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Sample records for 9-bromonoscapine-induced mitotic arrest

  1. Efficient Activation of Apoptotic Signaling during Mitotic Arrest with AK301

    PubMed Central

    Bleiler, Marina; Yeagley, Michelle; Wright, Dennis; Giardina, Charles

    2016-01-01

    Mitotic inhibitors are widely utilized chemotherapeutic agents that take advantage of mitotic defects in cancer cells. We have identified a novel class of piperazine-based mitotic inhibitors, of which AK301 is the most potent derivative identified to date (EC50 < 200 nM). Colon cancer cells arrested in mitosis with AK301 readily underwent a p53-dependent apoptosis following compound withdrawal and arrest release. This apoptotic response was significantly higher for AK301 than for other mitotic inhibitors tested (colchicine, vincristine, and BI 2536). AK301-treated cells exhibited a robust mitosis-associated DNA damage response, including ATM activation, γH2AX phosphorylation and p53 stabilization. The association between mitotic signaling and the DNA damage response was supported by the finding that Aurora B inhibition reduced the level of γH2AX staining. Confocal imaging of AK301-treated cells revealed multiple γ-tubulin microtubule organizing centers attached to microtubules, but with limited centrosome migration, raising the possibility that aberrant microtubule pulling may underlie DNA breakage. AK301 selectively targeted APC-mutant colonocytes and promoted TNF-induced apoptosis in p53-mutant colon cancer cells. Our findings indicate that AK301 induces a mitotic arrest state with a highly active DNA damage response. Together with a reversible arrest state, AK301 is a potent promoter of a mitosis-to-apoptosis transition that can target cancer cells with mitotic defects. PMID:27097159

  2. Pharicin A, a novel natural ent-kaurene diterpenoid, induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function.

    PubMed

    Xu, Han-Zhang; Huang, Ying; Wu, Ying-Li; Zhao, Yong; Xiao, Wei-Lie; Lin, Qi-Shan; Sun, Han-Dong; Dai, Wei; Chen, Guo-Qiang

    2010-07-15

    In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.

  3. Bcl-xL controls a switch between cell death modes during mitotic arrest

    PubMed Central

    Bah, N; Maillet, L; Ryan, J; Dubreil, S; Gautier, F; Letai, A; Juin, P; Barillé-Nion, S

    2014-01-01

    Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy. PMID:24922075

  4. Phosphorylation of Lte1 by Cdk prevents polarized growth during mitotic arrest in S. cerevisiae

    PubMed Central

    Spanos, Adonis; Jensen, Sanne; Sedgwick, Steven G.

    2010-01-01

    Lte1 is known as a regulator of mitotic progression in budding yeast. Here we demonstrate phosphorylation-dependent inhibition of polarized bud growth during G2/M by Lte1. Cla4 activity first localizes Lte1 to the polarity cap and thus specifically to the bud. This localization is a prerequisite for subsequent Clb–Cdk-dependent phosphorylation of Lte1 and its relocalization to the entire bud cortex. There, Lte1 interferes with activation of the small GTPases, Ras and Bud1. The inhibition of Bud1 prevents untimely polarization until mitosis is completed and Cdc14 phosphatase is released. Inhibition of Bud1 and Ras depends on Lte1’s GEF-like domain, which unexpectedly inhibits these small G proteins. Thus, Lte1 has dual functions for regulation of mitotic progression: it both induces mitotic exit and prevents polarized growth during mitotic arrest, thereby coupling cell cycle progression and morphological development. PMID:21149565

  5. Cell fate after mitotic arrest in different tumor cells is determined by the balance between slippage and apoptotic threshold

    SciTech Connect

    Galán-Malo, Patricia; Vela, Laura; Gonzalo, Oscar; Calvo-Sanjuán, Rubén; Gracia-Fleta, Lucía; Naval, Javier; Marzo, Isabel

    2012-02-01

    Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x{sub L} and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x{sub L} gene was silenced in A549 cells, vincristine induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x{sub L} or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x{sub L} switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x{sub L}/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types. Highlights: ► Vincristine induces cell death by apoptosis or mitotic catastrophe. ► Apoptosis-proficient cells die by apoptosis during mitosis upon vincristine treatment. ► p53wt apoptosis-deficient cells undergo apoptosis from a G1-like tetraploid state. ► p53mt apoptosis-deficient cells can survive and divide giving rise to 8N cells.

