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At least 1,747 records · Page 97Linked to original sources
PubMed2002

HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic mitotic checkpoints.

HIV-1 Vpr induces cell cycle G2/M arrest in both human and fission yeast cells, suggesting a highly conserved activity of this viral protein. In this review, we summarize the current understanding of Vpr-induced G2 arrest based on studies from both mammalian cells and the fission yeast (Schizosaccharomyces pombe) model system. Fission yeast has proven to be an excellent model system to investigate cell cycle G2/M control of eukaryotic cells. Similarly, fission yeast has also been instrumental in defining the molecular mechanism underlying the G2 arrest induced by Vpr. We have compared the classic DNA-damage and DNA-replication checkpoint controls of the cell cycle G2/M transition to the G2 arrest conferred by Vpr. Based on the current findings, we hypothesize that Vpr induces cell cycle G2 arrest through an alternative novel cellular pathway(s) rather than through the classic mitotic checkpoint controls. A number of cellular proteins which may be involved in this new cellular pathway(s) have been identified and are discussed.

Animals↗
PubMed2001

Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G(1)/S checkpoint activation in response to DNA damage.

ATR [ataxia-telangiectasia-mutated (ATM)- and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM- and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser(635) and Ser(645) in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late G(1), S, and G(2)/M, but not in early-mid G(1). Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid G(1). Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of Ser(645) was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the G(1)/S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells.

Amino Acid Sequence↗
PubMed2005

From oogenesis through gastrulation: developmental regulation of apoptosis.

Apoptosis is a mechanism employed by multicellular organisms throughout development as a means of eliminating damaged or otherwise unwanted cells. From oogenesis through fertilization and gastrulation, organisms use an array of cell- and tissue-specific mechanisms to regulate the apoptotic program in response to stress or developmental cues. Since cell death regulation is tightly interwoven with cell cycle and checkpoint controls, and embryos of the fly, fish and frog exhibit unique embryonic cell cycle regulation, it is of great interest to understand how early embryos coordinate these cellular functions.

Animals↗
PubMed2004

MAD2 dependent mitotic checkpoint defects in tumorigenesis and tumor cell death: a double edged sword.

The failure of cell cycle regulatory checkpoints is a common event in human cancer. Defects at the G(1)-S transition have been widely characterized, but only more recently has aberrant checkpoint signaling during mitotic progression been identified as playing a role in cancer. The metaphase to anaphase transition is regulated by multiple proteins that together comprise the mitotic checkpoint. Previously it has been shown that loss of one copy of MAD2, a mitotic checkpoint gene, results in aneuploidy and tumorigenesis arising from chromosome missegregation. More recently and quite surprisingly, MAD2 has been demonstrated to be an essential gene even in tumor cells such that near complete elimination of this protein from cancer cells results in p53 independent cell death. This is the first identification of a haploinsufficient tumor suppressor gene that is also required for tumor cell survival, and suggests that targeting this checkpoint in cancer might be a viable therapeutic strategy.

Animals↗
PubMed2000

Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress.

The eukaryotic cell cycle is overseen by regulatory mechanisms, termed checkpoints, that respond to DNA damage, mitotic spindle defects, and errors in the ordering of cell cycle events. The DNA replication and DNA damage cell cycle checkpoints of the fission yeast Schizosaccharomyces pombe require the hus1(+) (hydroxyurea sensitive) gene. To determine the role of the mouse homolog of hus1(+) in murine development and cell cycle checkpoint function, we produced a targeted disruption of mouse Hus1. Inactivation of Hus1 results in mid-gestational embryonic lethality due to widespread apoptosis and defective development of essential extra-embryonic tissues. DNA damage-inducible genes are up-regulated in Hus1-deficient embryos, and primary cells from Hus1-null embryos contain increased spontaneous chromosomal abnormalities, suggesting that loss of Hus1 leads to an accumulation of genome damage. Embryonic fibroblasts lacking Hus1 fail to proliferate in vitro, but inactivation of p21 allows for the continued growth of Hus1-deficient cells. Hus1(-/-)p21(-/-) cells display a unique profile of significantly heightened sensitivity to hydroxyurea, a DNA replication inhibitor, and ultraviolet light, but only slightly increased sensitivity to ionizing radiation. Taken together, these results indicate that mouse Hus1 functions in the maintenance of genomic stability and additionally identify an evolutionarily-conserved role for Hus1 in mediating cellular responses to genotoxins.

