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar kav after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (kav 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle). About 95% of monomers synthesized at the two stages of the cell cycle were eluted at kav 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

Animals↗
PubMed2004

[The characteristics of PSCL32.5 protein from Physarum polycephalum and the variation of the content through cell cycle].

Crude fraction of Arg/Ser-rich proteins (SR proteins) were isolated from plasmodia of Physarum polycephalum and immunoassayed by western blot with a monoclonal antibody against SC35 protein from HeLa cell. Two polypeptides were detected by the antibody, suggesting that they were SCL(SC35-like) proteins. The SCL proteins have their mass weight of 32.5 kD and 82.5 kD, respectively, and so were termed PSCL32.5 and PSCL82.5 in this paper. The PI of PSCI32.5 was ascertained as 6.19 by IEF after a further purification of the protein with SDS-PAGE. The densitometric scanning of the western blot bands of PSCI32.5 isolated at different phases of cell cycle of P. polycephalum demonstrated that the relative content of the protein varied through the cell cycle: it appeared as the lowest at early S phase, showed increases from S phase to G2 phase, and peaked at late G2 phase.

Animals↗
PubMed1981

Effects of aclacinomycin on cell survival and cell cycle progression of cultured mammalian cells.

The effects of aclacinomycin (ACM; NSC 208734) on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent (Chinese hamster ovary) cell systems. Cell cycle progression and the effect of the drug on various transition points in the cell cycle (i.e. G1 to S phase, through a window in early S phase and G2 phase to mitosis) were monitored by flow cytometry. Formation of Chinese hamster ovary cell colonies was inhibited by 50% following 24 hr of exposure to 0.05 micrograms ACM per ml whereas 1 hr of exposure to 1.0 micrograms ACM per ml reduced colony formation by only 30%. Stationary cultures required a drug concentration more than 5 times higher to reduce colony formation by an equivalent amount when present for 24 hr. Short-term (1-hr) exposure to drug concentrations up to 1.0 micrograms/ml had no effect on colony formation of stationary-phase Chinese hamster ovary cells. Cell growth was inhibited by 50% in suspension cultures of Friend leukemia and L1210 cells when exposed for 24 hr to 0.024 and 0.053 micrograms ACM per ml, respectively. Continuous drug exposure of Friend leukemia and L1210 cells to ACM concentrations of 0.05 to 0.1 micrograms/ml led to a slow down in cell progression manifested as an accumulation of cells in G2 + M phase by 24-hr and then in G1 phase by 48-hr culture. However, brief (1-hr) exposure of L1210 cells to 0.5 micrograms/ml resulted in an irreversible accumulation of cells in G2 + M phase. A more detailed examination of drug effects on the cell cycle determined that 0.1 micrograms ACM per ml resulted in a slow down in L1210 cells leaving G1 phase and entering mitosis and an accumulation of cells in G2 phase, although early S-phase cells appeared unaffected. At a 5 times higher drug concentration, exit of cells from G1 was almost completely halted, passage of cells through early S was slowed, and the entrance of cells into mitosis plateaued 3.5 hr after addition of the drug; G2-phase cells were only mildly affected. The RNA content of all cells examined was reduced by 35 to 50% depending upon dose and time of exposure. These findings are discussed in terms of the known biochemical effects of ACM on RNA and protein synthesis.

Aclarubicin↗
PubMed1986

Evidence of mouse epidermal subpopulations with different cell cycle times.

In order to obtain information on the distribution of total cell cycle times in hairless mouse epidermis, basal cells were isolated and prepared for DNA flow cytometry at intervals after a pulse labeling with 50 microCi of thymidine. The DNA distributions were recorded, and cells were sorted from windows in the S, G2, and G1 phases of the cell cycle, collected on glass slides, and subjected to autoradiography. The proportions of labeled cells were scored in each fraction, and the percentage of labeled mitoses was determined in histologic sections from the same animals. Grain count distributions were recorded at selected time points over labeled cells in sorted fractions and over labeled mitoses. The movement of the labeled S-phase cohort was thus followed through all cell cycle phases. Peaks in labeled cells were observed at about 36 h in S phase, G2 phase, and mitosis, and high levels of labeled G2 cells and mitoses were seen at about 80 h. These results indicate the existence of one rapidly cycling subpopulation of keratinocytes with a cell cycle time slightly less than 30 h, in addition to keratinocytes with considerably longer cell cycle times. The first peak of labeled G2 cells reached only about 30%. This is consistent with earlier findings of about 30% G2 cells with a rapid traverse, and 70% with a considerably delayed traverse through G2 phase. The proportion of labeled G1 cells reached a value corresponding to twice the initial labeling index at 8 h after pulse labeling. This is consistent with previously obtained phase durations, indicating an unperturbed cell cycle traverse of labeled cells from S phase through G2 and mitosis.

Animals↗
PubMed1986

Histone acetylation in replication and transcription: turnover at specific acetylation sites in histone H4 from Physarum polycephalum.

Histone H4 from growing cells is partially acetylated at lysines 5, 8, 12, and 16. The turnover rate at each of these sites was investigated by pulse-labeling plasmodia of Physarum polycephalum with [3H]acetate for 55 min in either S phase or G2 phase of the cell cycle. Labeled histone H4 was purified and digested with a protease which cleaves on the carboxyl side of arginine residues. The peptide containing the acetylation sites was purified by high-performance liquid chromatography. Subfractions of the peptide were obtained due to differences in acetyllysine content. Each subfraction was subjected to automated Edman degradation and the radioactivity released after each cycle was determined. Histone H4 was acetylated uniformly in vitro and acetylated peptide 1-23 was used as a control. The results show a very striking preference for turnover on lysine-5 in the "low acetyl" subfraction from cells in S phase; the "high acetyl" subfraction showed turnover at all four sites. The peptides labeled in G2 phase showed turnover mainly at positions -8, -12, and -16. The data imply that the patterns of histone acetyl turnover associated with replication and transcription are nonrandom and distinct. The results have implications for nucleosome structure particularly the possible role of lysine-5 in chromosome maturation and for the design of experiments to test chromatin function in vitro.