  6. Mitotic arrest-associated apoptosis induced by sodium arsenite in A375 melanoma cells is BUBR1-dependent

    SciTech Connect

    McNeely, Samuel C.; Taylor, B. Frazier; States, J. Christopher

    2008-08-15

    A375 human malignant melanoma cells undergo mitotic arrest-associated apoptosis when treated with pharmacological concentrations of sodium arsenite, a chemotherapeutic for acute promyelocytic leukemia. Our previous studies indicated that decreased arsenite sensitivity correlated with reduced mitotic spindle checkpoint function and reduced expression of the checkpoint protein BUBR1. In the current study, arsenite induced securin and cyclin B stabilization, BUBR1 phosphorylation, and spindle checkpoint activation. Arsenite also increased activating cyclin dependent kinase 1 (CDK1) Thr{sup 161} phosphorylation but decreased inhibitory Tyr15 phosphorylation. Mitotic arrest resulted in apoptosis as indicated by colocalization of mitotic phospho-Histone H3 with active caspase 3. Apoptosis was associated with BCL-2 Ser70 phosphorylation. Inhibition of CDK1 with roscovitine in arsenite-treated mitotic cells inhibited spindle checkpoint maintenance as inferred from reduced BUBR1 phosphorylation, reduced cyclin B expression, and diminution of mitotic index. Roscovitine also reduced BCL-2 Ser70 phosphorylation and protected against apoptosis, suggesting mitotic arrest caused by hyperactivation of CDK1 directly or indirectly leads to BCL-2 phosphorylation and apoptosis. In addition, suppression of BUBR1 with siRNA prevented arsenite-induced mitotic arrest and apoptosis. These findings provide insight into the mechanism of arsenic's chemotherapeutic action and indicate a functional spindle checkpoint may be required for arsenic-sensitivity.

  7. Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to Induce Mitotic Arrest in Tumor Cells.

    PubMed

    Qin, Tan; Chen, Fangjin; Zhuo, Xiaolong; Guo, Xiao; Yun, Taikangxiang; Liu, Ying; Zhang, Chuanmao; Lai, Luhua

    2016-08-11

    Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 μM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

  8. The SUMO protease SENP1 is required for cohesion maintenance and mitotic arrest following spindle poison treatment

    SciTech Connect

    Era, Saho; Abe, Takuya; Arakawa, Hiroshi; Kobayashi, Shunsuke; Szakal, Barnabas; Yoshikawa, Yusuke; Motegi, Akira; Takeda, Shunichi; Branzei, Dana

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer SENP1 knockout chicken DT40 cells are hypersensitive to spindle poisons. Black-Right-Pointing-Pointer Spindle poison treatment of SENP1{sup -/-} cells leads to increased mitotic slippage. Black-Right-Pointing-Pointer Mitotic slippage in SENP1{sup -/-} cells associates with apoptosis and endoreplication. Black-Right-Pointing-Pointer SENP1 counteracts sister chromatid separation during mitotic arrest. Black-Right-Pointing-Pointer Plk1-mediated cohesion down-regulation is involved in colcemid cytotoxicity. -- Abstract: SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1{sup -/-} cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2{alpha}{sup +/-} mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2{alpha} is SUMOylated during mitosis, the TOP2{alpha}{sup +/-} mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1{sup -/-} cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.

  9. Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

    PubMed

    Zhang, Zhong Rong; Al Zaharna, Mazen; Wong, Matthew Man-Kin; Chiu, Sung-Kay; Cheung, Hon-Yeung

    2013-01-01

    Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

  10. Involvement of p53 in cell death following cell cycle arrest and mitotic catastrophe induced by rotenone.

    PubMed

    Gonçalves, António Pedro; Máximo, Valdemar; Lima, Jorge; Singh, Keshav K; Soares, Paula; Videira, Arnaldo

    2011-03-01

    In order to investigate the cell death-inducing effects of rotenone, a plant extract commonly used as a mitochondrial complex I inhibitor, we studied cancer cell lines with different genetic backgrounds. Rotenone inhibits cell growth through the induction of cell death and cell cycle arrest, associated with the development of mitotic catastrophe. The cell death inducer staurosporine potentiates the inhibition of cell growth by rotenone in a dose-dependent synergistic manner. The tumor suppressor p53 is involved in rotenone-induced cell death, since the drug treatment results in increased expression, phosphorylation and nuclear localization of the protein. The evaluation of the effects of rotenone on a p53-deficient cell line revealed that although not required for the promotion of mitotic catastrophe, functional p53 appears to be essential for the extensive cell death that occurs afterwards. Our results suggest that mitotic slippage also occurs subsequently to the rotenone-induced mitotic arrest and cells treated with the drug for a longer period become senescent. Treatment of mtDNA-depleted cells with rotenone induces cell death and cell cycle arrest as in cells containing wild-type mtDNA, but not formation of reactive oxygen species. This suggests that the effects of rotenone are not dependent from the production of reactive oxygen species. This work highlights the multiple effects of rotenone in cancer cells related to its action as an anti-mitotic drug.

