5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase-mitotic cells in apical root meristems of Allium cepa.

KEY MESSAGE: Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

Sanguinarine-induced oxidative stress and apoptosis-like programmed cell death(AL-PCD) in root meristem cells of Allium cepa.

A vast number of studies on plant cell systems clearly indicate that various biotic and abiotic stresses give rise to the uncontrolled increase in the level of reactive oxygen species (ROS). Excess concentrations of ROS result in damage to proteins, lipids, carbohydrates, and DNA, which may lead, in consequence, to the apoptotic cell death. The current study investigates the effects of sanguinarine (SAN), a natural alkaloid derived from the roots of Sanguinaria canadensis, on root apical meristem cells of Allium cepa. It is shown that SAN treatment generated large amounts of hydrogen peroxide (H2O2) and superoxide anion (O2·-). Oxidative stress induced in SAN-treated cells was correlated with DNA fragmentation, formation of micronuclei (MN), altered and 'degenerated' chromatin structures characteristic of apoptosis-like programmed cell death (AL-PCD). The experiments with SAN + MG132 (a proteasome inhibitor engaged in Topo II-mediated formation of cleavable complexes) and SAN + ascorbic acid (AA; H2O2 scavenger) seem to suggest, however, that the high level of H2O2 is not the only factor responsible for changes observed at the chromatin level and for the consequent cell death. Our findings imply that Topo II-DNA covalent complexes and 26S proteasomes are also involved in SAN-induced DNA damage.

The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa.

Long-term treatment of Allium cepa seedlings with low concentration of hydroxyurea (HU) results in a disruption of cell cycle checkpoints, leading root apex meristem (RAM) cells to an abnormal organization of nuclear structures forming interphase (I) and mitotic (M) domains of chromatin at opposite poles of the nucleus. Thus far, both critical cell length and an uneven distribution of cyclin B-like proteins along the nuclear axis have been recognized as essential factors needed to facilitate the formation of biphasic interphase-mitotic (IM) cells. Two new aspects with respect to their emergence are investigated in this study. The first concerns a relationship between the polarity of increasing chromatin condensation (IM orientation) and the acropetal (base→apex) alignment of RAM cell files. The second problem involves the effects of auxin (IAA), on the frequency of IM cells. We provide evidence that there is an association between the advanced M-poles of the IM cell nuclei and the polarized accumulation sites of auxin efflux carriers (PIN2 proteins) and IAA. Furthermore, our observations reveal exclusion regions for PIN2 proteins in the microtubule-rich structures, such as preprophase bands (PPBs) and phragmoplast. The current and previous studies have prompted us to formulate a hypothetical mechanism linking PIN2-mediated unilateral localization of IAA and the induction of bipolar IM cells in HU-treated RAMs of A. cepa.

DNA topoisomerase II-dependent control of the cell cycle progression in root meristems of Allium cepa.

The catalytic ability of DNA topoisomerases (Topo) to generate short-term DNA breaks allow these enzymes to play crucial functions in managing DNA topology during S-phase replication, transcription, and chromatin-remodelling processes required to achieve commitment for the onset and transition through mitosis. Our experiments on root meristem cells of onion (Allium cepa) were designed to gain insight into the contribution of Topo II to plant-specific progression throughout interphase and mitosis. Irrespective of the position of the cell in interphase, the immunofluorescence of Topo II revealed similar nuclear labelling pattern with well defined signals dispersed in the nucleoplasm and the cortical zone of the nucleolus. Only weak labelling was detected in metaphase and anaphase chromosomes. Experiments with two potent anti-Topo II agents, doxorubicin (DOX, an anthracycline) and a bisdioxopiperazine derivative, ICRF-193, suggest that the inhibition-mediated increase in Topo II immunofluorescence may represent a compensatory mechanism, by which an up-regulated expression of the enzyme tends to counteract the drug-induced loss of indispensable catalytic and relaxation functions. γ-H2AX immunolabelling seems to indicate that both DOX- and ICRF-193-induced alterations in cell cycle progression reflect primarily the activity of the G2/M DNA damage checkpoint. Our findings provide evidence for the plant-specific cell cycle control mechanism induced by Topo II inhibitors under DNA stress conditions.

Dissimilar effects of ß-lapachone- and hydroxyurea-induced DNA replication stress in root meristem cells of Allium cepa.

Two anticancer drugs, ß-lapachone (ß-lap, a naphthoquinone) and hydroxyurea (HU, an inhibitor of ribonucleotide reductase), differently affect nuclear morphology and cell cycle control mechanisms in root meristem cells of Allium cepa. The 18 h treatment with 100 µM ß-lap results in a lowered number of M-phase cells, increased occurrence of mitotic abnormalities, including over-condensation of chromosomes, their enhanced stickiness, formation of anaphase bridges, micronucleation and reduced mitotic spindles. Following prolonged incubations using high doses of ß-lap, cell nuclei reveal dark-red fluorescence evenly distributed in chromatin surrounding the unstained regions of nucleoli. Both drugs generate H2O2 and induce DNA double strand breaks, which is correlated with γ-phoshorylation of H2AX histones. However, the extent of H2AX phosphorylation (including the frequency of γ-H2AX foci and the relative number cells creating phospho-H2AX domains) is considerably reduced in root meristem cells treated jointly with the ß-lap/HU mixture. Furthermore, various effects of caffeine (an inhibitor of ATM/ATR cell cycle checkpoint kinases) on ß-lap- and HU-induced γ-phoshorylation of H2AX histones and the protective activity of HU against ß-lap suggest that their genotoxic activities are largely dissimilar. ß-Lap treatment results in the induction of apoptosis-like programmed cell death, while HU treatment leads to cell adaptation to replication stress and promotion of abnormal nuclear divisions with biphasic interphase/mitotic states of chromatin condensation.

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 H(2)O(2) 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 H(2)O(2), 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.

Inter- and intrachromosomal asynchrony of cell division cycle events in root meristem cells of Allium cepa: possible connection with gradient of cyclin B-like proteins.

Alternate treatments of Allium cepa root meristems with hydroxyurea (HU) and caffeine give rise to extremely large and highly elongated cells with atypical images of mitotic divisions, including internuclear asynchrony and an unknown type of interchromosomal asynchrony observed during metaphase-to-anaphase transition. Another type of asynchrony that cannot depend solely on the increased length of cells was observed following long-term incubation of roots with HU. This kind of treatment revealed both cell nuclei entering premature mitosis and, for the first time, an uncommon form of mitotic abnormality manifested in a gradual condensation of chromatin (spanning from interphase to prometaphase). Immunocytochemical study of polykaryotic cells using anti-beta tubulin antibodies revealed severe perturbations in the microtubular organization of preprophase bands. Quantitative immunofluorescence measurements of the control cells indicate that the level of cyclin B-like proteins reaches the maximum at the G2 to metaphase transition and then becomes reduced during later stages of mitosis. After long-term incubation with low doses of HU, the amount of cyclin B-like proteins considerably increases, and a significant number of elongated cells show gradients of these proteins spread along successive regions of the perinuclear cytoplasm. It is suggested that there may be a direct link between the effects of HU-mediated deceleration of S- and G2-phases and an enhanced concentration of cyclin B-like proteins. In consequence, the activation of cyclin B-CDK complexes gives rise to an abnormal pattern of premature mitotic chromosome condensation with biphasic nuclear structures having one part of chromatin decondensed, and the other part condensed.

Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum.

Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of gamma-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G(2) phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human gamma-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA.