Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle

Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse.     

ERK1 activation is required for S-phase onset and cell cycle progression after fertilization in sea urchin embryos

Fertilization of sea urchin eggs results in a large, transient increase in intracellular free Ca2+ concentration that is responsible for re-initiation of the cell division cycle. We show that activation of ERK1, a Ca2+-dependent MAP kinase response, is required for both DNA synthesis and cell cycle progression after fertilization. We combine experiments on populations of cells with analysis at the single cell level, and develop a proxy assay for DNA synthesis in single embryos, using GFP-PCNA. We compare the effects of low molecular weight inhibitors with a recombinant approach targeting the same signalling pathway. We find that inhibition of the ERK pathway at fertilization using either recombinant ERK phosphatase or U0126, a MEK inhibitor, prevents accumulation of GFP-PCNA in the zygote nucleus and that U0126 prevents incorporation of [3H]-thymidine into DNA. Abrogation of the ERK1 signalling pathway also prevents chromatin decondensation of the sperm chromatin after pronuclear fusion, nuclear envelope breakdown and formation of a bipolar spindle.     

Cell progression after selective irradiation of DNA during the cell cycle

Chinese hamster ovary cells were labeled with [125I]iododeoxyuridine (125IUdR, 0.1184 MBq/ml for 20 min) and the labeled mitotic cells were collected by selective detachment ("mitotic shake off"). The cells were pooled, plated into replicate flasks, and allowed to progress through the cell cycle. At several times after plating, corresponding to G1, S, late S, and G2 plus M, cells were cooled to stop cell cycle progression and to facilitate accumulation of 125I decays. Evaluation of cell progression into the subsequent mitosis indicated that accumulation of additional 125I decays during G1 or S phase was eight to nine times less effective in inducing progression delay than decays accumulated during G2. The results support our previous hypothesis that DNA damage per se is not responsible for radiation-induced progression delay. Instead, 125I-labeled DNA appears to act as a source of radiation that associates during the G2 phase of the cell cycle with another radiosensitive structure in the cell nucleus, and damage to the latter structure by overlap irradiation is responsible for progression delay (M. H. Schneiderman and K. G. Hofer, Radiat. Res. 84, 462-476 (1980].     

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos.

We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polyermase alpha. These cells fail to proliferate in the presence of aphidicolin. In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo. In continuous exposure to aphidicolin (2 micrograms/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.     

The origin recognition complex in silencing, cell cycle progression, and DNA replication.

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.     

Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression

Background  

Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase α primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC), and as being important for sister chromatid cohesion. As such, understanding the role of Ctf4 within the context of a multicellular organism will be integral to our understanding of its potential role in developmental and disease processes.     

Visualizing DNA replication sites in the cell nucleus.

Studies of DNA replication associated with the nuclear matrix have led to a radically new view of replication at the macroscopic level. It is proposed that individual replicons and their associated replicational assemblies (replisomes) are clustered together during active replication by attachment to the nuclear matrix at special sites termed 'clustersomes'. Direct visualization of replication sites in permeabilized cells by fluorescence microscopy following biotin-11-dUTP incorporation provides support for this model. Discrete replication granules are observed with sizes and numbers consistent with each granule being a site of replicon cluster synthesis. Distinct patterns of these sites are seen in different periods of S-phase. Both the individual granules and their early and late S-phase dependent patterns are strikingly maintained following extraction of the cells for in situ nuclear matrix structures. Similar results were obtained when probing in vivo sites of replication following incorporation of 5-bromodeoxyuridine. The three-dimensional organization of these replicational granules (clustersomes) is studied using confocal light microscopy and an appropriate multidimensional image analysis system.     

The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest

Werner syndrome is an autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several lines of evidence have suggested that the Werner syndrome protein WRN plays a role in DNA replication and S-phase progression. In order to define the exact role of WRN in genomic replication we examined cell cycle kinetics during normal cell division and after methyl-methane-sulfonate (MMS) DNA damage or hydroxyurea (HU)-mediated replication arrest following acute depletion of WRN from human fibroblasts. Loss of WRN markedly extended the time cells needed to complete the cell cycle after either of these genotoxic treatments. Moreover, replication track analysis of individual, stretched DNA fibers showed that WRN depletion significantly reduced the speed at which replication forks elongated in vivo after MMS or HU treatment. These results establish the importance of WRN during genomic replication and indicate that WRN acts to facilitate fork progression after DNA damage or replication arrest. The data provide a mechanistic basis for a better understanding of WRN-mediated maintenance of genomic stability and for predicting the outcomes of DNA-targeting chemotherapy in several adult cancers that silence WRN expression.     

Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.     

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.     

DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication during cell cycle progression

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.     

Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint

Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1(-) fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G(2)/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

BRCA1-dependent Chk1 phosphorylation triggers partial chromatin disassociation of phosphorylated Chk1 and facilitates S-phase cell cycle arrest

Chk1 phosphorylation by the PI3-like kinases ATR and ATM is critical for its activation and its role in prevention of premature mitotic entry in response to DNA damage or stalled replication. The breast and ovarian tumor suppressor, BRCA1, is among several checkpoint mediators that are required for Chk1 activation by ATM and ATR. Previously we showed that BRCA1 is necessary for Chk1 phosphorylation and activation following ionizing radiation. BRCA1 has been implicated in S-phase checkpoint control yet its mechanism of action is not well characterized. Here we report that BRCA1 is critical for Chk1 phosphorylation in response to inhibition of replication by either cisplatin or hydroxyurea. While Chk1 phosphorylation of S317 is fully dependent on BRCA1, additional proteins may mediate S345 phosphorylation at later time points. In addition, we show that a subset of phosphorylated Chk1 is released from the chromatin in a BRCA1-dependent manner which may lead to the phosphorylation of Chk1 substrate, Cdc25C, on S216 and to S-phase checkpoint activation. Inhibition of Chk1 kinase by UCN-01 or expression of Chk1 phosphorylation mutants in which the serine residues were substituted with alanine residues abrogates BRCA1-dependent cell cycle arrest in response replication inhibition. These data reveal that BRCA1 facilitates Chk1 phosphorylation and its partial chromatin dissociation following replication inhibition that is likely to be required for S-phase checkpoint signaling.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.