Extract of bulbus of Fritillaria cirrhosa induces spindle multipolarity in human-derived colonic epithelial NCM460 cells through promoting centrosome fragmentation.

Bulbus of Fritillaria cirrhosa D. Don (BFC), an outstanding antitussive and expectorant herbal drug used in China and many other countries, has potential but less understood genotoxicity. Previously, we have reported that aqueous extract of BFC compromised the spindle assembly checkpoint and cytokinesis in NCM460 cells. Here, we found that one remarkable observation in BFC-treated NCM460 cells was multipolar mitosis, a trait classically compromises the fidelity of chromosome segregation. More detailed investigation revealed that BFC-induced spindle multipolarity in metaphases and ana-telophases in a dose- and time-dependent manner, suggesting BFC-induced multipolar spindle conformation was not transient. The frequency of multipolar metaphase correlated well to that of multipolar ana-telophases, indicating that BFC-induced multipolar metaphases often persisted through anaphase. Unexpectedly, BFC blocked the proliferation of binucleated cells, suggesting spindle multipolarity was not downstream of BFC-induced cytokinesis failure. Exposure of BFC to early mitotic cells, rather than S/G2 cells, contributed greatly to spindle multipolarity, indicating BFC might disrupt centrosome integrity rather than induce centrosome overduplication. The immunofluorescence results showed that the centrosomes were severely fragmented by a short-term treatment of BFC and the extent of centrosome fragmentation in early mitotic cells was larger than this in S/G2 cells. Consistently, several genes (e.g. p53, Rb centrin-2, Plk-4, Plk-1 and Aurora-A) involved in regulating centrosome integrity were significantly deregulated by BFC. Together, our results suggest that BFC causes multipolar spindles primarily by inducing centrosome fragmentation. Coupling these results to our previous observations, we recommend the risk/benefit ratio should be considered in the practical use of BFC.

The Yin and Yang of Autosomal Recessive Primary Microcephaly Genes: Insights from Neurogenesis and Carcinogenesis.

The stem cells of neurogenesis and carcinogenesis share many properties, including proliferative rate, an extensive replicative potential, the potential to generate different cell types of a given tissue, and an ability to independently migrate to a damaged area. This is also evidenced by the common molecular principles regulating key processes associated with cell division and apoptosis. Autosomal recessive primary microcephaly (MCPH) is a neurogenic mitotic disorder that is characterized by decreased brain size and mental retardation. Until now, a total of 25 genes have been identified that are known to be associated with MCPH. The inactivation (yin) of most MCPH genes leads to neurogenesis defects, while the upregulation (yang) of some MCPH genes is associated with different kinds of carcinogenesis. Here, we try to summarize the roles of MCPH genes in these two diseases and explore the underlying mechanisms, which will help us to explore new, attractive approaches to targeting tumor cells that are resistant to the current therapies.

Centrosomes: Til O-GlcNAc Do Us Apart.

The centrosome apparatus is vital for spindle assembly and chromosome segregation during mitotic divisions. Its replication, disjunction and separation have to be fine-tuned in space and time. A multitude of post-translational modifications (PTMs) have been implicated in centrosome modulation, including phosphorylation, ubiquitination and acetylation. Among them is the emerging O-linked N-acetylglucosamine (O-GlcNAc) modification. This quintessential PTM has a sole writer, O-GlcNAc transferase (OGT), and the only eraser, O-GlcNAcase (OGA). O-GlcNAc couples glucose metabolism with signal transduction and forms a yin-yang relationship with phosphorylation. Evidence from proteomic studies as well as single protein investigations has pinpointed a role of O-GlcNAc in centrosome number and separation, centriole number and distribution, as well as the cilia machinery emanating from the centrosomes. Herein we review our current understanding of the sweet modification embedded in centrosome dynamics and speculate that more molecular details will be unveiled in the future.

The Centrosome and the Primary Cilium: The Yin and Yang of a Hybrid Organelle.

Centrosomes and primary cilia are usually considered as distinct organelles, although both are assembled with the same evolutionary conserved, microtubule-based templates, the centrioles. Centrosomes serve as major microtubule- and actin cytoskeleton-organizing centers and are involved in a variety of intracellular processes, whereas primary cilia receive and transduce environmental signals to elicit cellular and organismal responses. Understanding the functional relationship between centrosomes and primary cilia is important because defects in both structures have been implicated in various diseases, including cancer. Here, we discuss evidence that the animal centrosome evolved, with the transition to complex multicellularity, as a hybrid organelle comprised of the two distinct, but intertwined, structural-functional modules: the centriole/primary cilium module and the pericentriolar material/centrosome module. The evolution of the former module may have been caused by the expanding cellular diversification and intercommunication, whereas that of the latter module may have been driven by the increasing complexity of mitosis and the requirement for maintaining cell polarity, individuation, and adhesion. Through its unique ability to serve both as a plasma membrane-associated primary cilium organizer and a juxtanuclear microtubule-organizing center, the animal centrosome has become an ideal integrator of extracellular and intracellular signals with the cytoskeleton and a switch between the non-cell autonomous and the cell-autonomous signaling modes. In light of this hypothesis, we discuss centrosome dynamics during cell proliferation, migration, and differentiation and propose a model of centrosome-driven microtubule assembly in mitotic and interphase cells. In addition, we outline the evolutionary benefits of the animal centrosome and highlight the hierarchy and modularity of the centrosome biogenesis networks.

