Nocodazole-arrested Cells Research Articles

Abstract B177: Synthetic lethal approach identifies potent and selective TTK and CLK inhibitor with preclinical anticancer activity in triple-negative breast cancer model

Abstract Historically, synthetic lethal-based drug discovery has yielded a high percentage of novel, first-in-class drugs while uncovering new tumor biology. Using a synthetic lethal approach, we identified one compound that preferentially induced apoptosis in triple-negative breast cancer (TNBC) cell lines while sparing luminal breast cancer cell lines. The synthetic lethal approach evolved into a targeted approach following the identification of the two kinases that were selectively inhibited by CC-671 in enzyme assays: human protein kinase monopolar spindle 1 (hMps1), also known as TTK, and CDC2-like kinase (CLK2). Detailed CC-671 cellular mechanism of action studies showed phosphorylation inhibition of the direct substrates of TTK and CLK2. The CC-671 IC50 concentration that inhibited phosphorylation of the CLK2 substrate SR protein ranged between 530 and 553 nM in CAL51 cells. Gene Set Enrichment Analyses (GSEA) of RNAseq data suggest that CC-671 CLK2 inhibition induces apoptosis by reducing expression of antiapoptotic isoforms via splicing modification. Cellular CC-671 TTK inhibition was demonstrated by significant reduction of TTK autophosphorylation at T686 and phosphorylation of KNL1, one of the well-characterized TTK substrates. CC-671 treatment also led to a decrease of cyclin B and securin protein level. As a result, the percentage of mitotic cells in nocodazole-arrested CAL51 cells was substantially reduced by CC-671. Furthermore, data from time-lapse video microscopy experiments confirmed the mitotic phase length was reduced by approximately half upon CC-671 treatment. Inhibition of phospho-Histone H3 (Ser 10) following a single CC-671 administration was observed in TNBC in vivo tumor model. More importantly, significant CC-671 in vivo efficacy was demonstrated in a cell line-derived TNBC xenograft model with weekly dosing. The data described here suggest that dual inhibition of TTK and CLK2 represents an attractive therapeutic approach for triple-negative breast cancer. CC-671 presents a unique profile for an anticancer kinase inhibitor. Citation Format: Dan Zhu, Gordafaried Deyanat-Yazdi, David Mikolon, Rama K. Narla, Sophie X. Peng, Yuhong Ning, Jennifer R. Riggs, John F. Boylan. Synthetic lethal approach identifies potent and selective TTK and CLK inhibitor with preclinical anticancer activity in triple-negative breast cancer model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B177.

Abstract 3770: The spindle assembly checkpoint requires phosphatase activity

Abstract The spindle assembly checkpoint (SAC) prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. A defective SAC contributes to errors in chromosome segregation, which promotes aneuploidy and may eventually lead to cancer. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein cdc20 until the chromosomes are properly aligned, at which point the MCC dissociates and releases cdc20 to activate the APC/C. Once the APC/C is activated, it targets many substrates for degradation, including cyclin B and securin, and the cell progresses into anaphase. The precise molecular mechanisms regulating SAC inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases in the SAC. To address this question, we treated mitotic cells with various phosphatase inhibitors to examine their effect on APC/C activation. Using this approach we identified two inhibitors, calyculin A and okadaic acid (1uM), that cause MCC dissociation and APC/C activation leading to cyclin A and cyclin B degradation in nocodazole-arrested cells. Although the cells are able to degrade cyclin B, they do not exit mitosis as evidenced by high levels of cdk1 substrate phosphorylation and chromosome condensation. Our results demonstrate that a calyculin A and okadaic acid (1uM) sensitive phosphatase is required for SAC activation. Citation Format: Kristen Foss, Liguo Zhang, Alexander C. Robeson, Sally Kornbluth. The spindle assembly checkpoint requires phosphatase activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3770. doi:10.1158/1538-7445.AM2015-3770

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G1/S boundary) or the Cdk1 inhibitor, RO3306 (G2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex.

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.

Abstract A239: Preclinical characterization of ABT-348, a kinase inhibitor targeting the Aurora, VEGFR/PDGFR, and SRC kinase families.

