Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots.

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.

LKB1 inactivation leads to centromere defects and genome instability via p53-dependent upregulation of survivin.

Inactivating mutations in the liver kinase B1 (LKB1) tumor suppressor gene underlie Peutz-Jeghers syndrome (PJS) and occur frequently in various human cancers. We previously showed that LKB1 regulates centrosome duplication via PLK1. Here, we report that LKB1 further helps to maintain genomic stability through negative regulation of survivin, a member of the chromosomal passenger complex (CPC) that mediates CPC targeting to the centromere. We found that loss of LKB1 led to accumulation of misaligned and lagging chromosomes at metaphase and anaphase and increased the appearance of multi- and micro-nucleated cells. Ectopic LKB1 expression reduced these features and improved mitotic fidelity in LKB1-deficient cells. Through pharmacological and genetic manipulations, we showed that LKB1-mediated repression of survivin is independent of AMPK, but requires p53. Consistent with the key influence of LKB1 on survivin expression, immunohistochemical analysis indicated that survivin is highly expressed in intestinal polyps from a PJS patient. Lastly, we reaffirm a potential therapeutic avenue to treat LKB1-mutated tumors by demonstrating the increased sensitivity to survivin inhibitors of LKB1-deficient cells.

A new tool for genotoxic risk assessment: Reevaluation of the cytokinesis-block micronucleus assay using semi-automated scoring following telomere and centromere staining.

BACKGROUND: The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS: Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS: The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION: These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.

3D genomic architecture reveals that neocentromeres associate with heterochromatin regions.

The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere-heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres.

DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes.

Mouse oocytes carrying DNA damage arrest in meiosis I, thereby preventing creation of embryos with deleterious mutations. The arrest is dependent on activation of the spindle assembly checkpoint, which results in anaphase-promoting complex (APC) inhibition. However, little is understood about how this checkpoint is engaged following DNA damage. Here, we find that within minutes of DNA damage checkpoint proteins are assembled at the kinetochore, not at damage sites along chromosome arms, such that the APC is fully inhibited within 30 min. Despite this robust response, there is no measurable loss in k-fibres, or tension across the bivalent. Through pharmacological inhibition we observed that the response is dependent on Mps1 kinase, aurora kinase and Haspin. Using oocyte-specific knockouts we find the response does not require the DNA damage response kinases ATM or ATR. Furthermore, checkpoint activation does not occur in response to DNA damage in fully mature eggs during meiosis II, despite the divisions being separated by just a few hours. Therefore, mouse oocytes have a unique ability to sense DNA damage rapidly by activating the checkpoint at their kinetochores.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the 'hot spots' and 'cold spots' of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.

Premature chromatid separation and altered proliferation of human leukocytes treated with vanadium (III) oxide.

Vanadium is a widely distributed metal in the Earth's surface and is released into the environment by either natural or anthropogenic causes. Vanadium (III) oxide (V2O3) is present in the environment, and many organisms are exposed to this compound; however, its effects at the cellular and genetic levels are still unknown. Therefore, in this study, the ability of V2O3 to induce chromosomal damage and impair cell proliferation was tested on human leukocytes in vitro. The cultures cells were treated for 48 h with different concentrations 2, 4, 8 or 16 µg/mL of V2O3, and we use the sister chromatid exchange's (SCE) test and the viability assay to evaluate the effects. In the results, no change was observed in either the viability or the frequency of SCE; however, a significant increase was observed in the incidence of premature chromatid separation (PCS), and a decrease was observed in both the mitotic index (MI) and the replication index (RI). Therefore, it can be suggested that V2O3 induces a genotoxic effect at the centromere level, indicating that it is a cause of aneuploidy that is capable of altering cell cycle progression.

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Analysis of HDACi-Induced Changes in Chromosomal Passenger Complex Localization.

The chromosomal passenger complex (CPC) is a key regulator of cell division. Its proper localization during the different phases of mitosis and cytokinesis is crucial for the exertion of its various functions. HDACi treatment has been demonstrated to disturb the centromeric localization of the CPC in tumor cells, thus leading to severe mitotic defects often followed by apoptosis. In this chapter, we describe how HDACi-induced changes of the CPC localization can be analyzed by indirect immunofluorescence using CPC-specific primary and fluorophore-coupled secondary antibodies followed by confocal microscopy.