Animals↗
PubMed2006

Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit.

In higher eukaryotic organisms, the checkpoint kinase 1 (Chk1) contributes essential functions to both cell cycle and checkpoint control. Chk1 executes these functions, in part, by targeting the Cdc25A protein phosphatase for ubiquitin-mediated proteolysis. In response to genotoxic stress, Chk1 is phosphorylated on serines 317 (S317) and 345 (S345) by the ataxia-telangiectasia-related (ATR) protein kinase. Phosphorylation of Chk1 on these C-terminal serine residues is used as an indicator of Chk1 activation in vivo. Here, we report that inhibition of Chk1 kinase activity paradoxically leads to the accumulation of S317- and S345-phosphorylated Chk1 in vivo and that ATR catalyzes Chk1 phosphorylation under these conditions. We demonstrate that Chk1 phosphorylation by ATR is antagonized by protein phosphatase 2A (PP2A). Importantly, dephosphorylation of Chk1 by PP2A is regulated, in part, by the kinase activity of Chk1. We propose that the ATR-Chk1-PP2A regulatory circuit functions to keep Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 primed to respond rapidly in the event that cells encounter genotoxic stress.

Carbazoles↗
PubMed2004

chk-1 is an essential gene and is required for an S-M checkpoint during early embryogenesis.

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe.(1) Genetic analysis revealed that chk1 is involved in a DNA damage G(2)-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G(2)-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide.(2) In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles.(3,4) What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M checkpoint hence causing premature entry into M-phase.

Animals↗
PubMed2002

Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin.

BACKGROUND: In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G2/M checkpoint control pathways. Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9. Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA. To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA. METHODS: Transfection of 2'-O-methoxyethyl-modified Hus1 antisense oligoribonucleotides into human H1299 nonsmall lung carcinoma cells was performed using Lipofectin as the carrier. The authors prepared RNA from transfected cells, and levels of Hus1 expression were analyzed by real time polymerase chain reaction. The growth and viability of cells treated with Hus1 antisense oligonucleotides in the presence or absence of cisplatin were analyzed and compared to controls. RESULTS: Transfection of selected Hus1 antisense oligonucleotides into p53 deficient H1299 cells resulted in significant downregulation of Hus1 mRNA, up to 80%; RNA analyses reveal a maximal Hus1 antisense activity at a concentration of 200 nM with an IC50 determined to be 90 nM. The design and transfection of oligonucleotides containing three mismatches to their corresponding antisense counterparts had no or only minor effects on Hus1 mRNA levels, showing the specificity of Hus1 mRNA downregulation. The cisplatin IC50 in untransfected H1299 cells was found to be 20 microM and could be reduced significantly to only 7 microM after transfection of a Hus1 antisense oligonucleotide. CONCLUSIONS: Experiments addressing the proliferation and viability of transfected H1299 cells suggest that downregulation of Hus1 by specific antisense oligonucleotides sensitizes human cells to treatment with the DNA damaging agent cisplatin.

Antineoplastic Agents↗
PubMed2003

Coupling the cell cycle to cell growth.

In order to multiply, both prokaryotic and eukaryotic cells go through a series of events that are collectively called the cell cycle. However, DNA replication, mitosis and cell division may also be viewed as having their own, in principle independent, cycles, which are tied together by mechanisms extrinsic to the cell cycle--the checkpoints--that maintain the order of events. We propose that our understanding of cell-cycle regulation is enhanced by viewing each event individually, as an independently regulated process. The nature of the parameters that regulate cell-cycle events is discussed and, in particular, we argue that cell mass is not such a parameter.

Cell Culture Techniques↗
PubMed2004

Dietary isothiocyanates inhibit Caco-2 cell proliferation and induce G2/M phase cell cycle arrest, DNA damage, and G2/M checkpoint activation.

