STATIONARY PHASE OF CULTURED MAMMALIAN CELLS (L5178Y)

The stationary phase of the mammalian cells L5178Y in culture can be divided into two stages: (a) an early phase characterized by the decline of mitotic index, followed by a stabilization of the cell number, and (b) a late stage, occurring several hours after the flattening of the growth curve, during which dead or dying cells appear in the cultures. The estimates of rates of cell progress showed that the rates from G₁ to S and from G₂ to M were affected in the early stationary phase. The main cause of cessation of increase in cell number in the early stationary phase is resulted from the decline in mitotic index, which is caused by prolongation of the G₂ period. The importance of the G₂ stage in regulating the cell growth is discussed in relation to other known situations in the literature.     

Differential sensitivity of muntjac lymphocyte chromosomes to mitomycin C,bromodeoxyuridine and hydroxylamine at different cell-cylce stages

Quantitative and qualitative analyses were made of aberrations induced by 3 hitherto well-known mutagens, mitomycin C (MC), 5-bromodeoxyuridine (BUdR and hydroxylamine hydrocholride (HA), in muntjac chromosomes, during different stages of the cell cycle. The sensitivity ro MC was increased in G₁, reached its maximum in early S and was considerably decreased in late S and G₂ stage treated cells. BUdR induced maximal aberrations when given during the synthetic phase and the cells in G₁ and G₂ were least affected. The sensitivity of the cells to HA in terms of induced chromosomal aberrations increased as they moved through the cell cycle, i.e. more damage was observed in cells treated in late S and G₂ stages than in those treated at G₁ and early S stages. While there were defined patterns of cell-cylce stage-dependent sensitivity for all 3 chemicals, the chromosomal sites being preferentially affected by each were found to be specific and invariant at different stages. Thus, it is presumed that the functional state of such “preferred sites” at one or other stage of the cell cycle is the factor responsible for the stage-dependent sensitivity of a cell towards these chemicals.     

Synthesis of glycoprotein, glycolipid, protein, and lipid in synchronized L5178Y cells

Synthesis of four macromolecular classes found in membranes—glycoprotein, glycolipid, protein, and lipid—was measured as a function of time of the cell cycle in synchronized L5178Y cells. Incorporation of leucine, choline, fucose, glucosamine, or thymidine into the cells, protein, nucleic acid, or lipid was measured by pulse-labeling for ½ hr at ½ hr intervals after release from the mitotic block. The amount of protein, lipid, glycoprotein, or glycolipid released or secreted into the medium by the L5178Y cells was also measured as a function of time of the cell cycle. Cellular protein was found to be synthesized throughout the cell cycle, with the highest synthesis occurring in the S period; synthesis was depressed in the M period. Cellular glycoprotein was synthesized at approximately the same times as protein, except that the rates of glycoprotein synthesis in the S period relative to other periods were much greater than for protein. Secreted protein was synthesized throughout the cell cycle without any general pattern, except that secretion was elevated in the late S and G₂ periods. Secreted glycoprotein was similar to secreted protein. Cellular lipid and cellular glycolipid were synthesized almost exclusively in the G₂ and M periods; there was no synthesis in the G₁ and S periods. Release or secretion of glycolipid and lipid also occurred in the G₂ and M periods.     

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G₁, DNA synthesis, S, the second growth stage, G₂, mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4′, 6-diamidino-2-phenylindole dichloride.

Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G_q, an hypothesized side loop from G₁. All morphological stages, not only division, appear to be phased by the circadian clock.

     

Immunocytochemical localization of callose in root cortical cells parasitized by the ring nematodeCriconemella xenoplax

Summary Under hypoxia (10 and 5% partial oxygen tension) meristematic cells ofAllium cepa L. roots acquired new cycle kinetics, characterized by reduced but constant rates of root growth. Under these conditions, there was preferential lengthening of G₁ and of the last third of the S period, S₃. Since hyperoxygenation shortened S₃ but not G₁ in these cells, the high sensitivity of late replication to environmental oxygen is demonstrated. The preferential depression of the replication rate when those cells replicated the last third of their DNA was not associated with diminished cell size. Rather, the lower the oxygen level the larger the mean size of the cycling cells. Under anoxia (0% oxygen tension) the rate of growth slowed, accompanied by preferential accumulation of cells in G₁. However, steady state kinetics of root growth was not achieved under these extreme conditions.Abbreviations Mean cell length - LI labelling index or frequency of cells with labelled nuclei after [³H]thymidine - G₁, S, G₂ pre-replicative, replicative, and post-replicative periods of the interphase of cycling cells - M mitosis     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