Transcriptional Activation of Pericentromeric Satellite Repeats and Disruption of Centromeric Clustering upon Proteasome Inhibition.

Heterochromatinisation of pericentromeres, which in mice consist of arrays of major satellite repeats, are important for centromere formation and maintenance of genome stability. The dysregulation of this process has been linked to genomic stress and various cancers. Here we show in mice that the proteasome binds to major satellite repeats and proteasome inhibition by MG132 results in their transcriptional de-repression; this de-repression is independent of cell-cycle perturbation. The transcriptional activation of major satellite repeats upon proteasome inhibition is accompanied by delocalisation of heterochromatin protein 1 alpha (HP1α) from chromocentres, without detectable change in the levels of histone H3K9me3, H3K4me3, H3K36me3 and H3 acetylation on the major satellite repeats. Moreover, inhibition of the proteasome was found to increase the number of chromocentres per cell, reflecting destabilisation of the chromocentre structures. Our findings suggest that the proteasome plays a role in maintaining heterochromatin integrity of pericentromeres.

OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases.

OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays.

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis.

Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis.

Analysis of Genetic Damage in Lymphocytes of Former Uranium Processing Workers.

The frequency of cells containing micronuclei (MN) and the presence of centromeres in these MN were analyzed in lymphocytes of 98 men from Southern Bohemia. Forty-six of them had worked at the uranium processing plant 'MAPE Mydlovary' which was closed in 1991, and 52 men were controls from the same area. FISH using human pan-centromeric chromosome paint was employed to detect centromere-positive (CEN+) and -negative (CEN-) MN. A total of 1,000 binucleated cells (BNC) per participant were analyzed after cytochalasin B treatment. All BNC with MN (CEN+ or CEN-) were recorded. No differences were found between formerly exposed workers and the control group, neither in the total frequency of cells with MN per 1,000 BNC (mean levels ± SD, 9.1 ± 3.1 and 9.8 ± 2.5, respectively) nor in the percentage of CEN- MN, which were equal (50 ± 18 and 49 ± 17, respectively). Also, there was no difference between individuals living in the 3 villages closest to the uranium processing plant and those living further away. Considering the fact that effective doses of the workers at MAPE Mydlovary were overall similar to those of former uranium miners in whom higher frequencies of CEN- MN have been found more than 10 years after they had finished working underground, these results are somewhat surprising. A more detailed analysis of the exposures indicates that uranium miners received a greater percentage of their effective dose from the inhalation of radon and its daughters, whereas uranium processing workers received it from the incorporation of long-lived radioactive nuclides such as uranium. If, as has been suggested before, the higher level of DNA damage in miners is due to induced genomic instability, then this phenomenon may be related to radon exposure rather than exposure to uranium.

Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes) subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo) repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe) mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

Mutagenic potential of the isoflavone irilone in cultured V79 cells.

After consumption of red clover-based dietary supplements, plasma concentrations of the isoflavone irilone (IRI) equal that of the well-investigated daidzein. Since some isoflavones are genotoxic, the potential of IRI to induce mutations was investigated. Gene mutations were determined by hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay and sequencing of mutant cDNA, chromosome and genome mutations by micronucleus assay complemented by immunochemical staining of centromere proteins and microtubules in cultured V79 cells. Cell proliferation was monitored by electronic cell counting, flow cytometry and fluorescence microscopy. IRI did not affect the mutant frequency in the Hprt locus but altered the mutation spectrum by increasing the proportion of deletions and decreasing that of base pair substitutions. Induction of chromosome mutations was supported by a slight but significant increase in the number of micronucleated cells containing chromosomal fragments despite activation of three cell cycle checkpoints possibly interfering with micronuclei formation. Moreover, IRI exhibited a strong aneugenic potential characterized by disrupted mitotic spindles, mitotic arrest, and asymmetrical cell divisions leading to chromosome loss, nuclear fragmentation as well as mitotic catastrophe. Thus, IRI might be another isoflavone to be taken into account in safety assessment of dietary supplements.

