The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells.

Olaparib is a poly (ADP-ribose) polymerase inhibitor (PARPi) that inhibits PARP1/2, leading to replication-induced DNA damage that requires homologous recombination repair. Olaparib is often insufficient to treat BRCA-mutated (BRCAMUT) and BRCA wild-type (BRCAWT) high-grade serous ovarian carcinomas (HGSOCs). We examined the short-term (up to 48 h) efficacy of PARPi treatment on a DNA damage response pathway mediated by ATR and CHK1 kinases in BRCAMUT (PEO-1) and BRCAWT (SKOV-3 and OV-90) cells. The combination of ATRi/CHK1i with PARPi was not more cytotoxic than ATR and CHK1 monotherapy. The combination of olaparib with inhibitors of the ATR/CHK1 pathway generated chromosomal abnormalities, independent on BRCAMUT status of cells and formed of micronuclei (MN). However, the beneficial effect of the PARPi:ATRi combination on MN was seen only in the PEO1 BRCAMUT line. Monotherapy with ATR/CHK1 inhibitors reduced BrdU incorporation due to a slower rate of DNA synthesis, which resulted from elevated levels of replication stress, while simultaneous blockade of PARP and ATR caused beneficial effects only in OV-90 cells. Inhibition of ATR/CHK1 increased the formation of double-strand breaks as measured by increased γH2AX expression at collapsed replication forks, resulting in increased levels of apoptosis. Our findings indicate that ATR and CHK1 inhibitors provoke premature mitotic entry, leading to genomic instability and ultimately cell death.

The Role of Lysine-Specific Demethylase 1 (LSD1) in Shaping the Endothelial Inflammatory Response.

BACKGROUND/AIMS: Inflammation is the body's natural response to stress in the broadest sense. The regulatory mechanisms that control this process, some of which are still unclear, are needed to balance the immune response, but also when insufficient, can cause immunodeficiency resulting in infection, cancer, neurodegeneration or other serious disorders. In this study, we focused on defining the role of lysine-specific demethylase 1 (LSD1), an enzyme involved in modulating the methylation state of lysine, including histone and non-histone proteins, in shaping the inflammatory profile of endothelial cells. METHODS: To determine the role of LSD1 in the inflammatory response of ECs, cells were stimulated with lipopolysaccharide (100 ng/ml LPS) in the presence and absence of an LSD1 inhibitor (2-PCPA). A transcription model of LSD1 deficient cells (HMEC-1 LSD1 KD) obtained by lentiviral shRNA transduction was also used. The indicated cellular models were analyzed by gene profiling, monitoring of p65 shuttling by Western blotting and immunofluorescence staining. Also chromatin immunoprecipitation (ChIP) was performed to identify the interactions between selected: IL-6/p65 and LSD1. RESULTS: Analysis of both experimental models revealed an altered inflammatory response following both LSD1 inhibition and LSD1 silencing. We observed decreased U-937 monocytes recruitment to LPS-activated endothelial cells and decreased extracellular secretion of many proinflammatory cytokines, also confirmed at the transcript level by RT-qPCR. Monitoring of the LPS-induced p65 translocation revealed inhibition of the NF-kB subunit in LSD1 KD vs nonT as well as due to pretreatment of 2-PCPA cells. Gene profiling performed with RNA microarrays confirmed the obtained biochemical data at the transcript level. CONCLUSION: In conclusion, the conducted studies showed a proinflammatory profile of LSD1 activity in endothelial cells, revealed by the inhibition of the enzyme activity and confirmed at the transcriptional level by the inhibition of its expression. Although we found significant changes in the modification of interactions between monocytes and endothelial cells as well as in cytokine/chemokine release and expression that were consistent with the altered NF-κB-p65 translocation into the nucleus, we did not identify a direct interaction between LSD1 and the transcription factor. Our finding may have important implications for prevention of cardiovascular diseases at their first stage - activation of the endothelium as well as for tumor cell biology, providing evidence for the use of LSD1 inhibitors to reduce the inflammatory response, which enhances tumor tissue remodeling, angiogenesis and metastasis.