  11. The apoptotic mechanisms of MT-6, a mitotic arrest inducer, in human ovarian cancer cells.

    PubMed

    Chen, Mei-Chuan; Kuo, Yi-Chiu; Hsu, Chia-Ming; Chen, Yi-Lin; Shen, Chien-Chang; Teng, Che-Ming; Pan, Shiow-Lin

    2017-04-07

    Patients with ovarian cancer are typically diagnosed at an advanced stage, resulting in poor prognosis since there are currently no effective early-detection screening tests for women at average-risk for ovarian cancer. Here, we investigated the effects of MT-6, a derivative of moscatilin, in ovarian cancer cells. Our investigation showed that MT-6 inhibited the proliferation and viability of ovarian cancer cells with submicromolar IC50 values. MT-6-treated SKOV3 cells showed significant cell cycle arrest at G2/M phase, followed by an increase in the proportion of cells in a sub-G1 phase. In addition, MT-6 induced a concentration-dependent increase in mitotic markers, mitotic kinases, cell cycle regulators of G2/M transition, and apoptosis-related markers in ovarian cancer cells. MT-6 treatment also induced mitochondrial membrane potential loss, JNK activation, and DR5 expression. Cotreatment of cells with the JNK inhibitor SP600125 considerably attenuated MT-6-induced apoptosis, mitochondria membrane potential loss, DR5 upregulation, and suppression of cell viability. MT-6 also inhibited tumor growth in an SKOV3 xenograft model without significant body weight loss. Together, our findings suggest that MT-6 is a potent anticancer agent with tumor-suppressive activity in vitro and in vivo that could be further investigated for ovarian cancer therapy in the future.

  12. The apoptotic mechanisms of MT-6, a mitotic arrest inducer, in human ovarian cancer cells

    PubMed Central

    Chen, Mei-Chuan; Kuo, Yi-Chiu; Hsu, Chia-Ming; Chen, Yi-Lin; Shen, Chien-Chang; Teng, Che-Ming; Pan, Shiow-Lin

    2017-01-01

    Patients with ovarian cancer are typically diagnosed at an advanced stage, resulting in poor prognosis since there are currently no effective early-detection screening tests for women at average-risk for ovarian cancer. Here, we investigated the effects of MT-6, a derivative of moscatilin, in ovarian cancer cells. Our investigation showed that MT-6 inhibited the proliferation and viability of ovarian cancer cells with submicromolar IC50 values. MT-6–treated SKOV3 cells showed significant cell cycle arrest at G2/M phase, followed by an increase in the proportion of cells in a sub-G1 phase. In addition, MT-6 induced a concentration-dependent increase in mitotic markers, mitotic kinases, cell cycle regulators of G2/M transition, and apoptosis-related markers in ovarian cancer cells. MT-6 treatment also induced mitochondrial membrane potential loss, JNK activation, and DR5 expression. Cotreatment of cells with the JNK inhibitor SP600125 considerably attenuated MT-6–induced apoptosis, mitochondria membrane potential loss, DR5 upregulation, and suppression of cell viability. MT-6 also inhibited tumor growth in an SKOV3 xenograft model without significant body weight loss. Together, our findings suggest that MT-6 is a potent anticancer agent with tumor-suppressive activity in vitro and in vivo that could be further investigated for ovarian cancer therapy in the future. PMID:28387244

  13. A Novel Resveratrol Based Tubulin Inhibitor Induces Mitotic Arrest and Activates Apoptosis in Cancer Cells

    PubMed Central

    Thomas, Elizabeth; Gopalakrishnan, Vidya; Hegde, Mahesh; Kumar, Sujeet; Karki, Subhas S.; Raghavan, Sathees C.; Choudhary, Bibha

    2016-01-01

    Resveratrol is one of the most widely studied bioactive plant polyphenols which possesses anticancer properties. Previously we have reported synthesis, characterization and identification of a novel resveratrol analog, SS28. In the present study, we show that SS28 induced cytotoxicity in several cancer cell lines ex vivo with an IC50 value of 3–5 μM. Mechanistic evaluation of effect of SS28 in non-small cell lung cancer cell line (A549) and T-cell leukemic cell line (CEM) showed that it inhibited Tubulin polymerization during cell division to cause cell cycle arrest at G2/M phase of the cell cycle at 12–18 h time period. Immunofluorescence studies confirmed the mitotic arrest upon treatment with SS28. Besides, we show that SS28 binds to Tubulin with a dissociation constant of 0.414 ± 0.11 μM. Further, SS28 treatment resulted in loss of mitochondrial membrane potential, activation of Caspase 9 and Caspase 3, leading to PARP-1 cleavage and finally cell death via intrinsic pathway of apoptosis. Importantly, treatment with SS28 resulted in regression of tumor in mice. Hence, our study reveals the antiproliferative activity of SS28 by disrupting microtubule dynamics by binding to its cellular target Tubulin and its potential to be developed as an anticancer molecule. PMID:27748367

