Effect of abscisic acid on the cell cycle in the growing maize root

The mechanism by which the rate of cell proliferation is regulated in different regions of the root apical meristem is unknown. The cell populations comprising the root cap and meristem cycle at different rates, proliferation being particularly slow in the quiescent centre. In an attempt to detect the control points in the cell cycle of the root apical meristem of Zea mays L. (cv. LG 11), quiescent-centre cells were stimulated to synthesise DNA and to enter mitosis either by decapping or by immersing intact roots in an aqueous 3,3-dimethyl-glutaric acid buffer solution. From microdensitometric and flow-cytometric data, we conclude that, upon immersion, the G₂ phase of the cell cycle of intact roots was shortened. However, when 50 M abscisic acid (ABA) was added to the immersion buffer, parameters of the cell cycle were restored to those characteristic of intact roots held in a moist atmosphere. On the other hand, decapping of primary roots preferentially shortened the G₁ phase of the cell cycle in the quiescent centre. When supplied to decapped roots, ABA reversed this effect. Therefore, in our model, applied ABA retarded the completion of the cell cycle and acted upon the exit from either the G₁ or the G₂ phase. Immersion of roots in buffer alone seems to trigger cells to more rapid cycling and may do so by depleting the root of some ABA-like factor.Abbreviations ABA cis-abscisic acid - DGA 3,3-dimethyl-glutaric acid - DAPI 4,6-diamidino-2-phenylindole - LI labelling index We thank Pierre Zaech of the Ludwig Institute, Epalinges, Switzerland, for expert assistance in flow cytometry and Dr. Jean-Marcel Ribaut of our Institute for providing data on exodiffusion and metabolism of ABA.     

Radiosensitivity and recovery of mouse L cells during the cell cycle

Summary Mouse fibroblasts, subline L-929 F were synchronized by mitotic detachment. The synchronized cell cultures were irradiated with 200 kVp X-rays at different time after mitosis, and age reponse functions and dose effect curves were determined using the colony test. The cell age in the mitotic cycle was obtained from a computer analysis of flow cytometric DNA histograms. Both intrinsic radiosensitivity 1/D ₀ and extrapolation numbern were found to vary during the cell cycle. TheD ₀ has a maximum value of 176 ± 1 rad in the middle ofG ₁ phase and a minimum of 71 ± 1 rad at theS/G ₂ transition, while the extrapolation number is rather constant from the beginning ofG ₁ phase (1.9 ± 0.1) to the middle ofS phase (2.3 ± 0.1) and reaches a steep maximum of 9.3 ± 1.1 atS/G ₂ transition. The values ofn in the various phases of cell cycle are compared with the respective values of the recovery factor determined after fractionated irradiation. - Cell survival after a single dose of 616 rad has minima for irradiation atG ₁/S transition and in earlyG ₂ phase; the survival in earlyG ₂ being about 40 times smaller than in earlyG ₁ phase. Implications for a cell cycle specific therapy are discussed.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg     

Cell cycle duration in antheridial filaments ofChara spp. (Characeae) with different genome size and heterochromatin content

The relationship between nuclear 1 C DNA content and cell cycle progression throughout successive stages of antheridial filaments were studied among five taxa ofChara: two dioecious species (n = 14):C. aspera (7.2 pg DNA),C. tomentosa (7.4 pg DNA), and three monoecious species (n = 28):C. vulgaris (13.5 pg DNA),C. fragilis (19.3 pg DNA), andC. contraria (19.6 pg DNA). With the use of double³H-thymidine labelling and morphometry a number of characteristics common to all of the investigated species were determined within the proliferative periods preceding spermiogenesis. These include: (1) simplified type of the cell cycle (S + G₂ + M), due to complete lack of G₁ intervals, (2) constant duration of S phase, (3) progressive shortening of G₂ + M periods, and (4) gradual reduction of cell lengths at successive mitotic divisions. Nucleotypic dependence was found between genome size and several time parameters estimated for consecutive stages of antheridial filaments: the higher the DNA C-value, the longer the cell cycles, their component phases, the total duration of the proliferative period, as well as the lower the rate of growth of interphase cells. Differential Giemsa staining of late G₂ phase nuclei revealed that the higher content of C-heterochromatin is connected with prolonged cell cycle durations in species with similar DNA C-values.     

