Analysis of the interphase accumulation induced by hydroxyurea on proliferating plant cells.

Cell distribution in different compartments of the cell cycle (G1, early, middle and late S, G2 and mitosis) has been studied during continuous treatments with hydroxyurea (HU) in onion root meristems by cytophotometric and autoradiographic methods. A sublethal dosis of HU (0.75 mM) has been chosen to allow a good wave of mitotic synchrony during recovery, with a negligible level of chromosomal aberrations. Proliferating cells begin the S period in the presence of HU and are accumulated in early S, where the maximum value (60%) is reached after 8 h of treatment; at the same time middle and late S are practically empty. In the presence of the drug, residual DNA synthesis allows a slow but continuous progress of cells throughout the S period. Differential sensitivity of S cells to HU can be observed; replication is more affected in early S (85% inhibition) than in the second half of the period (70% inhibition). On the other hand, G1 cells are not apparently affected by HU, while cells in G2 show a delay in their entrance into mitosis.     

Hydroxyurea-induced mitotic synchronization in Allium sativum root meristems

The synchronized divisions following a treatment with hydroxyurea (HU) — an inhibitor of DNA synthesis — were studied in root meristems of Allium sativum using two methods: autoradiography of median sections and morphological labeling with a cytokinesis inhibitor. It is shown that the second wave of mitoses is heterogeneous: it is composed mostly of cells which have been synchronized in the S phase by the HU treatment, of cells coming from the quiescent center stimulated to enter DNA synthesis and of cells which were not blocked by the 23 h HU treatment (slow cycling cells). It is also shown that the cell cycle following the first synchronized division is considerably shortened by the synchronization procedure.Abbreviations QC quiescent center - HU hydroxyurea - MHQD methyl-3 hydroxy-6 quinazoline dione 2–4     

Induction of foci of phosphorylated H2AX histones and premature chromosome condensation after DNA damage in <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem

Immunocytochemical analysis using antibody raised against human H2AX histones phosphorylated at serine 139 (γ-H2AX) demonstrates that root meristem cells of Vicia faba exposed to UV-radiation or incubated with hydroxyurea (HU) reveal discrete foci at the border of the nucleolus and perinucleolar chromatin or scattered over the whole area of cell nucleus. Western blots detected only one protein band at the position expected for the phosphorylated form of H2AX. The dose-effect relationship was demonstrated following treatment with 2.5 and 10 mM HU. Proteins extracted from root meristems incubated for 2 h either with HU and caffeine or with HU and sodium metavanadate showed unchanged amounts of bound γ-H2AX antibodies, as compared to root meristems treated with 2.5 mM HU. Higher quantities of phosphorylated H2AX histones were detected in proteins extracted from roots treated with HU and 2-aminopurine. All treatments were effective in producing evident aberrations of premature mitosis: broken and lagging chromatids, acentric fragments, chromosomal bridges and micronuclei. Our results show that phosphorylation of H2AX at the carboxy-terminal Ser-Gln-Glu sequence is among the earliest responses to double-strand breaks and, presumably, one of the key ATM/ATR-dependent signals indispensable for the repair of spontaneous and induced DNA damage in plant cells.     

An in vivo study on the synchronizing effect of hydroxyurea

The effect of hydroxyurea (HU; 0.5 mg/g body wt) on L 1210 ascites tumor cells has been studied using various cell kinetic methods. In contrast to the general assumption that HU blocks cells at the G1/S boundary [J. Brachet (1985) Molecular Cytology, Vol. I, p. 266, Academic Press, New York], the present results show that the cells are not held at G1/S but enter S at about the normal rate and are accumulated in early S phase due to a dose-dependent inhibiting effect of HU on DNA synthesis. Partial synchronization of the cells demonstrated by a distinct mitotic peak 10 h after HU application is not due to a G1/S block of the cells and their subsequent synchronous passage through the cycle after release from the block but is due to rather complex mechanisms of action of HU: a differential cytocidal effect and an effect on the passage of the cells through the cycle, both depending on the position of the cells throughout the cycle. HU kills S-phase cells, mainly cells in early S phase; i.e., a great portion of the cells "accumulated" in early S phase is killed by the drug, while G1-phase cells are almost not affected by the lethal effect of HU. These G1-phase cells pass through the cycle more rapidly after cessation of the HU effect. The same is true for the surviving cells accumulated in early S phase, while part of the cells in the remaining S phase are delayed in their passage through the cycle. This causes partial synchronization, since a great portion of all cells that survive HU treatment reach mitosis at the same time.     