  14. Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest

    PubMed Central

    Dikovskaya, Dina; Cole, John J.; Mason, Susan M.; Nixon, Colin; Karim, Saadia A.; McGarry, Lynn; Clark, William; Hewitt, Rachael N.; Sammons, Morgan A.; Zhu, Jiajun; Athineos, Dimitris; Leach, Joshua D.G.; Marchesi, Francesco; van Tuyn, John; Tait, Stephen W.; Brock, Claire; Morton, Jennifer P.; Wu, Hong; Berger, Shelley L.; Blyth, Karen; Adams, Peter D.

    2015-01-01

    Summary Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells. PMID:26299965

  15. Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

    PubMed

    Dikovskaya, Dina; Cole, John J; Mason, Susan M; Nixon, Colin; Karim, Saadia A; McGarry, Lynn; Clark, William; Hewitt, Rachael N; Sammons, Morgan A; Zhu, Jiajun; Athineos, Dimitris; Leach, Joshua D G; Marchesi, Francesco; van Tuyn, John; Tait, Stephen W; Brock, Claire; Morton, Jennifer P; Wu, Hong; Berger, Shelley L; Blyth, Karen; Adams, Peter D

    2015-09-01

    Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

  16. Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors

    PubMed Central

    Salum, Lívia B.; Altei, Wanessa F.; Chiaradia, Louise D.; Cordeiro, Marlon N.S.; Canevarolo, Rafael R.; Melo, Carolina P.S.; Winter, Evelyn; Mattei, Bruno; Daghestani, Hikmat N.; Santos-Silva, Maria Cláudia; Creczynski-Pasa, Tânia B.; Yunes, Rosendo A.; Yunes, José A.; Andricopulo, Adriano D.; Day, Billy W.; Nunes, Ricardo J.; Vogt, Andreas

    2013-01-01

    Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays. PMID:23524161

  17. Anthelmintic drug albendazole arrests human gastric cancer cells at the mitotic phase and induces apoptosis

    PubMed Central

    Zhang, Xuan; Zhao, Jing; Gao, Xiangyang; Pei, Dongsheng; Gao, Chao

    2017-01-01

    As microtubules have a vital function in the cell cycle, oncologists have developed microtubule inhibitors capable of preventing uncontrolled cell division, as in the case of cancer. The anthelmintic drug albendazole (ABZ) has been demonstrated to inhibit hepatocellular, ovarian and prostate cancer cells via microtubule targeting. However, its activity against human gastric cancer (GC) cells has remained to be determined. In the present study, ABZ was used to treat GC cells (MKN-45, SGC-7901 and MKN-28). A a CCK-8 cell proliferation assay was performed to assess the effects of ABZ on cell viability and cell cycle changes were assessed using flow cytometry. SGC-7901 cells were selected for further study, and flow cytometry was employed to determine the apoptotic rate, immunofluorescence analysis was employed to show changes of the microtubule structure as well as the subcellular localization and expression levels of cyclin B1, and western blot analysis was used to identify the dynamics of microtubule assembly. The expression levels of relevant proteins, including cyclin B1 and Cdc2, the two subunits of mitosis-promoting factor as well as apoptosis-asociated proteins were also assessed by western blot analysis. The results showed that ABZ exerted its anti-cancer activity in GC cell lines by disrupting microtubule formation and function to cause mitotic arrest, which is also associated with the accumulation of cyclin B1, and consequently induces apoptosis. PMID:28352336

  18. Effect of caffeine on radiation-induced mitotic delay: delayed expression of G/sub 2/ arrest

    SciTech Connect

    Rowley, R.; Zorch, M.; Leeper, D.B.

    1984-01-01

    In the presence of 5 mM caffeine, irradiated (1.5 Gy) S and G/sub 2/ cells progressed to mitosis in register and without arrest in G/sub 2/. Caffeine (5 mM) markedly reduced mitotic delay even after radiation doses up to 20 Gy. When caffeine was removed from irradiated (1.5 Gy) and caffeine-treated cells, a period of G/sub 2/ arrest followed, similar in length to that produced by radiation alone. The arrest expressed was independent of the duration of the caffeine treatment for exposures up to 3 hr. The similarity of the response to the cited effects of caffeine on S-phase delay suggests a common basis for delay induction in S and G/sub 2/ phases.