Characterization of centromere arrangements and test for random distribution in G0, G1, S,G2, G1, and early S′ phase in human lymphocytes

Summary The arrangement of centromeres, cluster formation and association with the nucleolus and the nuclear membrane were characterized in human lymphocytes during the course of interphase in a cell-phase-dependent manner. We evaluated 3 893 cell nuclei categorized by five parameters. The centromeres were visualized by means of indirect immunofluorescent labeling with anti-centromere antibodies (ACA) contained in serum of patients with CREST syndrome. The cell nuclei were classified as G₀, G₁, S, G₂, Gl₁ and early S phase by comparing microscopically identified groups of cell nuclei with flow cytometric determination of cell cycle stage of synchronized and unsynchronized lymphocyte cell cultures. Based on a discrimination analysis, a program was devised that calculated the probability for any cell nucleus belonging to the G₀, G₁, S, G₂, G₁ and early S phase using only two microscopic parameters. Various characteristics were determined in the G₀, S, and G₂ stages. A transition stage to S phase within G₁ was detected. This stage shows centromere arrangements not repeated in later cell cycles and which develop from the dissolution of centromere clusters in the periphery of the nucleus during G₀ and G₁. S phase exhibits various non-random centromere arrangements and associations of centromeres with the nucleolus. G₁ and early S phase of the second cell cycle display no characteristic centromere arrangement. The duplication of centromeres in G₂ is asynchronous in two phases. For all cell phases a test for random distribution of the centromeres in the cell nucleus was performed. There is a distinct tendency for centromeres to be in a peripheral position during Go and G₁; this tendency becomes weaker in S phase. Although the visual impression is a seemingly random distribution of centromeres in G₂ and G₁ statistical analysis still demonstrates a significant deviation from random distribution in favor of a peripheral location. Only the early S phase of the second cell cycle shows no significant deviation from a random distribution.     

Etoposide (VP-16) sensitizes p53-deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis

Human non-small-cell-lung-cancer (NSCLC) cells of _p53-null genotype were exposed to low-dosage topoisomearse II inhibitor etoposide (VP-16). The cellular proliferation rate could be effectively inhibited by VP-16 in dose-dependent manner. The effective drug concentration for growth inhibition could be as low as 0.5 M and the apoptotic phenotype became evident 48 h later. In H1299 cells, VP-16-induced cytotoxic effect was demonstrated associated with apoptosis that disappeared when restored with wild-type p53. Cell cycle analysis revealed that, upon VP-16 induction, cell death began with growth arrest by accumulating cells at the G₂-M phase. The cells at sub-G₁ phase increased at the expense of those at G₂-M transition state. To assess the regulation of cell cycle modulators, western blot analysis of H1299 cell lysates showed the release of apoptosis initiator, cytochrome c and apaf-1 hours following drug induction. The cleavage of downstream effectors, procaspase-9 and procaspase-7, but not procaspase-3, was accompanied with proteolysis of poly-(ADP-ribose) polymerase (PARP). VP-16-activated procaspase-7 cleavage was abrogated in cells with ectopically expressed p53.On the other hand, the inhibited procaspase-7 fragmentation by caspase-specific inhibitor reversed apoptotic phenotype caused by drug induction. Thus, VP-16-induced apoptotic cell death was contributed by caspase-7 activation in_p53-deficient human NSCLC cells.     