Abstract ABT-348 is a novel ATP-competitive inhibitor of Aurora kinases (Aurora B, C and A, IC50 values of 7, 1 and 120 nM, respectively). The activity against Aurora B is reflected in cellular assays of inhibition of histone H3 phosphorylation (IC50 value of 21 nM), induction of polyploidy (IC15 value of 12 nM) and inhibition of colony formation of human pancreatic carcinoma cells (MiaPaCa, IC50 value of 4 nM). Consistent with enzyme inhibition, ABT-348 inhibited the autophosphorylation of the respective enzyme in nocodazole-arrested cells (IC50 values of 13, 13 and 189 nM for Aurora B, C and A). Potency (IC50 values) in the cellular assays also correlated with inhibition of proliferation of cell lines derived from leukemia (0.3 nM, MV-4–11), lymphoma (4 nM, DOHH2), and solid tumors (MiaPaCa, 4 nM; SW620, 6 nM; OVCAR5, 7 nM; and HCT-15, 6 nM). Inhibition of Aurora B activity in vivo by ABT-348 was confirmed by measuring phosphorylation of histone H3 in circulating tumor cells obtained from an engrafted leukemia model (RS4;11). Inhibition of histone H3 phosphorylation was observed 4 hours after dosing that persisted in a dose-dependent manner for at least 8 hours. The extent of inhibition at 4 hours was related to the plasma concentration of ABT-348 (IC50: 3.6 μM), which was in close agreement with the value determined for inhibition of histone H3 phosphorylation in cells in the presence of mouse plasma (3.3 μM). In addition to its Aurora enzyme activity, evaluation of ABT-348 for inhibitory activity across 128 kinases revealed a unique kinome profile “signature” by virtue of its potent binding activity (Ki values <30 nM) against the VEGFR/PDGFR families and the SRC family of cytoplasmic tyrosine kinases (LYN, BLK, LCK, ABL, FYN, and SRC). The potent VEGFR/PDGFR binding activity was reflected in enzyme assays (IC50 values <10 nM) performed at 1 mM ATP and also correlated with inhibition of RTK auto-phosphorylation in cells for KDR, FLT3, CSF-1R, KIT, PDGFR and. These activities translated into potent inhibition of VEGF-stimulated endothelial cell proliferation (IC50 value <0.3 nM). Evidence that ABT-348 blocked VEGF-mediated responses in vivo was provided by the molecule's potent activity (ED50 value of 0.2 mg/kg, IV) in blocking VEGF-mediated vascular permeability in the uterus and its ability to induce dose-dependent increases in plasma PIGF in mice. Activity against the Src kinase family was evident in the anti-proliferative activity of ABT-348 against BCR ABL expressing tumor cells and cells expressing the gleevec-resistant BCR-ABL T315I mutation (IC50 values of 47 and 260 nM). Based upon these favorable in vitro and in vivo activities, ABT-348 was evaluated and found to be effective in leukemia (RS4;11, regression) xenografts. These results provide the rationale for clinical assessment of ABT-348 as a therapeutic agent in the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A239.

Cdk1 promotes kinetochore bi-orientation and regulates Cdc20 expression during recovery from spindle checkpoint arrest

The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore-microtubule attachment. Removal of the checkpoint-activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi-oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC-induced arrest. We show that Cdk1 promotes kinetochore bi-orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC-induced mitotic arrest by regulating bi-orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti-mitotic drugs.

Hyperphosphorylation by Cyclin B/CDK1 in Mitosis Resets CUX1 DNA Binding Clock at Each Cell Cycle*

The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).

Identification of Myb-binding Protein 1A (MYBBP1A) as a Novel Substrate for Aurora B Kinase

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.

Escaping the firing squad: acetylation of BubR1 protects it from degradation in checkpoint cells

Escaping the firing squad: acetylation of BubR1 protects it from degradation in checkpoint cells

Recruitment of separase to mitotic chromosomes is regulated by Aurora B

Accurate segregation of chromosomes, initiated by abrupt and irreversible dissolution of sister-chromatid cohesion at anaphase, is crucial for the faithful inheritance of parental genomes during eukaryotic cell division. The dissolution of sister-chromatid cohesion is catalyzed by separase after the destruction of securin by the anaphase-promoting complex. However, separase was localized to the mitotic centrosome, raising the question how separase hydrolyzes sister-chromatid cohesion at the centromere at anaphase onset. Here we show that separase is associated with mitotic chromosomes and this association is regulated by Aurora B kinase. Using a panel of separase antibodies, we found that separase protein accumulated in mitosis and was degraded at the end of telophase. To study the spatiotemporal distribution of separase in mitosis, we carried out immunofluorescence microscopic analyses. Surprisingly, separase was found to be associated with mitotic chromosomes from prophase to metaphase and dissociated from the chromosomes in anaphase right after sister chromatid separation. Staining of isolated mitotic chromosomes from Nocodazole-arrested cells revealed that separase is concentrated at the centromere. To examine if any mitotic kinases are responsible for chromosomal localization of separase in mitosis, we carried out RNAi-mediated knockdown and found that association of separase with mitotic chromosomes was a function of Aurora B. Consistent with the phenotype seen in the Aurora B-repressed cells, inhibition of Aurora B kinase by hersperadin prevented the association of separase with chromosomes. Our results suggest that Aurora B kinase activity helps coordinate the association of separase with chromosomes and the initiation of sister-chromatid separation.

Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells.

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post-translational modifications control diverse chromatin functions. Here, we report our findings from a large-scale screen for DNA-damage-responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA-damage-induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell-cycle re-positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl-transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.

Factors Affecting the Diversity of DNA Replication Licensing Control in Eukaryotes

Replication of eukaryotic genomes is limited to once per cell cycle, by a two-step mechanism. DNA replication origins are first "licensed" during G1 phase by loading of an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). Initiation then occurs during S phase, triggered by cyclin-dependent kinases (CDKs), which promote recruitment of proteins required for helicase activation and replisome assembly. CDKs and the anaphase promoting complex/cyclosome (APC/C) restrict licensing to G1 phase by directly and indirectly regulating pre-RC components, including ORC, Cdc6, Cdt1, and Mcm2-7. Despite the fundamental importance of licensing regulation, the mechanisms by which pre-RC components are regulated differ widely across Eukarya. Here we show that even within the genus Saccharomyces, Cdc6 is regulated differently in different species. We propose that two factors contribute to the rapid evolution of licensing regulation. The first is redundancy: eliminating any single pre-RC-regulatory mechanism has very little affect on viability. The second is interchangeability: we show that regulatory mechanisms can be swapped between pre-RC components without compromising the block to re-replication. These experiments provide a framework for understanding the diversity of licensing regulation in eukaryotes and provide new tools for manipulating the chromosome-replication cycle.

Modulation of the Mitotic Regulatory Network by APC-Dependent Destruction of the Cdh1 Inhibitor Acm1

The completion of mitosis depends on protein ubiquitination by the anaphase-promoting complex (APC). The APC is activated by association with Cdc20 in midmitosis and Cdh1 in late mitosis and G1. Here, we show that in budding yeast the activation of APC(Cdh1) is controlled in part by destruction of the Cdh1 inhibitor Acm1. We find that Acm1 uses pseudosubstrate and other sequence motifs to bind and inhibit Cdh1, but not Cdc20. Acm1 also contains a destruction sequence that promotes its ubiquitination by APC(Cdc20), resulting in the disappearance of Acm1 in early anaphase. Later in mitosis, Acm1 destruction is also promoted by APC(Cdh1). Finally, Cdk1-dependent phosphorylation of Acm1 modulates its localization and destruction. We conclude that ubiquitination of a Cdh1 inhibitor by APC(Cdc20) helps establish the order of activation of the two APC isoforms. We also speculate that the ability of APC(Cdh1) to target its own inhibitor enhances the bistability of the late mitotic regulatory system.

The complete removal of cohesin from chromosome arms depends on separase

Cohesin needs to be removed from chromosomes to allow sister chromatid separation in mitosis. In vertebrates, two pathways contribute to this process. The prophase pathway, which requires phosphorylation of the cohesin subunit SA2 and a cohesin-binding protein, called Wapl, removes the bulk of cohesin from the chromosome arms in early mitosis and allows the resolution of the chromosome arms. At anaphase onset, the protease separase removes centromere-enriched cohesin by proteolytic cleavage of another cohesin subunit, Scc1 (Rad21, Mcd1), which allows the separation of sister chromatids. When anaphase onset is delayed by the spindle-assembly checkpoint, the complete removal of cohesin from chromosome arms but not from centromeres generates typical X- or V-shaped chromosomes. Here, we found that cohesion between chromosome arms is preserved if mitosis is arrested with the proteasome inhibitor MG132. This arm cohesion depends on cohesin complexes that are protected by the shugoshin protein Sgo1, which appears to be distributed on chromosome arms as well as on centromeres in early mitosis. In cells lacking separase or expressing non-cleavable Scc1, arm cohesion was not efficiently removed during nocodazole arrest. Our observations suggest that a fraction of arm cohesin is protected by Sgo1, which prevents cohesin from being removed by the prophase pathway, and that separase is partly activated in nocodazole-arrested cells and removes the arm cohesin protected by Sgo1.