Hydroxyurea-The Good, the Bad and the Ugly.

Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and-recently-a promising factor in preventing cognitive decline in Alzheimer's disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.

Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba's ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Kinetics of DNA Repair in Vicia faba Meristem Regeneration Following Replication Stress.

The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even "chaotic" events are dealt with in a proper degree of order.

Silencing Lysine-Specific Histone Demethylase 1 (LSD1) Causes Increased HP1-Positive Chromatin, Stimulation of DNA Repair Processes, and Dysregulation of Proliferation by Chk1 Phosphorylation in Human Endothelial Cells.

: The methylation of histone lysine residues modifies chromatin conformation and regulates the expression of genes implicated in cell metabolism. Lysine-specific demethylase 1 (LSD1) is a flavin-dependent monoamine oxidase that can demethylate mono- and dimethylated histone lysines 4 and 9 (H3K4 and H3K9). The removal of methyl groups from the lysine residues of histone and non-histone proteins was found to be an important regulatory factor of cell proliferation. However, its role has not been fully elucidated. In this study, we assessed LSD1-mediated cell cycle progression using a human endothelial cell model. The short hairpin RNA knockdown of LSD1 inhibits the G2/M phase of cell cycle progression by checkpoint kinase 1 (Chk1) phosphorylation (S137). We observed elevated DNA damage, which was consistent with the increased detection of double-strand breaks as well as purines and pyrimidines oxidation, which accompanied the activation of ATR/ATRIP signaling by H2AXS139 phosphorylation. The irreversible pharmacological inhibition of LSD1 by 2-phenylcyclopropylamine (2-PCPA) inactivated its enzymatic activity, causing significant changes in heterochromatin and euchromatin conformation assessed by chromatin assembly factor 1 subunit A (CAF1A) and heterochromatin protein 1 isoform α and γ (HP1α/γ) immunofluorescence analysis. We conclude that the knockdown of LSD1 in endothelial cells leads to increased HP1-positive chromatin, the stimulation of DNA repair processes, and the dysregulation of proliferation machinery.

Pharmacological and transcriptional inhibition of the G9a histone methyltransferase suppresses proliferation and modulates redox homeostasis in human microvascular endothelial cells.

Epigenetic mechanisms, including histone post-translational modifications, are central regulators of cell cycle control. The euchromatic G9a histone methyltransferase (G9a HMT) is a key enzyme catalyzing histone H3 methylation on lysines 9 and 27, and its dysregulation has been linked to uncontrolled proliferation of tumor cells. Here, we have investigated the effect of G9a HMT silencing on cell proliferation of microvascular endothelial cells, a process necessary to sustain tumor growth through the formation of the vascular capillary network. Inhibition of G9a HMT activity in human microvascular endothelial cells (HMEC-1) was performed either pharmacologically, by treatment of cells with BIX-01294 or chaetocin, or transcriptionally, using shRNA. Cell viability and proliferation were examined using the resazurin reduction assay, flow cytometry and immunostaining of phosphorylated checkpoint kinase 1 (pSer317Chk1). Expression of cell cycle- and redox homeostasis-related genes was determined by quantitative PCR. Reactive oxygen species production was measured by oxidation of the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and the cell's total antioxidant capacity by using the ABTS assay. Inhibition of G9a HMT activity by BIX-01294 treatment or by shRNA attenuated the proliferation of HMEC-1, nuclear localization of phosphorylated Chk1, and induced cell cycle arrest in G1 phase. Transcriptional analysis demonstrated increased gene expression of the cyclin-dependent kinase (CDK) inhibitor p21, and also of Rb1, in BIX-01294 treated cells. Decreased proliferation rate was accompanied by enhanced antioxidant potential of HMEC-1 cells, as demonstrated by reduced production of reactive oxygen species, increased total antioxidant capacity and expression of the antioxidant enzymes catalase and superoxide dismutase 1. Collectively, our results demonstrate of the central role of G9a HMT in the promotion of endothelial cells proliferation, and suggest that endothelial G9a HMT may be a target in the treatment of vascular proliferative disorders and tumor neovascularization.