Effects of growth temperature upshift on cell cycle progression in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cell cycle progression of Cryptococcus neoformans was studied for cells grown exponentially at 15°, 24°, and 30°C. Except for speed, cell cycle progression was similar. In particular, budding occurred relatively soon after initiation of DNA synthesis at 15°, 24°, and 30°C. After growth temperature was shifted from 15° to 30°C, cells were transiently arrested before initiation of DNA synthesis. Thus, similar to Saccharomyces erevisiae, Start was the main susceptible cell cycle controlling point in C. neoformans. However, after spontaneous release from arrest as above, cells were further arrested in the unbudded state. Thus, the timing of budding was delayed just before the G₂ phase, or even until after entering the G₂ phase, but it was also transient, and 5h after the shift buds emerged relatively soon after initiation of DNA synthesis. Thus, C. neoformans cells can respond adaptively to mild stress by delaying budding. The existence of the second susceptible cell cycle control point, i.e., budding, appears to endow C. neoformans with a unique characteristic of stronger inhibition of multiplication than growth. A model of the C. neoformans cell cycle is also presented.     

COX-2 expression and cell cycle progression in human fibroblasts

Cyclooxygenase-2 (COX-2) is continuously expressed in mostcancerous cells where it appears to modulate cellular proliferation andapoptosis. However, little is known about the contribution oftransient COX-2 induction to cell cycle progression or programmed celldeath in primary cells. In this study we determined whether COX-2regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E₂(PGE₂) were not detected in quiescent cells but wereexpressed during the G₀/G₁ phase of the cellcycle induced by serum. Inhibition of COX-2 did not alter G₀/G₁ to S phase transition or induceapoptosis at concentrations that diminished PGE₂.Addition of interleukin-1 to serum enhanced COX-2 expression andPGE₂ synthesis over that by serum alone but had no effecton the progression of these cells into S phase. Furthermore,platelet-derived growth factor drove the G₀ fibroblasts into the cell cycle without inducing detectable levels of COX-2 orPGE₂. Collectively, these data show that transient COX-2expression in primary human fibroblasts does not influence cell cycle progression.

     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

Regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1

Background

The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G₁ phase.

Methodology/Principal Findings

In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G₀-G₁ phase arrest, but also reduced the number of cells at S and G₂-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G₀-G₁ phase despite the reduction of G₀-G₁ regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G₀-G₁ regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC.

Conclusions/Significance

This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G₀-G₁ phase.     

Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia Mill. and its synchronization

Using a ¹⁴C/³H double-labelling technique, the influence of kinetic on the length of the cell cycle of meristematic cells in haploid and diploid callus cultures of Datura innoxia was determined. The total length of the cell cycle of haploid cells as compared to that of diploid cells was reduced by 2.3 h (-kinetin) or 1.4 h (+kinetin). Furthermore, the addition of kinetin to the nutrient solution also reduces cell cycle duration at both ploidy levels. For synchronization of the cell cycle, a fluorodesoxyuridine/thymidine system was successfully employed. Apparently, the reduction of total cell cycle duration of cycling cells due to treatment with kinetin occurred at the expense of the G₁phase. Nevertheless, kinetin seems to exert an influence on the transition of cells from the G₂ into the M phase as well.Abbreviations FUdR fluorodeoxyuridine - HU hydroxyurea - IAA nidole acetic acid     

The effect of ethylene on root meristems of Pisum sativum and Zea mays

Summary Ethylene at a concentration of 100 l l^–1 causes a slight increase in the duration of the mitotic cycle in the primary root meristems of both Pisum sativum L. and Zea mays L. This is due to a lengthening of the G ₁ phase; other phases of the cycle are unaffected. Autoradiography and microdensitometry show that the rate of ³H-thymidine incorporation into nuclei of Pisum is maximal when about half the DNA has been replicated, and that ethylene has no effect upon this rate. Ethylene causes a reduction of the number of dividing cells in the root meristem, particularly in Pisum.Abbreviations Duration of the S phase, the G ₁ phase, the G ₂ phase of the mitotic cell cycle, respectively - T _C Duration of the complete mitotic cell cycle - QC Quiescent centre - LI, MI Labelling index, Mitotic index (i.e. fraction of the population labelled or in mitosis, respectively) - PF Proliferative fraction (i.e. fraction of the population making progress towards mitosis) - [³H]dT tritiated thymidine     

Regulation of energy flow and control of the cell cycle inSaccharomyces cerevisiae