The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.     

The response of apical meristems of primary roots of Vicia faba L. to colchicine treatments

The effects of 0.5% and 0.025% solutions of colchicine on the passage of cells through the mitotic cycle in apical meristems of primary roots of Vicia faba have been examined. Both treatments affected cell progression through the mitotic cycle in the same way: S and G1 were shorter, and G2 and mitosis longer, than the corresponding control values. The duration of the various phases of the mitotic cycle were similar to those reported previously for apical meristems of lateral roots though cycle time itself was longer. Recovery of root proliferating tissues from colchicine-induced inhibition of growth is correlated with the presence of quiescent cells. Meristems which have no quiescent cells do not recover from eolchicine treatment, while meristems which contain many quiescent cells recover faster than those which contain few. The growth fraction and the proportion of proliferating cells with a short cycle time are linearly related to the duration of the S period in root meristems.     

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

Background and Aims

Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis.

Methods

Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H₂O₂ production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation).

Key Results

Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H₂O₂, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants.

Conclusions

The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.     

Immunochemical detection of cell cycle synchronization in a human erythroleukemia cell line, K562

Cells of the human erythroleukemic line K562 can be induced by manipulation of culture conditions to arrest within the G1 phase of the cell cycle, and subsequently to enter S phase synchronously after release from G1. Cell cultures subjected to serum deprivation and hydroxyurea (HU) treatment demonstrated less than 5% of the cells to be in S phase. Four hours after release from HU, 63% of the cells were in S phase, as detected by immunofluorescent staining. This protocol offers a method for synchronization of K562 cells at the G1/S border and a technique for detection of S-phase cells without the use of radioisotopes or flow cytometry instrumentation.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

High sensitivity of DNA replication to 5-aminouracil in the middle of the S period

Summary Cell distribution in different compartments of the cell cycle (G₁, early, middle and late S, G₂ and mitosis) has been studied during treatment with 0.5 mM 5-aminouracil and recovery inAllium cepa L. root meristems by cytophotometric and autoradiographic methods. At optimum conditions for obtaining mitotic synchronization, 5-aminouracil gives rise to cell accumulation in the S period, preferentially in its middle zone where the relative DNA content is 2.8 ± 0.1 C. After a 14-hour treatment 33% of the proliferative population is accumulated in this particular region.During recovery, a drastic reduction of the S phase and a clear increase of the mitotic frequency are the most important events observed. Apparently, the removal of the drug frees the blockage and the accumulated cells complete their interphase making up the mitotic wave.     

Water Deficit in Pea Root Tips: Effects on the Cell Cycle and on the Production of Dehydrin-like Proteins

Dehydrin-like proteins have been detected in nuclei and cytoplasmof meristematic root tip cells from pea seedlings subjectedto slow dehydration at 90% relative humidity for 48 h or more.Evidence was gained from Western blotting and immunocytochemicalexperiments using an antibody raised against the conserved domainof dehydrin proteins. Flow cytometer analysis has shown thatcycling cells of root tip meristems from dehydrated seedlingsare mostly arrested in G2 phase. Other stress treatments thoughtto involve water depletion (osmotic stress, cold treatment)or to modulate cell response to water deficit (abscisic acid)gave less clear-cut results with all treatments lowering theproportion of cells entering the S phase, but without a definiteand persistent arrest in any preferential phase of the cycle.Possible interrelationships between G2 arrest and dehydrin productionare discussed. Cell cycle; dehydrins; flow cytometry; nuclei; pea; Pisum sativum L.; water stress     

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     

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G₁/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 M). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×10⁵/root and 1.4×10⁵/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.     