Peculiarities of modification by astaxanthin of radiation-induced damages in the genome of human blood lymphocytes exposed in vitro on different stages of the mitotic cycle

The features of astaxanthin impact (20 μg/mL) in the culture of human blood lymphocytes exposed to γ-radiation (1.0 Gy) on the G₀, S, and G₂ phases of the cell cycle were studied using Comet assay. Decrease in the level of DNA damages (Tail Moment index) under astaxanthin influence on lymphocytes irradiated in all stages of cell division was established, while, as a result of previous cytogenetic investigations, lack of the modifying action of astaxanthin after irradiation of cells in the G₂ stage and radioprotective effect in the G0 stage of the mitotic cycle had been revealed. In G₀ phase, the activation of the processes of apoptosis by astaxanthin in irradiated cells with high levels of genomic damages was found. The obtained data demonstrate that astaxanthin has a powerful radioprotective potential, mainly due to its apoptogenic properties.     

Analytical expressions for PLM(t), CL(t) and CLM(t) without use of Laplace transforms

Based on the age density functions for each phase of the cell life cycle (G₁, S, G₂ and M) in an exponentially growing steady state population derived by Trucco &; Brockwell (1968), the expressions for the percentage labeled mitoses curve [PLM(t)], the continuous labeling curve [CL(t)] and the continuous labeled mitotic curve [CLM(t)] are obtained explicitly without use of Laplace transforms. This approach is useful in describing the cell population when the steady state is disturbed due to, for example, irradiation. The mitotic index [MI(t)] for this case is considered.     

Relationships between Cell Cycle and Conjugation in 3 Hypotrichs*

SYNOPSIS. Relationships between the cell cycle and the beginning of conjugation were analyzed for 3 hypotrichs: Diophrys scutum, Oxytricha bifaria, and Euplotes crassus. The first 2 species enter conjugation with micronuclei in G₁; the latter species with a micronucleus in G₂. The 1st micronuclear division of conjugating E. crassus is mitotic. Thus meiotic DNA replication occurs when the cells of each species have already entered the mating process. Cells from asynchronous populations start conjugation with their macronuclei primarily in G₁ or more rarely at the beginning of the S stage in a percentage significantly different from that expected on the basis of random mating among all cells in the population. Also, macronuclear replication, when already begun, was blocked in cells undergoing conjugation. Therefore only the G₁ or the very early S stages of the cell cycle are compatible with conjugation in the 3 analyzed species.     

Characteristics of γ-ray-induced chromosomal aberrations in mutagen-sensitive mutants of L5178Y cells

We have examined the chromosomal radiosensitivities of an ionizing-radiation- and MMS-sensitive mutant (M10), and a UV- and 4NQO-sensitive mutant (Q31), isolated from mouse lymphoma L5178Y cells, with regard to killing effects. In the first mitoses after 100 R γ-irradiations, it was found that M10 cells were highly radiosensitive in terms of chromosomal aberrations accompanying longer mitotic delay (3 h); the frequencies of both chromatid-type and chromosome-type aberrations were, respectively, about 7 and 4 times higher than that of wild-type L5178Y cells. Furthermore, chromatid exchanges, particularly triradials, isochromatid breaks with sister union, and chromatid gaps and breaks were markedly enhanced at G₁ phase of M10 cells. In contrast, the chromosomal radiosensitivity of Q31 cells after 100 R irradiation was similar to that of L5178Y cells. On the other hand, spontaneous aberration frequencies (overall breaks per cell) of M10 and Q31 cells were, respectively, 5.1 and 2.2 times higher than that of wild-type L5178Y cells. The chromosomal hypersensitivity to γ-rays in M10 cells is discussed in the light of knowledge obtained from ataxia telangiectasia cells.     