Acetylation↗
PubMed1977

Synthesis and transport of myosin in Physarum polycephalum.

Immunological techniques have been used to study the rate of synthesis and intracellular transport of myosin in the slime mould Physarum polycephalum. Quantitative precipitation of myosin in homogenates of Physarum was achieved using an antimyosin antibody produced in rabbit in response to purified Physarum myosin. Dodecylsulphate-gel electrophoresis revealed that about 50% of the precipitated material is myosin. The rates of synthesis of total cellular protein and myosin were measured over the mitotic cycle. Both were found to increase exponentially or linearly between two successive nuclear divisions. Similarly, no difference in the proportion of myosin-synthesising polysomes, assayed by precipitation with antimyosin serum, could be detected between the S phase and G2 phase of the mitotic cycle. Myosin makes up nearly 2% of total plasmodial proteins. Its transport into the nucleus occurs predominantly during the G2 phase.

Animals↗
PubMed1992

Circadian variations in cell cycle phase progression of mouse epidermal cells measured directly by bivariate BrdUrd/DNA flow cytometry.

Circadian stage-dependent variations in cell cycle traverse of mouse epidermal cells in vivo were investigated. The fate of cohorts of basal cells pulse-labelled with bromodeoxyuridine (BrdUrd) at different times of the day were studied by bivariate BrdUrd/DNA flow cytometry of isolated epidermal basal cells. Basal cells were tracked through the cell cycle up to 96 h after intraperitoneal injection of BrdUrd at 0800 and 2000, or followed for 6 h after BrdUrd injection at 0400, 1200, 1600 and 2400. The results confirmed our previous assumption that the cell cycle progression through S phase and G2 phase is considerably delayed at night, i.e. from 1600 to 0400, compared with daytime. The results indicate variations in G1 phase as well. The data strongly support the hypothesis that the main parameters responsible for circadian fluctuations in mitotic activity are variations in the S and G2 phase durations. The data are also consistent with the notion of proliferative heterogeneity among basal cells as described by a hierarchical proliferation model.

Animals↗
PubMed1986

Serum-dependent control of entry into S phase of next generation in rat 3Y1 fibroblasts. Effect of large T antigen of simian virus 40.

When rat 3Y1 fibroblasts are deprived of serum in S phase and/or G2 phase in the first generation, the cells delay entry into S phase in the second generation for the duration of the serum deprivation. We can now show that when resting 3Y1 cells are infected with Simian virus 40 (SV40), the removal of serum through S and G2 phases in the first generation does not markedly delay entry into S phase in the second generation. These observations suggest that the serum-dependent control of entry into S phase of the second generation continues from the first generation, and that the abolition of this control by infection with SV40 in the first generation involves the mechanism operative when the resting cells are stimulated to enter S phase (of the first generation) by infection with SV40.

Alpha-Globulins↗
PubMed1984

Mitotic delays and macromolecular synthesis in G2 phase-irradiated plasmodia of Physarum polycephalum.

Mitotically synchronous surface plasmodia of Physarum polycephalum were irradiated at different times of G2 phase with doses of UV ranging from 350 to 2800 Jm-2. The UV sensitivity, measured in terms of delay in progression towards mitosis, gradually declined through G2 phase to almost zero in early prophase, when irradiated with 350 Jm-2 of UV. However, with higher doses, 700 and 1400 Jm-2, late G2 was found to be even more susceptible than early G2. The delay in these cases rises for the last 0.1 part of the cycle up to a UV-transition point in early prophase. Thus, processes which were not sensitive to a lower dose of UV are affected by higher doses. Above 700 Jm-2, delay was not directly proportional to dose. With doses above 1400 Jm-2, the delays obtained were more variable, although some tendency for an increased delay is observed in plasmodia irradiated in late G2 when compared with those irradiated in middle G2. The least UV-sensitive phase is between -0.2 and -0.4 part of the cycle in relation to mitosis (0 point). Both RNA synthesis and protein synthesis are inhibited in plasmodia irradiated at any time during G2 phase with 1400 Jm-2 of UV. However, on an average, irradiation in late G2 caused the most inhibitions.

Fungal Proteins↗
PubMed1997

p100: a novel proliferation-associated nuclear protein specifically restricted to cell cycle phases S, G2, and M.

By immunization with nuclear lysates of L428 cells, we raised a monoclonal mouse antibody, Ki-S2 (IgG1). In Western blots, this antibody recognizes a nuclear antigen with an apparent molecular mass of 100 kD, termed p100. Protein sequencing of p100 showed that this is a hitherto unknown protein. Immunohistochemical examination of cryostat and paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the nuclei of a fraction of proliferating cells. Cell sorting and fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition G1/S until the end of cytokinesis. During mitosis, this protein is strictly associated with the spindle pole and with the mitotic spindle, whereas during S and G2, p100 is diffusely distributed throughout the cell nucleus. Immediately after completion of cytokinesis, p100 was rapidly degraded. In L428 cells, p100 is phosphorylated at least during mitosis. It has a turnover time of about 1 hour. Studies on routinely processed paraffin sections of specimens of malignant lymphoma, benign and malignant nevocellular tumors, and breast cancer showed that in all cases less than 40% of the Ki-67-positive growth fraction expressed p100. Thus, p100 might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer prognosis, beyond its general biologic relevance as a cell cycle protein.

Animals↗
PubMed2003

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.

Apoptosis↗