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.     

MONOCLONAL ANTIBODY RAISED AGAINST HUMAN MITOTIC CYCLIN B1IDENTIFIES CYCLIN B-LIKE MITOTIC PROTEINS IN SYNCHRONIZED ONION (ALLIUM CEPA L.) ROOT MERISTEM

Cyclin B-like mitotic proteins have been detected in synchronized Allium cepa L. root tip cells by using mouse monoclonal anti-cyclin B₁antibody raised against human cyclin B₁Immunoblot shows two closely placed isoforms of cyclin B-like proteins having an apparent molecular weight around 54 kDa. In vivo [³⁵S]-methionine labelling followed by immunoprecipitation and autoradiography indicates that cyclin B-like proteins are mainly synthesized in the G2 phase of the cell cycle and destroyed in late mitosis. Immunoblotting data depict that the level of cyclin B-like proteins reaches the maximum at the late G2 to early M phase; and it becomes degraded in the late hours of mitosis. Moreover, the cyclin B isoforms are stabilized in colchicine-arrested metaphase cells as already reported in animal cells.     

The Roles of Cyclin A2, B1, and B2 in Early and Late Mitotic Events

Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (∼7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.     

MASTL is the human ortholog of Greatwall kinase that facilitates mitotic entry,anaphase and cytokinesis

Greatwall (Gwl) was originally discovered in Drosophila as an essential kinase for correct chromosome condensation and mitotic progression. In Xenopus, Gwl may influence the positive-feedback loop that directs cyclin B1-Cdk1 activation and the mitotic state by inhibiting the phosphatase PP2A. Here, we describe the human orthologue of Gwl called microtubule-associated serine/threonine kinase-like (MASTL). We found that MASTL localizes to the nucleus in interphase and re-localizes in part to centrosomes in mitosis, when it is active. Cells strongly depleted of MASTL by RNAi delay in G2 phase and reveal slow chromosome condensation. MASTL RNAi cells that enter and progress through mitosis often fail to completely separate their sister chromatids in anaphase. This causes chromatin to be trapped in the cleavage furrow, which may lead to formation of 4N G1 cells by cytokinesis failure. Further, our experiments indicate that MASTL supports the phosphorylation state of mitotic phospho-proteins downstream of cyclin B1-Cdk1, including the APC/C. Cyclin B1 destruction is incomplete when mitotic cells that are strongly depleted of MASTL exit mitosis. We propose that MASTL enhances cyclin B1-Cdk1-dependent mitotic phosphorylation-events, directing mitotic entry, anaphase and cytokinesis in human cells.     

The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases.

Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.     

Chk1-Dependent Regulation of Cdc25B Functions to Coordinate Mitotic Events

The coordination of mitotic spindle formation and chromatin condensation is an essential prerequisite for successful mitosis. Both events are thought to be initiated by cyclin B/Cdk1, whose initial activation occurs in late prophase at the centrosomes. Recently, we have shown that Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B/Cdk1. Here, we demonstrate that inhibition of Chk1 kinase induces mitotic entry with regular spindle assembly but aberrant and mislocalized chromatin. This effect, which we have termed the ‘paraspindle’ phenotype, was reverted by downregulation of Cdc25B phosphatase using siRNA, which restored normal mitosis with regular chromatin. Analogous to Chk1 inhibition, the ‘paraspindle’ phenotype was induced by overexpression of Cdc25B but not Cdc25A. Our results suggest that Chk1 functions to coordinate mitotic events through regulation of Cdc25B.     

Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells.

When BHK21 cells synchronized in early S phase were exposed to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, mitosis specific events such as premature chromosome condensation, the production of MPM-2 antigens, dispersion of nuclear lamins and the appearance of mitotic asters were induced, and then disappeared upon further incubation. These mitosis specific events occurred even in the presence of cycloheximide. Within 1 h of exposure to OA, cdc2/histone H1 kinase activity rose 10-fold compared with untreated controls, but returned to the control level upon further incubation. Using antibodies against either p34cdc2 or cyclin B it was found that p34cdc2 complexed with cyclin B was dephosphorylated after OA treatment concomitant with the activation of cdc2 kinase, and that cyclin B was subsequently degraded concomitant with a decrease in cdc2 kinase activity, as in normal mitosis. In contrast, when cells in G1 phase were treated with OA no increase in cdc2 kinase activity was observed. Moreover when cells in pseudo-metaphase induced by nocodazole were treated with OA, cdc2 kinase was inactivated. These results suggest that OA sensitive protein phosphatases control both the activation and inactivation of the p34cdc2 kinase.     