Integration of RNAi and Small Molecule Screens to Identify Targets for Drug Development.

Cellular models for siRNA and small molecule high-throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a twofold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readout/multiple screening strategy.

Environmental stresses induce karyotypic instability in colorectal cancer cells.

Understanding how cells acquire genetic mutations is a fundamental biological question with implications for many different areas of biomedical research, ranging from tumor evolution to drug resistance. While karyotypic heterogeneity is a hallmark of cancer cells, few mutations causing chromosome instability have been identified in cancer genomes, suggesting a nongenetic origin of this phenomenon. We found that in vitro exposure of karyotypically stable human colorectal cancer cell lines to environmental stress conditions triggered a wide variety of chromosomal changes and karyotypic heterogeneity. At the molecular level, hyperthermia induced polyploidization by perturbing centrosome function, preventing chromosome segregation, and attenuating the spindle assembly checkpoint. The combination of these effects resulted in mitotic exit without chromosome segregation. Finally, heat-induced tetraploid cells were on the average more resistant to chemotherapeutic agents. Our studies suggest that environmental perturbations promote karyotypic heterogeneity and could contribute to the emergence of drug resistance.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition.

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.

PLK1 and YY1 interaction in follicular lymphoma is associated with unfavourable outcome.

AIMS: Ying Yang 1 (YY1) is a transcription factor involved in both proliferation and apoptosis. It is prognostic in follicular lymphoma (FL), increased protein levels being associated with favourable outcome. PLK1 is a critical regulator of mitosis, playing a role in spindle formation and in regulation of the G2/M cell cycle checkpoint. PLK1 phosphorylates YY1 at the G2/M checkpoint with activation of YY1 and resultant progression from G2 into mitosis. METHODS: This study aims to investigate possible molecular coexpression and interaction of YY1 with PLK1 in FL using Duolink II in situ proximity ligation assay (PLA) in 51 FL samples in a tissue microarray. RESULTS: Positive PLA signals were present at variable frequency and Kaplan-Meier analysis showed association of signal frequency above the median with unfavourable outcome (p=0.0270). PLA signals were localised to the nuclear edge, with only one signal per cell, suggesting PLK1 and YY1 coexpression at the centrosome. In a minority of cells, two very close PLA signals were present in a single cell, and occasionally, there was a strong ring of semi-confluent fluorescent PLA signals round the nucleus of non-dividing cells, while rarely events were observed in the cytoplasm surrounding dividing cells. CONCLUSIONS: The results confirm association of YY1 and PLK1 with outcome in FL and suggest coexpression at the centrosome. Given the reported interaction of YY1 with PLK1 at the centriole and promotion of cell division at the G2/M checkpoint, the results would concord with the known association of higher proliferation with poor outcome in FL.

Paclitaxel-dependent cell lines reveal a novel drug activity.

We previously described the isolation of Tax 18 and Tax 11-6, two paclitaxel-dependent cell lines that assemble low amounts of microtubule polymer and require the drug for cell division. In the present studies, fluorescence time-lapse microscopy was used to measure microtubule dynamic instability behavior in these cells. The mutations were found to cause small decreases in microtubule growth and shortening, but the changes seemed unable to explain the defects in microtubule polymer levels or cell division. Moreover, paclitaxel further suppressed microtubule dynamics at low drug concentrations that were insufficient to rescue the mutant phenotype. Wild-type (WT) cells treated with similar low drug concentrations also had highly suppressed microtubules, yet experienced no problems with cell division. Thus, the effects of paclitaxel on microtubule dynamics seemed to be unrelated to cell division in both WT and mutant cell lines. The higher drug concentrations needed to rescue the mutant phenotype instead inhibited the formation of unstable microtubule fragments that appeared at high frequency in the drug-dependent, but not WT, cell lines. Live cell imaging revealed that the fragments were generated by microtubule detachment from centrosomes, a process that was reversed by paclitaxel. We conclude that paclitaxel rescues mutant cell division by inhibiting the detachment of microtubule minus ends from centrosomes rather than by altering plus-end microtubule dynamics.