The IKK Inhibitor BMS-345541 Affects Multiple Mitotic Cell Cycle Transitions

The IκB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFκB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKα also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell-cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell-cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS 345541 function could be useful for cell-cycle control studies and may provide valuable clues for the design of future therapeutics.

Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925-936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5'-(gamma-thio)triphosphate (ATP[gammaS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration.

DNA Degradation at Unprotected Telomeres in Yeast Is Regulated by the CDK1 (Cdc28/Clb) Cell-Cycle Kinase

In the absence of functional telomeric cap protection, the ends of eukaryotic chromosomes are subject to DNA damage responses that lead to cell-cycle arrest and, eventually, genomic instability. However, the controlling activities responsible for the initiation of genome instability on unprotected telomeres remained unclear. Here we show that in budding yeast, unprotected telomeres undergo a tightly cell-cycle-regulated DNA degradation. Ablation of the function of essential capping proteins Cdc13p or Stn1p only caused telomere degradation in G2/M, but not in G1 of the cell cycle. Accordingly, G1-arrested cells with unprotected telomeres remained viable, while G2/M-arrested cells failed to recover. The data also show that completion of S phase and the activity of the S-Cdk1 kinase were required for telomere degradation. These results strongly suggest that after a loss of the telomere capping function, telomere-led genome instability is caused by tightly regulated cellular DNA repair attempts.

Induction of Centrosome Amplification during Arsenite-Induced Mitotic Arrest in CGL-2 Cells

Arsenite-induced mitotic abnormalities result in mitotic death in several cancer cell lines. However, how arsenite induces these effects is not known. We have previously shown that arsenite induces mitotic arrest, mitotic abnormalities, and mitotic death in CGL-2 cells. To further delineate the mechanism of action of arsenite, we examined its effect on centrosome duplication and the possible link between centrosome dysregulation and arsenite-induced mitotic death. Immunofluorescence staining of gamma-tubulin revealed that centrosome amplification was induced in arsenite-arrested mitotic cells but not in nocodazole-arrested cells. When S phase-enriched cells were treated with arsenite, they progressed into and arrested at mitosis and then formed supernumerary centrosomes. A further increase in arsenite-induced centrosome amplification was seen during the prolonged mitotic arrest. The arsenite-induced supernumerary centrosomes might result from uneven fragmentation of centrosome, overexpression of pericentriolar materials, and inhibition of centrosomal coalescence during mitosis. Furthermore, termination of mitotic arrest by treatment of arsenite-arrested mitotic cells with cyclin-dependent kinase 1 inhibitors or by suppression of spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 or Mad2 markedly reduced the induction of centrosome amplification and mitotic death in arsenite-treated cells. These results indicate that centrosome amplification is induced in arsenite-arrested mitotic CGL-2 cells in a spindle checkpoint-dependent manner and is involved in the induction of arsenite-induced mitotic death.

The Transcriptional Co-Repressor C-Terminal Binding Protein (CtBP) Associates with Centrosomes During Mitosis

CtBP is a co-repressor of transcription that acts by inhibiting co-activators and recruiting to promoter control elements (via interactions with DNA-binding transcription factors) a complex of proteins that modify histones, repress transcription and silence gene expression. There are two highly homologous genes, CtBP1 and CtBP2 that encode CtBP. In addition, a CtBP1-related protein has been described that has 11 fewer amino acids at its N-terminus than CtBP1. This variant of CtBP1 – known as CtBP3 or BARS [Brefeldin A (BFA) ribosylated substrate] – plays a critical role in the fragmentation of the Golgi complex at the onset of mitosis. Although there are some reports of CtBP with a cytoplasmic distribution after transfection, it is unclear how in normal cells a nuclear co-repressor regulates the behaviour of the Golgi complex at the onset of mitosis. Using polyclonal rabbit antibodies that were raised against a human CtBP1-GST fusion-protein, here we show by immunofluorescence laser-scanning confocal microscopy that in mitotic cells a species of CtBP becomes associated with the centrosomes at the onset of prophase and then throughout mitosis. The association can be seen in both cycling mitotic cells and nocodazole-arrested cells. The interaction was confirmed by co-immunoprecipitation and centrosome isolation. Since centrosomes are considered to be the organising centre for Golgi morphogenesis, the interaction demonstrated here may explain how a nuclear co-repressor of transcription can exert a regulatory effect on the Golgi complex at a specific stage of the cell cycle.

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.