Premature chromatin condensation upon accumulation of NIMA.

The NIMA protein kinase of Aspergillus nidulans is required for the G2/M transition of the cell cycle. Mutants lacking NIMA arrest without morphological characteristics of mitosis, but they do contain an activated p37nimX kinase (the Aspergillus homologue of p34cdc2). To gain a better understanding of NIMA function we have investigated the effects of expressing various NIMA constructs in Aspergillus, fission yeast and human cells. Our experiments have shown that the instability of the NIMA protein requires sequences in the non-catalytic C-terminus of the protein. Removal of this domain results in a stable protein that, once accumulated, promotes a lethal premature condensation of chromatin without any other aspects of mitosis. Similar effects were also observed in fission yeast and human cells accumulating Aspergillus NIMA. This phenotype is independent of cell cycle progression and does not require p34cdc2 kinase activity. As gain of NIMA function by accumulation results in premature chromatin condensation, and loss of NIMA function results in an inability to enter mitosis, we propose that NIMA functions in G2 to promote the condensation of chromatin normally associated with entry into mitosis.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

The prolyl isomerase Pin1 functions in mitotic chromosome condensation

The prolyl isomerase Pin1 plays important roles in numerous cellular processes. Here we provide evidence that Pin1 has an important function in chromosome condensation during mitosis. We first demonstrate that the interaction of Pin1 with chromatin is greatly elevated in G2/M phase and that this correlates with the presence on chromosomes of several mitotic phosphoproteins, especially topoisomerase (Topo) IIalpha. Inducible overexpression of Pin1 was shown to result in higher M phase-specific phosphorylation, while downregulation of Pin1 by siRNA treatment reduced phosphorylation of TopoIIalpha and other mitotic proteins. Furthermore, immunodepletion of Pin1 from mitotic cell extracts prevented such extracts from inducing chromosome condensation when added to S phase nuclei. Indeed, purified Pin1 and cdc2/cyclin B kinase were by themselves sufficient to induce condensation. This reflects the ability of Pin1 to increase TopoIIalpha phosphorylation by cdc2/cyclin B in vitro, which in turn dramatically increased formation of a TopoIIalpha/Pin1/DNA complex.     

PLANT MITOSIS PROMOTING FACTOR DISASSEMBLES THE MICROTUBULE PREPROPHASE BAND AND ACCELERATES PROPHASE PROGRESSION IN TRADESCANTIA

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant ofChlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34^cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells ofTradescantia virginianain which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34^cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.     

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.     

Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.     

Depletion of BubR1 promotes premature centrosomal localization of cyclin B1 and accelerates mitotic entry

The role of BubR1 has been established mainly in mitosis as an essential mitotic checkpoint protein although it is expressed throughout the cell cycle. To explore a possible role of BubR1 in regulating the G2 phase of cell cycle, we have employed siRNA–mediated hBubR1 knockdown in HeLa cells. Here, we demonstrate that reducing BubR1 levels during the G2 phase causes accelerated mitotic entry. As expected, BubR1 depletion leads to degradation of cyclin B1 in the G2 phase. Intriguingly, cyclin B1 is prematurely targeted to centrosomes appearing at early G2 phase in BubR1-depleted cells despite its low levels. This is in contrast to control cells where cyclin B1 appears at the centrosomes in early prophase based on cell cycle-specific localization of CENP-F. Furthermore, cyclin B/Cdk1 kinase activity in early G2 is aberrantly high in BubR1-depleted cells. Together, our results indicate that hBubR1 depletion triggers premature centrosomal localization of cyclin B1 probably leading to premature mitotic entry. This study is the first to suggest a role of hBubR1 in controlling centrosome targeting of cyclin B1 and timing of mitotic entry.     