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in <Emphasis Type="Italic">Vicia faba</Emphasis> root meristems

According to the principal control point (PCP) hypothesis, experiments with excised, carbohydrate-starved stationary root meristems of Vicia faba var. minor have demonstrated that cells which previously divided asynchronously were preferentially blocked in G₁ (PCP1) and G₂ (PCP2) phases. When stationary phase meristems are supplied with exogenous carbohydrate (2 % sucrose), the G1- and G2-arrested cells start out DNA replication and mitotic divisions, respectively. The resumption of DNA synthesis and mitosis is not immediate and the delays of G₁- and G₂-arrested cells are found different. Using this model, we examined the effects of 4 pulse incubations with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, on the duration of intervals elapsing between the provision of sucrose and the first appearance of S- and M-phase cells. We have found that depending on the period during which OA had been applied, the release from G1 and G2 phase arrest-points becomes prolonged, showing different time-course modifications. The obtained data provide evidence that activation of PP1 and PP2A is required to allow the cells for both PCP1→S and PCP2→M transitions in root meristems of V. faba.     

NUCLEAR DNA CONTENT OF EMBRYONIC RADICLES AND CULTURED STATIONARY PHASE ROOT TIPS OF PEA (PISUM SATIVUM L.)

Cells in mature embryos and stationary phase (SP) root meristems of pea arrest in G1 and G2 of the cell cycle. The patterns of distribution of G2 nuclei in radicles and SP meristems, with and without G2 factor, were compared by using cytophotometric analysis of the relative amount of DNA/nucleus in sectioned material. Radicles and SP meristems were each divided into 5 zones and the ratio of G1 to G2 nuclei was determined for each zone. The G2 population in the radicle is restricted mainly to the embryonic cortex. A small part of the G2 population was located in the central cylinder and the root cap. In SP meristems without G2 factor, the pattern of distribution of G2 cells was similar to that in radicles. SP meristems with G2 factor contained G2 arrested nuclei in all regions of the root tip. In each region the percentage of G2 nuclei was higher than that in the same region of SP meristems without G2 factor. This indicates that the population of cells that responds to G2 factor is distributed throughout the root tip.     

Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1

Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.     

Effects of monocerin on cell cycle progression in maize root meristems synchronized with aphidicolin

Summary Monocerin is a benzopyran fungal toxin with broad activity on plants, fungi and insects. Its effect upon cell cycle progression has been analyzed in maize roots. Meristematic cells were synchronized by treatment with aphidicolin. Flow cytometric DNA analysis and mitotic indices indicated durations of 1.5 h, 5 h, 2 h and 1 h for respectively G1, S, G2 and M phases of the normal cell cycle at 25°C. Treatment of these synchronized meristems with 0.5 mM monocerin during release after an aphidicolin block produced a short delay in S phase and then a more important delay (about 2.5 h) in entry into mitosis. Treatments for similar durations (3 h) during progression through the cycle revealed two periods of action of monocerin. The first appears to be mid to late S and the second one G2, before the transition point between G2 and M. Action on either one of these target periods could lead to a delay in the G2/M transition, but these two responses did not appear to be additive.Abbreviations APH Aphidicolin - CV Coefficient of variation - DAPI Diamidinophenylindole - DMSO Dimethyl sulfoxide - EDTA Ethylenediaminetetraacetic acid - HPLC High pressure liquid chromatography - MI Mitotic index - SD Standard deviation - UV ultraviolet light     

IS THE NUCLEAR DNA CONTENT OF MATURE ROOT CELLS PRESCRIBED IN THE ROOT MERISTEM?

Cells of the mature root exhibit arrest within the G1 and G2 periods of the mitotic cycle. The number of cells arrested with a 2C or 4C DNA amount in mature tissue was compared with that in meristems of excised primary root tips deprived of carbohydrate. Results from four plant species are described. Cells in mature tissue of seedling roots of Vicia and Pisum exhibited arrest predominately at the 4C while those of Triticum and Helianthus arrested preponderantly at the 2C DNA level. The proportion of cells arrested at the 2C and 4C levels in mature root tissue was specific for each species tested. In each species the cycle stage where most cells arrested was the same in carbohydrate-deficient root meristems as in mature root tissue; consequently, most meristematic cells are preconditioned or predetermined to arrest in a specific mitotic period. A test system was developed in Pisum in which the predominant period of arrest was altered by the removal of the cotyledons. The predominant arrest period changed from 4C to 2C in both mature root tissue and carbohydrate-deficient root meristems with cotyledon removal and indicated that mature root cells are preconditioned while meristematic as to where they will eventually arrest in the mitotic cycle.