Chromosome 17 centromere duplication and responsiveness to anthracycline-based neoadjuvant chemotherapy in breast cancer.

Human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) genes have been proposed as predictive biomarkers of sensitivity to anthracycline chemotherapy. Recently, chromosome 17 centromere enumeration probe (CEP17) duplication has also been associated with increased responsiveness to anthracyclines. However, reports are conflicting and none of these tumor markers can yet be considered a clinically reliable predictor of response to anthracyclines. We studied the association of TOP2A gene alterations, HER2 gene amplification, and CEP17 duplication with response to anthracycline-based neoadjuvant chemotherapy in 140 patients with operable or locally advanced breast cancer. HER2 was tested by fluorescence in situ hybridization and TOP2A and CEP17 by chromogenic in situ hybridization. Thirteen patients (9.3%) achieved pathologic complete response (pCR). HER2 amplification was present in 24 (17.5%) of the tumors. TOP2A amplification occurred in seven tumors (5.1%). CEP17 duplication was detected in 13 patients (9.5%). CEP17 duplication correlated with a higher rate of pCR [odds ratio (OR) 6.55, 95% confidence interval (95% CI) 1.25-34.29, P = .026], and analysis of TOP2A amplification showed a trend bordering on statistical significance (OR 6.97, 95% CI 0.96-50.12, P = .054). TOP2A amplification and CEP17 duplication combined were strongly associated with pCR (OR 6.71, 95% CI 1.66-27.01, P = .007). HER2 amplification did not correlate with pCR. Our results suggest that CEP17 duplication predicts pCR to primary anthracycline-based chemotherapy. CEP17 duplication, TOP2A amplifications, and HER2 amplifications were not associated with prognosis.

Alisertib added to rituximab and vincristine is synthetic lethal and potentially curative in mice with aggressive DLBCL co-overexpressing MYC and BCL2.

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼ 10-20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼ 50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

COX-2 inhibitors arrest prostate cancer cell cycle progression by down-regulation of kinetochore/centromere proteins.

BACKGROUND: Previous studies have shown that COX-2 inhibitors inhibit cancer cell proliferation. However, the molecular mechanism remains elusive. METHODS: Prostate cancer LNCaP, 22Rv1, and PC3 cells were cultured and treated with the COX-2 inhibitors celecoxib and CAY10404. Knockdown of COX-2 in LNCaP cells was carried out using lentiviral vector-loaded COX-2 shRNA. Cell cycle progression and cell proliferation were analyzed by flow cytometry, microscopy, cell counting, and the MTT assay. The antagonists of EP1, EP2, EP3, and EP4 were used to examine the effects of the PGE2 signaling. The effect of COX-2 inhibitors and COX-2 knockdown on expression of the kinetochore/centromere genes and proteins was determined by RT-PCR and immunoblotting. RESULTS: Treatment with the COX-2 inhibitors celecoxib and CAY10404 or knockdown of COX-2 significantly inhibited prostate cancer cell proliferation. Flow-cytometric analysis and immunofluorescent staining confirmed the cell cycle arrested at the G2/M phase. Biochemical analysis showed that inhibition of COX-2 or suppression of COX-2 expression induced a dramatic down-regulation of key proteins in the kinetochore/centromere assembly, such as ZWINT, Cdc20, Ndc80, CENP-A, Bub1, and Plk1. Furthermore, the EP1 receptor antagonist SC51322, but not the EP2, EP3, and EP4 receptor antagonists, produced similar effects to the COX-2 inhibitors on cell proliferation and down-regulation of kinetochore/centromere proteins, suggesting that the effect of the COX-2 inhibition is through inactivation of the EP1 receptor signaling. CONCLUSIONS: Our studies indicate that inhibition of COX-2 can arrest prostate cancer cell cycle progression through inactivation of the EP1 receptor signaling and down-regulation of kinetochore/centromere proteins.

Cohesin removal precedes topoisomerase IIα-dependent decatenation at centromeres in male mammalian meiosis II.

Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.