Cell Cycle Arrest during Measles Virus Infection: a G0-Like Block Leads to Suppression of Retinoblastoma Protein Expression

One of the major mechanisms by which measles virus (MV) infection causes disease and death is suppression of the immune response. The nonresponsiveness of MV-infected human lymphocytes to mitogens and a partial block in the G₀/G₁ phase of the cell cycle observed in vitro is thought to reflect in vivo immunosuppression. In order to molecularly dissect MV-induced immunosuppression, we analyzed expression of surface activation markers and cell cycle-regulatory proteins in MV-infected human T lymphocytes. MV Edmonston (MV-Ed) could induce and maintain a high level of the early activation marker CD69 in the absence of proliferation. Expression of cyclins D3 and E, which positively control entry into S phase, was also significantly decreased. Analysis of inhibitors of progression into S phase showed that a high level of p27 was maintained in the G₀/G₁-blocked subpopulation of MV-Ed-infected cells compared to the proliferating MV-infected cells. Furthermore, cell cycle-related upregulation of retinoblastoma (Rb) protein synthesis did not occur in the MV-Ed-infected lymphocytes. Acridine orange staining, which distinguishes cells in G₀ from cells in G₁, showed that RNA levels were not upregulated following activation, which is consistent with cells remaining in a G₀ state. Although expression of surface activation markers indicated entry into the cycle, intracellular Rb and RNA levels suggested a quiescent state. These results indicate that MV can uncouple activation of T lymphocytes from transition of G₀ to G₁.     

Protein synthesis during the cell cycle of L5178Y cells

There are two peaks of ³H-leucine incorporation in the cell cycle of L5178Y cells. The first, during S stage, corresponds to a peak of ³H-leucine incorporation into the nuclear fraction. The second, during S or early G2, corresponds to a peak of ³H-leucine incorporation into the mitochondrial fraction. The rate of protein synthesis is unique for the proteins from each of the four fractions, nuclear, mitochondrial, microsomal, and soluble.The SDS polyacrylamide-gel electrophoretic patterns of ³H-leucine incorporation were different among three subcellular fractions: nuclear, mitochondrial, and microsomal + soluble. However, the incorporation pattern for each fraction remains qualitatively the same throughout the cell cycle.     

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Abstract. Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G₁ phase of the cell cycle. These include the predifferentiation arrest state (G_D) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (G_S), and the nutrient deficiency arrest state (G_N). Cells at states of reversible nonterminal differentiation (G_D?) and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at G_D, G_S and G_N states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [³⁵S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the G_S, G_D, G_D?. and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the G_D? and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.     

Activation of Src Family Members Is Not Required for the Platelet-Derived Growth Factor β Receptor To Initiate Mitogenesis

The basal activity of Src family kinases is readily detectable throughout the cell cycle and increases by two- to fivefold upon acute stimulation of cells with growth factors such as platelet-derived growth factor. Previous reports have demonstrated a requirement for Src activity for the G₁/S and G₂/M transitions. With a chimeric α-β PDGF receptor (PDGFR) expressed in fibroblasts, we have investigated the importance of the PDGF-mediated increase in Src activity at the G₀/G₁ transition for subsequent cell cycle events. A mutant PDGFR chimera that was not able to detectably associate with or activate Src was compromised in its ability to mediate tyrosine phosphorylation of receptor-associated signaling molecules and initiated a submaximal activation of Erk. In contrast to these early cell cycle events, later responses such as entry of cells into S phase and cell proliferation proceeded normally when Src activity did not increase following acute stimulation with PDGF. We conclude that the initial burst of Src activity is required for efficient tyrosine phosphorylation of receptor-associated proteins such as PLCγ, RasGAP, Shc, and SHP-2 and for maximal activation of Erk. Surprisingly, these events are not required for PDGF-dependent cell proliferation. Finally, later cell cycle events do not require that Src be activated at the G₀/G₁ transition and leave open the possibility that events such as the G₁/S transition require the basal Src activity and/or activation of Src at later times in G₁.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Cell cycle: Regulatory events in G1 → S transition of mammalian cells

A cell divides into two daughter cells by progressing serially through the precisely controlled G₁, S, G₂, and M phases of the cell cycle. The crossing of the G₁/S border, which is marked by the initiation of DNA synthesis, represents commitment to division into two complete cells. Beyond this critical point no further external signals are required. We now have more comprehensive knowledge of the temporal sequence of systems at this key transition from G₁ to S—growth factor responses, a cascade of kinase reactions, activation of cyclins and their associated kinases, and oncogene and tumor suppressor gene products. Furthermore, we know that the absolute requirement for calcium and the timing of events associated with calmodulin and the 68 kDa calmodulin-binding protein are consistent with overall Ca⁺⁺/calmodulin control of all steps from the response to growth factors in G₁ to DNA replication in S phase. We now have to sort out the inter-relationships of myriad control proteins and their relation to the Ca⁺⁺/calmodulin-dependent controls—Which are causes? Which are effects? And which are parallel processes? The answers will be important, as they represent both a much deeper understanding of this key process of life and an important opportunity for improving therapeutic medicine.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.