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with ³²P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.     

ATR (AT mutated Rad3 related) activity stabilizes Cdc6 and delays G2/M-phase entry during hydroxyurea-induced S-phase arrest of HeLa cells

The Cdc6 protein, a key DNA replication initiation factor, contributes to the long-term maintenance of the S-phase checkpoint by anchoring the Rad3–Rad26 complex to chromatin. Here, we demonstrate that ATR (AT mutated and Rad3 related) activity is essential for maintaining high chromatin levels of the Cdc6 protein, thereby delaying entry into mitosis during hydroxyurea (HU)-induced S-phase arrest of HeLa cells. Downregulation of ATR (AT mutated and Rad3 related) (i.e., using ATR-siRNA) reduced the protein levels of chromatin Cdc6 and significantly increased the cellular levels of phospho-histone H3 (Ser 10), an index of mitosis. Downregulation of Cdc6 was completely restored by pretreatment with MG132, a proteasome inhibitor. Moreover, mitotic entry of MG132-pretreated cells was significantly downregulated. Our results also show that ATR (AT mutated and Rad3 related) kinase phosphorylates Cdc6 at serine residue 6. Thus, this ATR (AT mutated and Rad3 related)-mediated phosphorylation of Cdc6 is likely associated with stabilization of Cdc6 protein, thereby maintaining high levels of chromatin Cdc6 and delaying premature mitotic entry. This novel mechanism likely contributes to the functional regulation of chromatin Cdc6, which delays the cell cycle of hydroxyurea-induced cells to enter mitosis at the S-phase checkpoint.     

Role of polyamines during chromosome condensation of mammalian cells

The objective of the present study was to investigate the role of polyamines in the process of chromosome condensation. The phenomenon of premature chromosome condensation (PCC) involving fusion between mitotic and interphase cells was used as the assay system. The factors present in the mitotic cells would bring about the breakdown of the nuclear membrane and condensation of the interphase chromatin into chromosomes, similar to that which occurs during the initiation of mitosis. Alpha-difluoromethyl ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase was used to deplete both mitotic and interphase cells of polyamines. The results indicate that the polyamine depleted mitotic cells have a diminished ability to induce PCC. This inhibition could easily be reversed by exogenous addition of polyamines at the time of fusion. Furthermore, exogenously added polyamines hastened the entry of exponentially growing cells into mitosis. These observations suggest an essential role for polyamines during the process of chromosome condensation of mammalian cells.     

Mammalian cell fusion. VI. Regulation of mitosis in binucleate HeLa cells.

In two different cell fusion experiments a synchronized population of HeLa cells, prelabeled with ³H-TdR, was fused with an unlabeled one using inactivated Sendai virus. In the first experiment, HeLa cells in early G2 phase which were exposed to either 4 °C, cycloheximide, actinomycin D or X-irradiation were fused separately with untreated and more advanced G2 cells. A comparison of the rates of mitotic accumulation (in the presence of Colcemid) for the various classes of mono- and binucleate cells revealed that the hybrid (binucleate) cells were intermediate between those of the advanced and the retarded parental types indicating that the chromosome condensing factors of the advanced component were diluted as a result of such fusion. The manner in which the retarding effects of actinomycin D and cycloheximide were reversed in the hybrid cells suggested that proteins had a major role as chromosome condensing factors in the G2 mitotic transition. In the second experiment, when S phase HeLa cells were fused with those in G2, the resulting heterophasic (S/G2) binucleate cells reached mitosis at about the same time as the homophasic (S/S) cells of the lagging parent indicating a complete dominance of the S over the G2 with regard to their progress towards mitosis. However, the addition of Mg²⁺ (2 × 10^?2 M of MgCl₂) to the medium helped the G2 nuclei to enter mitosis asynchronously, which consequently induced premature chromosome condensation (PCC) in the S phase component. These data suggested that in the heterophasic (S/G2) binucleate cells the S phase component caused decondensation of the G2 chromatin thus blocking it from entering into mitosis. This effect which did not appear to be dose-dependent could be neutralized and the G2 nuclei relieved from this repression by an external supply of Mg²⁺ ions.