Benzyl isothiocyanate and phenethyl isothiocyanate, two aromatic phytochemicals present in substantial concentrations in edible vegetables of the genus Brassica, were investigated for their effects on Caco-2 cell proliferation. Benzyl and phenethyl isothiocyanate inhibited DNA synthesis, with 50% inhibitory concentrations of 5.1 and 2.4 micromol/L, respectively, and significantly increased the doubling times of Caco-2 cells from 32 h to 220 and 120 h, respectively. There was no adverse effect of either chemical on cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, but benzyl isothiocyanate and phenethyl isothiocyanate both caused an accumulation of cells in the G(2)/M phase of the cell cycle, which was maintained for at least 48 h in cells synchronized at prometaphase with nocodazole and subsequently treated with 10 micromol/L benzyl isothiocyanate or phenethyl isothiocyanate. Both benzyl and phenethyl isothiocyanate increased DNA strand breakage, increased phosphorylation of the G(2)/M checkpoint enforcer Chk2, and induced p21 expression. These results suggest that the antiproliferative effects of benzyl and phenethyl isothiocyanates toward Caco-2 cells are due at least in part to the activation of the G(2)/M DNA damage checkpoint, and that sustained G(2)/M phase cell cycle arrest in response to benzyl and phenethyl isothiocyanates may be maintained through upregulation of p21. This study indicates that some dietary isothiocyanates may exert an antiproliferative effect through activation of the G(2)/M DNA damage checkpoint.

Brassica↗
PubMed2002

Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint.

PURPOSE: To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. MATERIALS AND METHODS: Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. RESULTS: Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. CONCLUSIONS: These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.

Apoptosis↗
PubMed2000

Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression.

Schizosaccharomyces pombe hus1 promotes radioresistance and hydroxyurea resistance, as well as S and G2 phase checkpoint control. We isolated a human cDNA homologous to hus1, called HUS1. The major focus of this report is on a detailed analysis of the physical interactions of the HUS1-encoded protein and two other checkpoint control proteins, RAD1p and RAD9p, implicated in the cellular response to DNA damage. We found that HUS1p interacts with itself and the N-terminal region of RAD1p. In contrast, the C-terminal portion of the checkpoint protein RAD9p is essential for interacting with HUS1p and the C-terminal region of RAD1p. Since the N-terminal portion of RAD9p was previously demonstrated to participate in apoptosis, this protein likely has at least two functional domains, one that regulates programmed cell death and another that regulates cell cycle checkpoint control. Truncated versions of HUS1p are unable to bind RAD1p, RAD9p, or another HUS1p molecule. RAD1p-RAD1p and RAD9p-RAD9p interactions can be demonstrated by coimmunoprecipitation, but not by two-hybrid analysis, suggesting that the proteins associate as part of a complex but do not interact directly. Northern blot analysis indicates that HUS1 is expressed in different tissues, but the mRNA is most predominant in testis where high levels of RAD1 and RAD9 message have been detected. These studies suggest that HUS1p, RAD9p, and RAD1p form a complex in human cells and may function in a meiotic checkpoint in addition to the cell cycle delays induced by incomplete DNA replication or DNA damage.

Binding Sites↗
PubMed2004

DNA replication checkpoint control mediated by the spindle checkpoint protein Mad2p in fission yeast.

The relationship between the DNA replication and spindle checkpoints of the cell cycle is unclear, given that in most eukaryotes, spindle formation occurs only after DNA replication is complete. Fission yeast rad3 mutant cells, which are deficient in DNA replication checkpoint function, enter, progress through, and exit mitosis even when DNA replication is blocked. In contrast, the entry of cds1 mutant cells into mitosis is delayed by several hours when DNA replication is inhibited. We show here that this delay in mitotic entry in cds1 cells is due in part to activation of the spindle checkpoint protein Mad2p. In the presence of the DNA replication inhibitor hydroxyurea (HU), cds1 mad2 cells entered and progressed through mitosis earlier than did cds1 cells. Overexpression of Mad2p or inactivation of Slp1p, a regulator of the anaphase-promoting complex, also rescued the checkpoint defect of HU-treated rad3 cells. Rad3p was shown to be involved in the physical interaction between Mad2p and Slp1p in the presence of HU. These results suggested that Mad2p and Slp1p act downstream of Rad3p in the DNA replication checkpoint and that Mad2p is required for the DNA replication checkpoint when Cds1p is compromised.

