Promotion of cell arrest in G2 in root and shoot meristems in Pisum by a factor from the cotyledons

Characterization of a factor within the cotyledons of Pisum that promotes cell arrest in G2 in mature root tissue and stationary phase root and shoot meristems is presented. Diffusion of the G2 factor into aseptic liquid and solid agar media enabled us to perform experiments focused on its cellular effect. The factor promotes cell arrest in G2 in shoots and roots of Pisum and roots of Vicia indicating a lack of species and organ specificity. In seedling roots of Pisum the factor promotes arrest in G2 in a large portion of the cell population. However, because pea cotyledons have a limited supply which is depleted 8–10 days after seed germination, cells previously responsive to the G2 factor in Pisum root meristems eventually assume preponderant arrest in G1. Once these cells arrest in G1 they are no longer influenced to arrest in G2. The G2 factor doss not promote arrest in G2 in meristem root cells of Helianthus and Triticum which normally show preponderant arrest in G1.     

Cell cycle suspension: A novel process lurking in G2 arrest

Cell cycle checkpoint is a self-protective mechanism for cells to monitor genome integrity and ensure the high-fidelity transmission of genetic information to daughter cells. Insufficient function of cell cycle checkpoints has been demonstrated to partially account for tumor initiation, promotion and progression. In the ten melanoma cell lines that we tested in preliminary experiments, two human uveal melanoma cell lines, 92-1 and OCM-1, were found to be significantly different in terms of radiosensitivity but similar in DNA repair ability. Evident G₂ arrest was induced in both cell types and the maximum was reached at 16 h after irradiation regardless of X-rays or high-LET carbon beams. OCM-1 cells overrode the G₂ arrest and reentered the cell cycle right after reaching the maximum, whereas 92-1 could not. Upon 10 Gy of radiation, the cell cycle of 92-1 was suspended and remained unchanged for up to 5 d. The cell cycle suspension is a unique process lurking in G₂ arrest and related to cellular radiosensitivity. Its induction is dose-dependent and there is a dose threshold for it. The degradation of Cyclin B1 has been found related to the cell cycle suspension though, the mechanism of cell cycle suspension is still under investigation. Basing on our knowledge, this is the first report on cell cycle suspension and we present here a de novo mechanism to cellular radiosensitivity. Further clarification of the mechanism underlying cell cycle suspension is believed to be of significance in tumor radiosensitization or even direct tumor control.     

Senescence from G2 arrest,revisited

Senescence was classically defined as an irreversible cell cycle arrest in G1 phase (G1 exit) triggered by eroded telomeres in aged primary cells. The molecular basis of this G1 arrest is thought to be due to a DNA damage response, resulting in accumulation of the cyclin dependent kinase (Cdk) inhibitors p21 and p16 that block the inactivating phosphorylation of the retinoblastoma tumor suppressor pRb, thereby preventing DNA replication. More than a decade ago, several studies showed that p21 also mediates permanent DNA damage-induced cell cycle arrest in G2 (G2 exit) by inhibiting mitotic Cdk complexes and pRb phosphorylation. The idea that the senescence program can also be launched after G2 arrest has gained support from several recent publications, including evidence for its existence in vivo.     

Cell plate development and delayed formation of the pectic middle lamella in root meristems

Summary The first stages of cell wall formation were followed in the root meristems of maize and French bean. Most of the primary wall components (hemicellulose, cellulose and highly methylated pectins) were laid down simultaneously along the cell plate. During young cell wall maturation within the meristem itself, significant topochemical alterations, coupled with the addition of new polysaccharides, produced complete redistribution of wall material leading to the progressive appearance of a proper middle lamella. Thus the formation of a pectic middle lamella does not precede the deposition of primary walls. It is delayed until the new partition joins to the mother cell wall.Abbreviations DMSO dimethylsulphoxide - EDTA ethylene diaminetetraacetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

Cell cycle age dependence for radiation-induced G2 arrest: evidence for time-dependent repair

Exponentially growing eucaryotic cells, irradiated in interphase, are delayed in progression to mitosis chiefly by arrest in G2. The sensitivity of Chinese hamster ovary cells to G2-arrest induction by X rays increases through the cell cycle, up to the X-ray transition point (TP) in G2. This age response can be explained by cell cycle age-dependent changes in susceptibility of the target(s) for G2 arrest and/or by changes in capability for postirradiation recovery from G2-arrest damage. Discrimination between sensitivity changes and repair phenomena is possible only if the level of G2-arrest-causing damage sustained by a cell at the time of irradiation and the level ultimately expressed as arrest can be determined. The ability of caffeine to ameliorate radiation-induced G2 arrest, while inhibiting repair of G2-arrest-causing damage makes such an analysis possible. CHO cell monolayers were irradiated (1.5 Gy), then exposed to 5 mM caffeine for periods of 0-10 hr. Cell progression was monitored by the mitotic cell selection procedure. In the presence of caffeine, progression of irradiated cells was relatively unperturbed, but on caffeine removal, G2 arrest was expressed. The duration of G2 arrest was independent of the length of the prior caffeine exposure and, since cells of all ages were ultimately examined, the duration of arrest was also independent of cell cycle age at the time of irradiation. This finding indicates that the target for G2-arrest induction is present throughout the cell cycle and that the level of G2-arrest damage incurred is initially constant for all cell cycle phases. The data are consistent with the existence of a time-dependent recovery mechanism to explain the age dependence for radiation induction of G2 arrest.     