Pharmacological inhibition of arginine and lysine methyltransferases induces nuclear abnormalities and suppresses angiogenesis in human endothelial cells.

Posttranslational modifications of histone tails can alter chromatin structure and regulate gene transcription. While recent studies implicate the lysine/arginine protein methyltransferases in the regulation of genes for endothelial metabolism, the role of AMI-1 and AMI-5 compounds in angiogenesis remains unknown. Here, we show that global inhibition of arginine and lysine histone methyltransferases (HMTs) by AMI-5 induced an angiostatic profile in human microvascular endothelial cells and human umbilical vein endothelial cells. Based on FACS analysis, we found that inhibition of HMTs significantly affects proliferation of endothelial cells, by suppressing cell cycle progression in the G0/G1 phase. Immunofluorescent studies of the endothelial cells replication pattern by 5-ethynyl-2'-deoxyuridine incorporation disclosed that AMI-5, and the arginine methyltransferase inhibitor AMI-1, induced heterochromatin formation and a number of nuclear abnormalities, such as formation of micronuclei (MNs) and nucleoplasmic bridges (NPBs), which are markers of chromosomal instability. In addition to the modification of the cell cycle machinery in response to AMIs treatment, also endothelial cells migration and capillary-like tube formation processes were significantly inhibited, implicating a stimulatory role of HMTs in angiogenesis.

Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression.

Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant-DSBs versus alkaline-DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

Behavior of replication origins in Eukaryota - spatio-temporal dynamics of licensing and firing.

Although every organism shares some common features of replication, this process varies greatly among eukaryotic species. Current data show that mathematical models of the organization of origins based on possibility theory may be applied (and remain accurate) in every model organism i.e. from yeast to humans. The major differences lie within the dynamics of origin firing and the regulation mechanisms that have evolved to meet new challenges throughout the evolution of the organism. This article elaborates on the relations between chromatin structure, organization of origins, their firing times and the impact that these features can have on genome stability, showing both differences and parallels inside the eukaryotic domain.

Replication and re-replication: Different implications of the same mechanism.

Replication is a process which provides two copies of genetic material to a mother cell that are essential for passing complete genetic information to daughter cells. Despite the extremely precise control of this process, regulation of replication can be impaired. This may trigger e.g. re-replication which leads to an increase in the total DNA content in a cell and, depending on the intensity, may result in gene amplification, genomic instability or apoptosis. Both replication and re-replication require pre-replication complex assembly, licensing, firing and initiation of DNA synthesis. Implications of each process in a cell are very different and all such possibilities are under intensive research because in both processes the same protein apparatus is used to carry out DNA synthesis. Therefore this article is meant to show the consequences of the same mechanism underlying two different processes.

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells.

KEY MESSAGE: PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used. Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).

Premature chromosome condensation induced by caffeine, 2-aminopurine, staurosporine and sodium metavanadate in S-phase arrested HeLa cells is associated with a decrease in Chk1 phosphorylation, formation of phospho-H2AX and minor cytoskeletal rearrangements.

Here, we demonstrate that in HeLa cells, Ser317 of Chk1 undergoes phosphorylation in response to replication stress induced by hydroxyurea. We also demonstrate the existence of constitutive (interphase and mitotic) Chk1 kinase phosphorylation, the translocation of its phosphorylated form from the nucleus to cytoplasm in prometaphase as well as strong labeling of apoptotic nuclei with α-Chk1(S317) antibodies. Additionally, we show that caffeine, 2-aminopurine, staurosporine and sodium metavanadate can induce premature chromosome condensation (PCC) by the abrogation of the S-M checkpoint. Staurosporine appeared to be the most effective PCC inductor, and as in the case of the remaining inductors, the addition of hydroxyurea each time brought about an increase in the number of cells showing PCC symptoms (synergic effect). The forced premature mitosis was accompanied by an increasing index of double-strand breaks marked by the phosphorylation of histone H2AX on Ser139. Moreover, we found that the chemicals used brought about minor actin and tubulin network rearrangements that occurred following either replication stress or drug-induced cell cycle delay. At the same time, it was found that the extent of the cytoskeleton rearrangement did not hinder PCC in all its subperiods, i.e., from PCC-type prophase to PCC-type telophase.