  19. Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo[S

    PubMed Central

    Varshney, Salil; Shankar, Kripa; Beg, Muheeb; Balaramnavar, Vishal M.; Mishra, Sunil Kumar; Jagdale, Pankaj; Srivastava, Shishir; Chhonker, Yashpal S.; Lakshmi, Vijai; Chaudhari, Bhushan P.; Bhatta, Rabi Shankar; Saxena, Anil Kumar; Gaikwad, Anil Nilkanth

    2014-01-01

    We developed a common feature pharmacophore model using known antiadipogenic compounds (CFPMA). We identified rohitukine, a reported chromone anticancer alkaloid as a potential hit through in silico mapping of the in-house natural product library on CFPMA. Studies were designed to assess the antiadipogenic potential of rohitukine. Rohitukine was isolated from Dysoxylum binacteriferum Hook. to ⬧95% purity. As predicted by CFPMA, rohitukine was indeed found to be an antiadipogenic molecule. Rohitukine inhibited lipid accumulation and adipogenic differentiation in a concentration- and exposure-time-dependent manner in 3T3-L1 and C3H10T1/2 cells. Rohitukine downregulated expression of PPARγ, CCAAT/enhancer binding protein α, adipocyte protein 2 (aP2), FAS, and glucose transporter 4. It also suppressed mRNA expression of LPL, sterol-regulatory element binding protein (SREBP) 1c, FAS, and aP2, the downstream targets of PPARγ. Rohitukine arrests cells in S phase during mitotic clonal expansion. Rohitukine was bioavailable, and 25.7% of orally administered compound reached systemic circulation. We evaluated the effect of rohitukine on dyslipidemia induced by high-fat diet in the hamster model. Rohitukine increased hepatic expression of liver X receptor α and decreased expression of SREBP-2 and associated targets. Rohitukine decreased hepatic and gonadal lipid accumulation and ameliorated dyslipidemia significantly. In summary, our strategy to identify a novel antiadipogenic molecule using CFPMA successfully resulted in identification of rohitukine, which confirmed antiadipogenic activity and also exhibited in vivo antidyslipidemic activity. PMID:24646949

  20. Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells

    SciTech Connect

    AbuBakar, S.; Au, W.W.; Legator, M.S.; Albrecht, T.

    1988-01-01

    Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.

  1. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium.

    PubMed

    Scaife, Robin M

    2004-10-01

    Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.

  2. Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype.

    PubMed

    Mosieniak, Grażyna; Sliwinska, Małgorzata A; Przybylska, Dorota; Grabowska, Wioleta; Sunderland, Piotr; Bielak-Zmijewska, Anna; Sikora, Ewa

    2016-05-01

    Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin.

  3. A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe.

    PubMed

    Martinez-Castillo, Macario; Bonilla-Moreno, Raul; Aleman-Lazarini, Leticia; Meraz-Rios, Marco Antonio; Orozco, Lorena; Cedillo-Barron, Leticia; Cordova, Emilio J; Villegas-Sepulveda, Nicolas

    2016-01-01

    Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562), were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB) occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562), which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP.

  4. A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    PubMed Central

    Martinez-Castillo, Macario; Bonilla-Moreno, Raul; Aleman-Lazarini, Leticia; Meraz-Rios, Marco Antonio; Orozco, Lorena; Cedillo-Barron, Leticia; Cordova, Emilio J.

    2016-01-01

    Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562), were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB) occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562), which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP. PMID:27832139

  5. Paclitaxel sensitivity of breast cancer cells requires efficient mitotic arrest and disruption of Bcl-xL/Bak interaction.

    PubMed

    Flores, M Luz; Castilla, Carolina; Ávila, Rainiero; Ruiz-Borrego, Manuel; Sáez, Carmen; Japón, Miguel A

