Contribution to the understanding of the mechanism of cytokinesis in plant cells: the action of deoxyguanosine on the kinetics of a root meristem cell population.

The kinetics of binucleate cells, formed by the action of deoxyguanosine, are studied using three methods: in a population synchronized with hydroxyurea, by autoradiography after pulse-labelling, and in a sample of a cell population morphologically located at the M--G1 limit. Deoxyguanosine induces a slowing down in S and G2, independent of the inhibition of cytokinesis. It is only when it takes effect during the G2 stage that deoxyguanosine brings about the formation of binucleate cells.     

Structural changes in chromatin during interphase

The structural organization of the dense chromatin in G₁, S and G₂ of onion meristem cells has been evaluated. A naturally synchronous subpopulation of caffeineinduced binucleate cells was employed. — Fibre size is positively correlated with cell stage in interphase. Fibre size distribution is unimodal in G₁ nuclei with the peak at 12.5 nm in diameter, while in G₂ the distribution is bimodal due to a new population of thicker fibres (22.5 nm in diameter). Separation between fibre centres takes place between mid G₁ and mid S. — Using stereological principles, the length of chromatin fibres integrated in the chromatin patches could be estimated. This length remains constant from mid G₁ to mid S, when a 1.5 fold increase in total DNA takes place. On the other hand, 40 mm/hour of chromatin fibre is integrated in the chromatin patches between mid S and mid G₂. Comparable data of the relative proportion of the different nuclear components have been obtained in each interphase period. — The reported changes provide evidence of a cyclic pattern of chromatin condensation, which may be the structural support for a model of chromatin function in these cycling cells.     

ACTIVITY OF TUBULOSINE ON THE KINETICS OF ROOT MERISTEM CELL POPULATION

The action of tubulosine on the mitotic cycle was studied using continuous labelling with tritiated thymidine. This alkaloid provokes a lengthening of the G₁ and S phases and a blocking of G₂ is totally reversible when the treatment is followed by recovery in normal medium. At a dose of tubulosine which induces a reversible mitostasis in the shortest possible time the lengthening of the phases of the cell cycle was estimated by three different techniques: labelled mitoses for the determination of G₂; labelling intensity for the determination of S; binucleate cells for the determination of T, and an original technique using labelling index of binucleate cells for the determination of G₁. The limits of the technique of labelled mitosis together with the interest of the technique aiming at the direct determination of G₁ in the case of a perturbed cycle are then discussed.     

Rate of DNA synthesis in binucleate cells

Summary The microspectrophotometric study of the amount of DNA in the course of the interphase of synchronous binucleate cells, experimentally induced in the roots of the onion (Allium cepa), at 30° C, showed that the rate of DNA synthesis does not appear to be constant throughout the interphase, being greater at the beginning and end of the S period and slower during the middle part of the period.We found that the G ₁ period occupies from 0% at 10% of the cell cycle, the S period about 81% and G ₂ period plus mitosis about 14%.Contrary to our expectations, highly significant differences were found in the DNA content of the two nuclei of more than 1% of the binucleate cells, which suggests that DNA synthesis may be asynchronous in the two nuclei within a common cytoplasm.     

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.     

Noninvasive estimation of cell cycle phase and proliferation rate of human mesenchymal stem cells by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent mesenchymal stem cells (MSCs) using a fluorescence microscope after staining with 4′,6-diamidine-2′-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by phase-shifting laser microscopy (PLM) revealed that the laser phase shift of cells in the G₂/M phase was markedly higher than that of cells in the G₀/G₁ phase. Even in the synchronous cultures to G₀/G₁ and G₂/M cell cycle phases, the laser phase shift of the cells in the G₂/M phase was markedly higher than that of the cells in the G₀/G₁ phase. The analysis of the cultures of MSCs from different donors with the addition of FGF2 at different concentrations revealed that there was a marked negative correlation between the average phase shift and mean generation time. In conclusion, it is possible to estimate noninvasively the proliferation activity of MSCs population by measuring the phase shift using PLM.     

Cytochemistry of histones throughout the division cycle in meristematic cells

Summary We have been able to follow the increase in nuclear histones along the division cycle, using a natural synchronous meristematic cell population labelled by caffeine as binucleate. We have thus found a parallel increase in DNA and histone along the S period. Some increase in nuclear histones during the G₁ and G₂ has also been detected. At the same time a high rise of stainable histone in the nuclei in prophase has been found.Changes in the composition of nuclear histones have been followed counting the percentage of arginine-rich histones in the total histone content. Two peaks located at the beginning and at the end of the S period as well as a sharp descent of the percentage of arginine-rich histone in the prophasic nucleus have been found.     

Sedimentation velocity characterisation of the cell cycle of granulocytic progenitor cells in monkey hemopoietic tissue

Sedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H³TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H³TdR exposure indicates that 55% of total CFC's in adult marrow are in G₀ or G₁ with a volume of 410 μ³, 42% are in S phase and of volume 450–950 μ³, and the remainder are in G₂ and mitosis with a volume of between 600–950 μ³. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G₂ component. H³TdR exposure confirmed the non-cycling status of these fetal progenitor cells.     

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G₂ phase, and underwent cell division simultaneously. Only 10% of the cells remained in G₀ or G₁. Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G₀ + G₁/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G₂ phase. However, reversal of G₁ arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G₀ + G₁ phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G₀. The frequency of cells entering G₀ was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.     