H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia.

Immunocytochemistry using alpha-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases alpha and delta, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX aggregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.

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.

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium.

Diphenyleneiodonium (DPI) inhibits activity of flavoenzymes like NADPH oxidase, the major source of superoxide anion in cardiovascular system, but affects also other oxidoreductases. Contradictory data have been published concerning the effect of diphenyleneiodonium on the production of reactive oxygen species in cells, both inhibitory and stimulatory action of DPI being reported. We have examined the effect of DPI on the cellular production of reactive oxygen and nitrogen species (ROS/RNS) and on the proliferation and apoptosis of human vascular endothelial cells. We found increased oxidation of ROS-sensitive probes (dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate) when DPI (20 microM-100 microM) was present in the treated cells. However, oxidation of the fluorogenic probes was inhibited if DPI (20 microM-100 microM) was removed from the reaction medium after cell preincubation. These results suggest an artifactual oxidation of the fluorogenic probes by DPI or its metabolites. A similar pattern of influence of DPI on the production of NO (measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) was observed. Modulation of generation of reactive oxygen and nitrogen species in DPI-treated cells influenced the nitration of tyrosine residues of cellular proteins, estimated by Western blotting. Decreased level of nitration generally paralleled the lowered production of ROS. A decreased 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium (MTT) reducing activity of cells for was observed immediately after 1h treatment of human endothelial cells with DPI (1 microM-100 microM), in spite of lack of changes in cell viability estimated by other methods. These results point to a next limitation of MTT in estimation of viability of cells treated with oxidoreductase inhibitors. DPI inhibited the proliferation of HUVECs as well as immortalized cell line HUVEC-ST, as assessed by acid phosphatase activity test and measurement of total nucleic acid content. Proapoptotic action of DPI was observed 12 h after incubation with this compound.

Induction of premature mitosis in root meristem cells of Vicia faba and Pisum sativum by various agents is correlated with an increased level of protein phosphorylation.

The intra-S-phase checkpoint response to hydroxyurea (HU)-mediated arrest of DNA replication was analysed in root meristems of two legumes, Pisum sativum and Vicia faba. The obtained results suggest that a molecular signal which invokes mechanisms allowing the cells to override the S-M dependency control system may be generated by caffeine (CF) and a number of alternative, yet related chemical agents, benzyl-6-aminopurine (BAP), 2-aminopurine (2-AP), and 6-dimethylaminopurine (DMAP). A variety of aberrant mitotic divisions included chromosomal breaks and gaps, lost and lagging chromatids and chromosomes, acentric fragments, chromosome bridges and micronuclei. Furthermore, similar effects induced by sodium vanadate, an inhibitor of protein phosphatases, extend the number of inhibitors capable of inducing premature chromosome condensation (PCC) in root meristem cells, as well as the range of possible regulatory pathways leading to the transition from S-phase arrest towards abnormal mitosis. Until preprophase, FITC-conjugated monoclonal antibodies (alpha-Y(a)b-FITC) that specifically recognize phosphorylated form of threonine indicate no evident cell cycle-dependent changes in an overall phosphorylation status of root meristem cells in the control plants. Irrespective of the stage of interphase, alpha-Y(p)ab-FITC was localized basically in the cytoplasm, whereas nuclear staining was considerably weaker, with a significant fluorescence confined merely to nucleolar regions. The intensity of alpha-Y(p)ab-FITC staining in HU/CF-treated seedlings was found higher than that in the control plants (with the exception of G2 cells), suggesting a general increase in the level of protein phosphorylation, a physiological response mediated probably by an enhanced activity of the cdc-like protein kinase(s).