Pharmacological screening of bryophyte extracts that inhibit growth and induce abnormal phenotypes in human HeLa cancer cells.

Antitumor activities of substances from natural sources apart from vascular plants and micro-organisms have been poorly investigated. Here we report on a pharmacological screening of a bryophyte extract library using a phenotypic cell-based assay revealing microtubules, centrosomes and DNA. Among the 219 moss extracts tested, we identified 41 extracts acting on cell division with various combinations of significant effects on interphasic and mitotic cells. Seven extracts were further studied using a cell viability assay, cell cycle analysis and the phenotypic assay. Three distinct pharmacological patterns were identified including two unusual phenotypes.

The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation.

HMN-176 is a potential new cancer therapeutic known to retard the proliferation of tumor cell lines. Here, we show that this compound inhibits meiotic spindle assembly in surf clam oocytes and delays satisfaction of the spindle assembly checkpoint in human somatic cells by inducing the formation of short and/or multipolar spindles. HMN-176 does not affect centrosome assembly, nuclear envelope breakdown, or other aspects of meiotic or mitotic progression, nor does it affect the kinetics of Spisula or mammalian microtubule (MT) assembly in vitro. Notably, HMN-176 inhibits the formation of centrosome-nucleated MTs (i.e., asters) in Spisula oocytes and oocyte extracts, as well as from isolated Spisula or mammalian centrosomes in vitro. Together, these results reveal that HMN-176 is a first-in-class anticentrosome drug that inhibits proliferation, at least in part, by disrupting centrosome-mediated MT assembly during mitosis.

High-content screening and profiling of drug activity in an automated centrosome-duplication assay.

Maintenance of centrosome number is essential for cell-cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image-based centrosome-duplication assay that is accurate and robust enough for both careful cell-biology studies and high-throughput screening, and employ this assay in a series of chemical-genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform-specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320-member library of uncharacterized small molecules, we identified five potent centrosome-duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.

Heat shock induces centrosomal dysfunction, and causes non-apoptotic mitotic catastrophe in human tumour cells.

Normal human diploid cells and various human tumour cells were heat shocked at 43 degrees C for 2h and allowed to recover at 37 degrees C. It was found that heat shock treatment transiently disrupted the immunostaining of centrosomes, and no centrosome staining was detected in either normal or tumour cells 24h after heat shock. Staining recovered thereafter in normal cells, but in tumour cells abnormal centrosomes, multiple and minute centrosomes were induced. While normal cells were arrested in G1 and G2 after heat shock, significant numbers of mitotic cells with multiple poles appeared in tumour cells. Subsequently, cells with multiple micronuclei increased in tumour cells with time after heat shock. Although the nuclear morphology of these cells was similar to that of the apoptotic cells, no DNA ladder formation was observed up to 4 days after heat shock. Furthermore, an in situ assay failed to detect signals representative of apoptosis, indicating that apoptosis did not appear to be involved in heat shock-induced cell death of human tumour cells. Instead, cell lethality was associated with mitotic catastrophe.

Possible roles of protein kinase A in cell motility and excystation of the early diverging eukaryote Giardia lamblia.

Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28- kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC(50) of 3 microm, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta.

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

Characterization of microsome-associated tobacco BY-2 centrins.

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.

Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds.

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gammaTuRC is required for microtubule nucleation at the centrosome.

An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Identification of a novel Ca(2+)-regulated protein that is associated with the marginal band and centrosomes of chicken erythrocytes.

We have identified a novel Ca(2+)-regulated protein, p23, that is expressed specifically in avian erythrocyte and thrombocyte lineages. Sequence analysis of this 23 kDa protein reveals that it bears no homology to any known sequence. In mature definitive erythrocytes p23 exists in equilibrium between a soluble and a cytoskeletal bound pool. The cytoskeletal fraction is associated with the marginal band of microtubules, centrosomes and nuclear membrane under conditions of low free [Ca2+]. An increase in free [Ca2+] to 10(-6) M is sufficient to induce dissociation of > 95% of bound p23 from its target cytoskeletal binding sites, yet this [Ca2+] has little effect on calmodulin-mediated MB depolymerization. Analysis of p23 expression and localization during erythropoiesis together with results from heterologous p23 expression in tissue cultured cells demonstrated that this protein does not behave as a bone fide microtubule-associated protein. In addition, the developmental analysis revealed that although p23 is expressed early in definitive erythropoeisis, its association with the MB, centrosome and nuclear membrane occurs only in the final stages of differentiation. This cytoskeletal association correlates with marked p23 stabilization and accumulation at a time p23 expression is being markedly downregulated. We hypothesize that the mechanism of p23 association to the MB and centrosomes may be induced in part by a decrease in intracellular [Ca2+] during the terminal stages of definitive erythropoiesis.