    2012-06-01

    Taxanes are being used for the treatment of breast cancer. However, cancer cells frequently develop resistance to these drugs with the subsequent recurrence of the tumor. MDA-MB-231 and T-47D breast cancer cell lines were used to assess the effect of paclitaxel treatment on apoptosis and cell cycle, the possible mechanisms of paclitaxel resistance as well as the enhancement of paclitaxel-induced apoptosis based on its combination with phenylethyl isothiocyanate (PEITC). T-47D cells undergo apoptosis in response to paclitaxel treatment. The induction of apoptosis was associated with a robust mitotic arrest and the disruption of Bcl-xL/Bak interaction. By contrary, MDA-MB-231 cells were insensitive to paclitaxel-induced apoptosis and this was associated with a high percentage of cells that slip out of paclitaxel-imposed mitotic arrest and also with the maintenance of Bcl-xL/Bak interaction. The sequential treatment of MDA-MB-231 cells with PEITC followed by paclitaxel inhibited the slippage induced by paclitaxel and increased the apoptosis induction achieved with any of the drugs alone. In breast cancer tissues, high Bcl-xL expression was correlated with a shorter time of disease-free survival in patients treated with a chemotherapeutic regimen that contains paclitaxel, in a statistically significant way. Thus, resistance to paclitaxel in MDA-MB-231 cells is related to the inability to disrupt the Bcl-xL/Bak interaction and increased slippage. In this context, the combination of a drug that induces a strong mitotic arrest, such as paclitaxel, with another that inhibits slippage, such as PEITC, translates into increased apoptotic induction.

  6. Sensitivity to sodium arsenite in human melanoma cells depends upon susceptibility to arsenite-induced mitotic arrest

    SciTech Connect

    McNeely, Samuel C.; Belshoff, Alex C.; Taylor, B. Frazier; Fan, Teresa W-M.; McCabe, Michael J.; Pinhas, Allan R.

    2008-06-01

    Arsenic induces clinical remission in patients with acute promyelocytic leukemia and has potential for treatment of other cancers. The current study examines factors influencing sensitivity to arsenic using human malignant melanoma cell lines. A375 and SK-Mel-2 cells were sensitive to clinically achievable concentrations of arsenite, whereas SK-Mel-3 and SK-Mel-28 cells required supratherapeutic levels for toxicity. Inhibition of glutathione synthesis, glutathione S-transferase (GST) activity, and multidrug resistance protein (MRP) transporter function attenuated arsenite resistance, consistent with studies suggesting that arsenite is extruded from the cell as a glutathione conjugate by MRP-1. However, MRP-1 was not overexpressed in resistant lines and GST-{pi} was only slightly elevated. ICP-MS analysis indicated that arsenite-resistant SK-Mel-28 cells did not accumulate less arsenic than arsenite-sensitive A375 cells, suggesting that resistance was not attributable to reduced arsenic accumulation but rather to intrinsic properties of resistant cell lines. The mode of arsenite-induced cell death was apoptosis. Arsenite-induced apoptosis is associated with cell cycle alterations. Cell cycle analysis revealed arsenite-sensitive cells arrested in mitosis whereas arsenite-resistant cells did not, suggesting that induction of mitotic arrest occurs at lower intracellular arsenic concentrations. Higher intracellular arsenic levels induced cell cycle arrest in the S-phase and G{sub 2}-phase in SK-Mel-3 and SK-Mel-28 cells, respectively. The lack of arsenite-induced mitotic arrest in resistant cell lines was associated with a weakened spindle checkpoint resulting from reduced expression of spindle checkpoint protein BUBR1. These data suggest that arsenite has potential for treatment of solid tumors but a functional spindle checkpoint is a prerequisite for a positive response to its clinical application.

  7. Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

    SciTech Connect

    Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

    2012-06-01

    Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

  8. Combination of {gamma}-radiation antagonizes the cytotoxic effects of vincristine and vinblastine on both mitotic arrest and apoptosis

    SciTech Connect

    Sui, Meihua; Fan Weimin . E-mail: fanw@musc.edu

    2005-03-15

    Purpose: Combination therapy with different modalities is a common practice in the treatment of cancer. The promising clinical profile of vincristine and vinblastine has promoted considerable interest in combining these vinca alkaloids with radiation therapy to treat a variety of solid tumors. However, the therapeutic efficacy and the interaction between the vinca alkaloids with radiation is not entirely clear. In this study, we assessed the potential interactions in the combination of vincristine or vinblastine with {gamma}-radiation against human tumor cells in vitro. Methods and materials: Vincristine or vinblastine and {gamma}-radiation were administrated at three different sequences designed as preradiated, coradiated, and postradiated combinations in human breast cancer cells and human epidermoid carcinoma cells. The cytotoxic interactions and mutual influences between these two modalities were analyzed by a series of assays including cytotoxic, morphologic, and biochemical examinations. Results: Our results showed that the combination of these two modalities did not produce any synergistic or additive effects. Instead, the clonogenic assays showed the survival rates of these combinations were increased up to 2.17-fold and 2.7-fold, respectively, of those treated with vincristine or vinblastine alone (p < 0.01). DNA fragmentation, T{alpha}T-mediated dUTP nick end labeling (TUNEL) assay, and flow cytometric assays also showed that the combination of {gamma}-radiation significantly interfered with the ability of these vinca alkaloids to induce apoptosis. Further analyses indicated that addition of {gamma}-radiation resulted in cell cycle arrest at the G{sub 2} phase, which subsequently prevented the mitotic arrest induced by vincristine or vinblastine. In addition, biochemical examinations revealed that {gamma}-radiation regulated p34{sup cdc2}/cyclin B1 and survivin, and inhibited I{kappa}B{alpha} degradation and bcl-2 phosphorylation. Conclusions: These