Summary Measurements of glucose utilization and ethanol production by a respiratory-deficient mutant ofS. cerevisiae in a batch culture show that during the phase of acceleration, the glucose utilization per newly formed cell,k, is higher than the average value and the fermentationF < 1. In the phase of retardation, on the other hand,k is lower andF > 1. Correlating with the changes in the physiological state of the cell population, these results indicate that a considerable fraction of the total glucose consumed is utilized for the synthesis of polysaccharides in the G₁-phase, whereas reserve carbohydrates are catabolised during (S + G₂ + M)-phases of the cell cycle.A cybernetic model for the regulation of the energy (ATP) flow during the cell cycle is presented. It is postulated that the coupling between the energy-yielding and energy-consuming processes is provided by (i) a feedback regulation of the rate of energy production by the energy level and (ii) formation and breakdown of an intracellular energy storage system with a control function in the G₁S transition during the cell cycle.     

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.     

Change in the rate of organelle movement during progression of the cell cycle inAdiantum protonemata

Summary The rate of organelle movement during progression of the cell cycle in single-celled protonemata of the fernAdiantum capillus-veneris is determined microscopically with a time-lapse video system. Under red light organelle movement is very slow (1.8 m/min) in early G₁ in the apical 100-m region. The rate of organelle movement becomes higher in proportion to distance from the nuclear region, reaching a plateau in the neighborhood of 300 m from the tip. Organelle movement during the progression of G₁ and S phases in the dark does not show a significant difference from that in early G₁ under red light. In M phase, however, organelle movement in the nuclear region slows down a few minutes after nucleolar disappearance and then stops until the beginning of cell plate formation. Organelle movement in the basal region of the protonema slows down, but does not stop, shortly after movement in the nuclear region has ceased. This indicates that a message is sent from the nuclear region to the basal region.     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

Differential stimulation of hepatitis C virus RNA translation by microRNA-122 in different cell cycle phases

Hepatitis C virus (HCV) replicates preferentially in the liver, and in most cases, the HCV infection becomes chronic and often results in hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation is directed by the internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR) of the viral RNA. Thereby, IRES activity is modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5′-UTR and stimulates HCV translation, thereby most likely contributing to HCV liver tropism. Here, we show that HCV IRES-dependent translation efficiency in the hepatoma cell line Huh7 is highest during the G₀ and G₁ phases of the cell cycle but significantly drops during S phase and even more in the G₂/M phase. The superimposed stimulation of HCV translation by ectopic miR-122 works best during G₀, G₁ and G₂/M phases but is lower during S phase. However, the levels of Ago2 protein do not substantially change during cell cycle phases, indicating that other cellular factors involved in HCV translation stimulation by miR-122 may be differentially expressed in different cell cycle phases. Moreover, the levels of endogenously expressed miR-122 in Huh7 cells are lowest in S phase, indicating that the predominant G₀/G₁ state of non-dividing hepatocytes in the liver facilitates high expression of the HCV genome and stimulation by miR-122, with yet-unknown factors involved in the differential extent of stimulation by miR-122.Key words: HCV, translation, miR-122, microRNA, miRNA, Ago, Ago2     

Cell cycle analysis of tumour-derived cultures ofCrepis capillaris: A kinetic analogy with proliferation of animal tumour cells

Summary Analysis of the cell cycle by three methods has revealed unusual kinetics of proliferation in tumour derived suspensions ofCrepis capillaris. The different methods of analysis yield different estimates of cycle phase durations, and such discrepancies have been explained in terms of low growth fractions with rapid total cycle traverse. Specifically, confidence in the estimation of G₂ duration by the fraction of labelled mitosis analysis, and comparison with shorter G₂ estimates obtained by the two other methods, suggests that cells drop out in G₁. However, cells which do not drop out of the proliferative compartment traverse G₁ extremely rapidly. Extremely short cell cycle durations in which the G₁ phase is virtually non-existent are uncharacteristic of plant cell suspension cultures, in which the G₁ phase has previously been shown to be extended as compared with meristematic root tip cells. A model has been proposed in which a central core of rapidly dividing cells continuously loses cells into a subpopulation of resting or G₀ cells with the G₁ DNA content. Similarities between plant and animal tumours with respect to cell growth and division are discussed.