Carrier Proteins↗
PubMed2006

Few crucial links assure checkpoint efficiency in the yeast cell-cycle network.

MOTIVATION: The ability of cells to complete mitosis with high fidelity relies on elaborate checkpoint mechanisms. We study S- and M-phase checkpoint responses in silico in the budding yeast with a stochastic dynamical model for the cell-cycle. We aim to provide an unbiased functional classification of network interactions that reflect the contribution of each link to checkpoint efficiency in the presence of cellular fluctuations. RESULTS: We developed an algorithm BNetDyn to compute stochastic dynamical trajectories for an input gene network and its structural perturbations. User specified output measures like the mutual information between trigger and output nodes are then evaluated on the stationary state of the Markov process. Systematic perturbations of the yeast cell-cycle model by Li et al. classify each link according to its effect on checkpoint efficiencies and stabilities of the main cell-cycle phases. This points to the crosstalk in the cascades downstream of the SBF/MBF transcription activator complexes as determinant for checkpoint optimality; a finding that consistently reflects recent experiments. Finally our stochastic analysis emphasizes how dynamical stability in the yeast cell-cycle network crucially relies on backward inhibitory circuits next to forward induction. AVAILABILITY: C++ source code and network models can be downloaded at http://www.vital-it.ch/Software/

Cell Cycle↗
PubMed2002

[Embryonal stem cells do not undergo cell cycle arrest upon exposure to damaging factors].

As shown recently (Malashicheva et al., 2000), embryonic teratocarcinoma F9 mouse cells do not stop on the G1/S border after the treatment with agents causing G1 arrest in normal fibroblast cells. Since after a prolonged cultivation in vitro F9 cells could lose some properties characteristic of the stem cells, we studied here the capability of mouse ES cells to undergo cell cycle blocks following gamma-irradiation, adriamycin and PALA treatment as well as upon cultivation in the presence of nocodazol, an inhibitor of spindle assembly. The results obtained show that ES cells, similarly as their tumorigenic derivative F9 cells, do not demonstrate any delay on the G1/S boundary of the cell cycle. Moreover, nocodazol treatment for 48 h leads to accumulation of polyploid cells. Immunoblot experiments reveal a low level of p21/Waf1 expression both in F9 and in ES cells. Interestingly, the content of p21/Waf1 has been found to increase after cell treatment with proteasome inhibitor lactacystin, implying that p21/Waf1 level is regulated by proteasomal degradation. Thus, the p21/Waf1--dependent mechanisms of cell cycle control (checkpoint control) do not function properly in embryonic stem cells.

Acetylcysteine↗
PubMed2002

Mutations in genes of Saccharomyces cerevisiae encoding pre-mRNA splicing factors cause cell cycle arrest through activation of the spindle checkpoint.

Previous work has identified a group of genes whose products play important roles in two seemingly unrelated processes: cell cycle progression and splicing. The products of these genes show a network of physical and genetic interactions suggestive of the existence of a protein complex, the cell cycle and splicing complex (CSC). Here we analyze the genetic interactions between ISY1, SYF2 and NTC20, three non-essential components of the CSC. We show that mutations in ISY1 cause lethality in the absence of Ntc20p, and that the double mutant isy1Delta syf2Delta shows a temperature-dependent cell cycle arrest. This arrest is due to lower levels of alpha-tubulin, a protein encoded by TUB1 and TUB3, two intron-containing genes. We show that the low levels of alpha-tubulin in isy1Delta syf2Delta trigger activation of the spindle checkpoint, causing cell cycle arrest. Thus, our results have uncovered an unexpected role for pre-mRNA splicing in the maintenance of the fidelity of chromosome transmission during cell division.

Biological Transport↗
PubMedDate not supplied

Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots.

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.

Antibodies, Monoclonal↗
PubMed1995

G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro.

In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.

Amino Acid Sequence↗