  9. Two p90 ribosomal S6 kinase isoforms are involved in the regulation of mitotic and meiotic arrest in Artemia.

    PubMed

    Duan, Ru-Bing; Zhang, Li; Chen, Dian-Fu; Yang, Fan; Yang, Jin-Shu; Yang, Wei-Jun

    2014-06-06

    There are multiple isoforms of p90 ribosomal S6 kinase (RSK), which regulate diverse cellular functions such as cell growth, proliferation, maturation, and motility. However, the relationship between the structures and functions of RSK isoforms remains undetermined. Artemia is a useful model in which to study cell cycle arrest because these animals undergo prolonged diapauses, a state of obligate dormancy. A novel RSK isoform was identified in Artemia, which was termed Ar-Rsk2. This isoform was compared with an RSK isoform that we previously identified in Artemia, termed Ar-Rsk1. Ar-Rsk2 has an ERK-docking motif, whereas Ar-Rsk1 does not. Western blot analysis revealed that Ar-Rsk1 was activated by phosphorylation, which blocked meiosis in oocytes. Knockdown of Ar-Rsk1 reduced the level of phosphorylated cdc2 and thereby suppressed cytostatic factor activity. This indicates that Ar-Rsk1 regulates the cytostatic factor in meiosis. Expression of Ar-Rsk2 was down-regulated in Artemia cysts in which mitosis was arrested. Knockdown of Ar-Rsk2 resulted in decreased levels of cyclin D3 and phosphorylated histone H3, and the production of pseudo-diapause cysts. This indicates that Ar-Rsk2 regulates mitotic arrest. PLK and ERK RNAi showed that Ar-Rsk2, but not Ar-Rsk1, could be activated by PLK-ERK in Artemia. This is the first study to report that RSK isoforms with and without an ERK-docking motif regulate mitosis and meiosis, respectively. This study provides insight into the relationship between the structures and functions of RSK isoforms.

  10. Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression

    PubMed Central

    1996-01-01

    The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H- ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest. PMID:8666924

  11. Akt activation suppresses Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence.

    PubMed

    Hirose, Yuchi; Katayama, Makoto; Mirzoeva, Olga K; Berger, Mitchel S; Pieper, Russell O

    2005-06-01

    Pharmacologic inhibition of the DNA signal transducers Chk1 and p38 blocks G2 arrest and sensitizes glioblastoma cells to chemotherapeutic methylating agent-induced cytotoxicity. Because Akt pathway activation has been suggested to also block G2 arrest induced by DNA-damaging agents and because glioma cells frequently have high levels of Akt activation, we examined the contribution of the Akt pathway to methylating agent-induced G2 arrest and toxicity. U87MG human glioma cells containing an inducible Akt expression construct were incubated with inducing agent or vehicle, after which the cells were exposed to temozolomide and assayed for activation of the components of the G2 arrest pathway and survival. Temozolomide-treated control cells activated the DNA damage signal transducers Chk1, Chk2, and p38, leading to Cdc25C and Cdc2 inactivation, prolonged G2 arrest, and loss of clonagenicity by a combination of senescence and mitotic catastrophe. Temozolomide-treated cells induced to overexpress Akt, however, exhibited significantly less drug-induced Cdc25C/Cdc2 inactivation and less G2 arrest. Akt-mediated suppression of G2 arrest was associated not with alterations in Chk1 or p38 activation but rather with suppression of Chk2 activation and reduced recruitment of Chk2 to sites of damage in chromatin. Unlike bypass of the G2 checkpoint induced by pharmacologic inhibitors of Chk1 or p38, however, Akt-induced bypass of G2 arrest suppressed, rather than enhanced, temozolomide-induced senescence and mitotic catastrophe. These results show that whereas Akt activation suppresses temozolomide-induced Chk2 activation and G2 arrest, the overriding effect is protection from temozolomide-induced cytotoxicity. The Akt pathway therefore represents a new target for the sensitization of gliomas to chemotherapeutic methylating agents such as temozolomide.