Sesquiterpene lactone dehydroleucodine selectively induces transient arrest in G2 in Allium cepa root meristematic cells

Dehydroleucodine is a sesquiterpene lactone recently isolated from aerial parts of a medicinal herb, Artemisia douglasiana Besser. We have previously shown that 25 and 100 microM dehydroleucodine slowed down onion root growth by 30 and 70%, respectively, affecting neither cell viability nor cell elongation. In the present study we analyze the effect of dehydroleucodine on cell cycle phases in onion (Allium cepa L.) root meristematic cells synchronized with caffeine or caffeine and hydroxyurea. Synchronized root cells treated with 100 and 200 microM dehydroleucodine present an interphase lengthening of 5.2 h and 8.2 h, respectively. The S-phase length, estimated by [3H]thymidine incorporation assay, was 6 h for both control roots and roots that had been immersed in dehydroleucodine. The peak of [3H]leucine incorporation was observed 6 h after release from synchronization in controls and in dehydroleucodine-treated roots, indicating that protein synthesis in G2 was not affected. Thus, these results show that dose-dependently dehydroleucodine selectively induces a transient arrest of meristematic cell in G2 and that dehydroleucodine can be used experimentally as a cell cycle suppressor.     

Cell cycle-independent expression of immediate-early gene 3 results in G1 and G2 arrest in murine cytomegalovirus-infected cells

The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G₀/G₁ phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G₁ DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G₁. Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G₂. Moreover, MCMV can also replicate in G₂ cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G₁ or G₂. Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.     

Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT

Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.     

Cell cycle arrest induced by theophylline in root meristem of Haplopappus gracilis

Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, induced a block of the cell cycle in G₁, a temporary arrest in G₂ and 70% decrease in the uptake of labelled thymidine in roots of Haplopappus. These effects are compared to those previously found with aminophylline and discussed in view of the possible involvement of cAMP in the regulation of the cell cycle in plants.     

Caffeine-inhibitable control of the radiation-induced G2 arrest in L5178Y-S cells deficient in non-homologous end-joining

The two L5178Y (LY) sublines bear a heterozygous Tp53 mutation that affects its transactivation function. LY-S (radiation-sensitive) cells are deficient in double strand break (DSB) repair by non-homologous end-joining (NHEJ) and do not express p21^WAF1 (Cdkna1) either constitutively or after x-irradiation, in contrast to their radiation-resistant counterpart LY-R cells, which express p21^WAF1 constitutively. Radiation-induced G2 arrest in LY-S cells is very long (11 h/Gy) but 2 mM caffeine treatment shortens it, decreases the fraction of G2 cells and increases the fraction of apoptotic cells. The treatment also increases the DNA damage that is estimated with the comet assay 18 h after irradiation with 5 Gy (ca. 23% of the initial value for x-rays and ca. 47% for x-rays plus caffeine). This indicates that either the repair has not been completed or the apoptotic DNA fragmentation has been initiated (or both). The same treatment applied to x-irradiated (5 Gy) LY-R cells (G2 arrest, 4 h/Gy) has no radiosensitising effect, induces no apoptosis and does not alter the amount of DNA damage left unrepaired (ca. 28%). The results are compatible with the assumption that inhibition of the Atm-dependent homologous recombination repair by caffeine, brings differential effects in LY sublines because of the defect of the alternative DNA repair system (NHEJ) in LY-S cells. Received: 23 June 2000 / Accepted: 5 January 2001     

Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis

In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G₂ phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.     

The age-distribution of cell cycle populations in plant root meristems : Complex tissues

The durations of the mitotic cycle periods, the proportions of cells in each cycle period of the proliferative and nonproliferative populations, and the rates of cell progression from G1-S and from G2-M were used to characterize the cytokinetics in root meristems of four plant species. The observed age-distribution of cells in the cycle of each meristem was not comparable to either a theoretical exponential or uniform age-distribution. A more exact fit of the observed age-distributions with theoretical distributions was hindered by proliferative cells which halted temporarily in G1 and/or in G2 in relative proportions similar to the nonproliferative cells in the meristem. Moreover, the preponderant cycle period in which cells halted temporarily differed among the species observed: it was G1 in Helianthus and Triticum and G2 in Pisum and Vicia. The cell populations of these complex tissues can be subdivided kinetically into three types: (1) rapidly proliferating cells; (2) slowly proliferating cells that halt temporarily in G1 and/or in G2; and (3) nonproliferating cells that halt in G1 and/or in G2.     

Induction of cell arrest in G2: structural specificity of trigonelline

Synthetic analogues of N-methyl nicotinic acid, trigonelline, were prepared to test the structural features necessary for the induction of cellular arrest in G2 in Pisum sativum. Analogues that (1) were regioisomers of trigonelline, (2) possessed different 1,3-substituents, and (3) contained additional substituents on the pyridine ring were tested for their ability to induce cell arrest in G2 and to antagonize trigonelline induced arrest in G2. Only N-methyi-3-quinoline-carboxylic acid and 1-methyl nicotinamide induced cell arrest in G2, and 1-methyl-4-pyridine carboxylic acid and 1-methyl-2-pyridine carboxylic acid were effective trigonelline antagonists. These data further support a specific role for trigonelline in the induction of cell arrest in G2.     

G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6- h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.