Cell cycles in embryos of the silkworm,Bombyx mori: G2-arrest at diapause stage

Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G₁, S and G₂ phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G₁, S and G₂ were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G₂ at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G₁ from G₂, suggesting that their G₁ phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G₁ fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G₂ at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G₁. Finally, most of the cells may be arrested in G₁, while there is only a small fraction of cells continuously cycling. Offprint requests to: T. Yaginuma     

HER2 as a potential therapeutic target on quiescent prostate cancer cells

Quiescent prostate cancer (PCa) cells are common in tumors but are often resistant to chemotherapy. Quiescent PCa cells are also enriched for a stem-like tumor initiating population, and can lead to recurrence after dormancy. Unfortunately, quiescent PCa cells are difficult to identify and / or target with treatment in part because the relevant markers are intracellular and regulated by protein stability. We addressed this problem by utilizing PCa cells expressing fluorescent markers for CDKN1B (p27) and CDT1, which can separate viable PCa cells into G₀, G₁, or combined S/G₂/M populations. We used FACS to collect G₁ and G₀ PC3 PCa cells, isolated membrane proteins, and analyzed protein abundance in G₀ vs G₁ cells by gas chromatography mass spectrometry. Enrichment analysis identified nucleocytoplasmic transport as the most significantly different pathway. To identify cell surface proteins potentially identifying quiescent PCa cells for future patient samples or for antibody based therapeutic research, we focused on differentially abundant plasma membrane proteins, and identified ERBB2 (HER2) as a cell surface protein enriched on G₀ PCa cells. High HER2 on the cell membrane is associated with quiescence in PCa cells and likely induced by the bone microenvironment. Using a drug conjugated anti-HER2 antibody (trastuzumab emtansine) in a mouse PCa xenograft model delayed metastatic tumor growth, suggesting approaches that target HER2-high cells may be beneficial in treating PCa. We propose that HER2 is deserving of further study in PCa as a target on quiescent cells to prevent recurrence, decrease chemotherapy resistance, or eradicate minimal residual disease.     

Commitment of Mitotic Cells to Meiosis during the G2 Phase of Premeiosis

In the developing anther, archesporial cells that proliferateby mitotic division are converted into meiotic cells duringthe premeiotic interphase. Experiments with explanted microsporocytesof Lilium and Trillium were made to obtain evidence for theconversion of mitotic to meiotic cells during the premeioticperiod. Explanted premeiotic cells were cultured through thedivision cycle at relatively high division frequencies and showeda variety of division types with respect to chromosomal events.The type of division depended on the premeiotic stage at whichthe cells were explanted. Cells in the G₁, S and early G² phasesunderwent mitotic division and formed a diad or binucleate monad.Cells explanted at the late G₂ phase were cultured throughoutthe normal meiotic cycle, which resulted in typical tetrad configuration. In microsporocytes explanted during the main part of the G₂interval, centromere behavior was meiotic, but chromosome pairingand chiasma formation were disturbed. Thus, she G₂ intervalwas shown to be critical for the commitment of mitotic cellsto meiotic division. Detailed analysis showed that the intracellularchanges that commit the cells to meiosis begin shortly aftercompletion of premeiotic DNA synthesis and that these changesare progressive and cumulative. (Received February 2, 1982; Accepted May 24, 1982)     

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

In vitro DNA synthesis as indicator of mammary epithelial cell division: [14C]thymidine uptake versus flow cytometry cell cycle analysis

Summary Mammary and adipose explants from eight mid-lactation Holstein cows were co-cultured for 24 h in the presence or absence of liver explants, 1 μg/ml pituitary bovine somatotrophin, or 100 ng/ml insulinlike growth factor-I. Liver explants in the media significantly depressed DNA and protein synthesis by mammary tissue as measured by [¹⁴C]-thymidine and amino acid incorporation. As measured by flow cytometry, the concentration of DNA in the G₀G₁ and G₂M cells and the percentage of cells in the G₀G₁ population of mammary tissue was also significantly depressed by liver tissue. Changes in the percentage of cells in the S and G₂M phases were not significant. Insulinlike growth factor-I in the presence of liver explants depressed protein synthesis, thymidine incorporation, and the concentration of DNA in the G₀G₁ and G₂M cells compared to control but did not affect the percentage of cells in the G₀G₁, S, or G₂M phases. Previously it was assumed that changes in [¹⁴C]thymidine incorporation indicated that changes in cell division were occurring. Flow cytometry revealed that changes in DNA content of mammary cells as a result of liver or hormonal stimulation were not due to changes in cell division. Indications are that differences in cellular DNA content result from changes in the rate of amplification of individual genes responsible for milk protein synthesis.     

Effects of Divalent Cations on the Glycolipids from Cultured Mouse Neuroblastoma Cells

Abstract: The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain G_M3, G_M2, G_M1, G_D3, and G_D1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GaINAc(β1→4) Gal(β1→4)Glc(β1→1)Cer (GgOse₃Cer), and GaINAc(β1→3)Gal(α1→4) Gal-(β1→4)Glc(β1→1)Cer (GbOse₃Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mm -EGTA showed a two- to threefold increase in G_M3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mm -EGTA plus 0.4 mm -EDTA a similar increase in G_M3 was observed but this change was now accompanied by decreases in G_M2, G_M1 GgOse₃Cer, and GbOse₄Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca²⁺ sufficient to bind all chelator. Mn²⁺ replacement reversed the chelator effects differentially; G_M2 and G_M1 levels were the most sensitive to increases in Mn²⁺ concentration; GgOse₃Cer and GbOse₄Cer were also sensitive, whereas G_M3 was the least affected. These results suggest calcium serves an important regulatory role on G_M3 levels and that manganese concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY I. AN IN VITRO STUDY USING MURINE TUMOUR CELL LINES

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G₁, S and (G₂+ M) DNA content in a population. As this method gives the relative (G₂+ M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G₂+ M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with the increase in the (G₂+ M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.