  12. Structurally simplified biphenyl combretastatin A4 derivatives retain in vitro anti-cancer activity dependent on mitotic arrest

    PubMed Central

    Tarade, Daniel; Ma, Dennis; Pignanelli, Christopher; Mansour, Fadi; Simard, Daniel; van den Berg, Sean; Gauld, James; McNulty, James; Pandey, Siyaram

    2017-01-01

    The cis-stilbene, combretastatin A4 (CA4), is a potent microtubule targeting and vascular damaging agent. Despite promising results at the pre-clinical level and extensive clinical evaluation, CA4 has yet to be approved for therapeutic use. One impediment to the development of CA4 is an inherent conformational instability about the ethylene linker, which joins two aromatic rings. We have previously published preliminary data regarding structurally simplified biphenyl derivatives of CA4, lacking an ethylene linker, which retain anti-proliferative and pro-apoptotic activity, albeit at higher doses. Our current study provides a more comprehensive evaluation regarding the anti-proliferative and pro-apoptotic properties of biphenyl CA4 derivatives in both 2D and 3D cancerous and non-cancerous cell models. Computational analysis has revealed that cytotoxicity of CA4 and biphenyl analogues correlates with predicted tubulin affinity. Additional mechanistic evaluation of the biphenyl derivatives found that their anti-cancer activity is dependent on prolonged mitotic arrest, in a similar manner to CA4. Lastly, we have shown that cancer cells deficient in the extrinsic pathway of apoptosis experience delayed cell death following treatment with CA4 or analogues. Biphenyl derivatives of CA4 represent structurally simplified analogues of CA4, which retain a similar mechanism of action. The biphenyl analogues warrant in vivo examination to evaluate their potential as vascular damaging agents. PMID:28253265

  13. The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells.

    PubMed

    Li, S; Szymborski, A; Miron, M-J; Marcellus, R; Binda, O; Lavoie, J N; Branton, P E

    2009-01-22

    The human adenovirus E4orf4 protein, when expressed alone, induces p53-independent death in a wide range of cancer cells. Earlier studies by our groups suggested that although in some cases cell death can be associated with some hallmarks of apoptosis, it is not always affected by caspase inhibitors. Thus it is unlikely that E4orf4-induced cell death occurs uniquely through apoptosis. In the present studies using H1299 human lung carcinoma cells as a model system we found that death is induced in the absence of activation of any of the caspases tested, accumulation of reactive oxygen species, or release of cytochrome c from mitochondria. E4orf4 caused a substantial change in cell morphology, including vigorous membrane blebbing, multiple nuclei in many cells and increased cell volume. Most of these characteristics are not typical of apoptosis, but they are of necrosis. FACS analysis and western blotting for cell cycle markers showed that E4orf4-expressing cells became arrested in G(2)/M and also accumulated high levels of cyclin E. The presence of significant numbers of tetraploid and polyploid cells and some cells with micronuclei suggested that E4orf4 appears to induce death in these cells through a process resulting from mitotic catastrophe.

  14. Airborne urban particles (Milan winter-PM2.5) cause mitotic arrest and cell death: Effects on DNA, mitochondria, AhR binding and spindle organization.

    PubMed

    Gualtieri, Maurizio; Ovrevik, Johan; Mollerup, Steen; Asare, Nana; Longhin, Eleonora; Dahlman, Hans-Jørgen; Camatini, Marina; Holme, Jørn A

    2011-08-01

    Airborne particulate matter (PM) is considered to be an important contributor to lung diseases. In the present study we report that Milan winter-PM2.5 inhibited proliferation in human bronchial epithelial cells (BEAS-2B) by inducing mitotic arrest. The cell cycle arrest was followed by an increase in mitotic-apoptotic cells, mitotic slippage and finally an increase in "classical" apoptotic cells. Exposure to winter-PM10 induced only a slight effect which may be due to the presence of PM2.5 in this fraction while pure combustion particles failed to disturb mitosis. Fewer cells expressing the mitosis marker phospho-histone H3 compared to cells with condensed chromosomes, suggest that PM2.5 induced premature mitosis. PM2.5 was internalized into the cells and often localized in laminar organelles, although particles without apparent plasma membrane covering were also seen. In PM-containing cells mitochondria and lysosomes were often damaged, and in mitotic cells fragmented chromosomes often appeared. PM2.5 induced DNA strands breaks and triggered a DNA-damage response characterized by increased phosphorylation of ATM, Chk2 and H2AX; as well as induced a marked increase in expression of the aryl hydrocarbon receptor (AhR)-regulated genes, CYP1A1, CYP1B1 and AhRR. Furthermore, some disturbance of the organization of microtubules was indicated. It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.

  15. Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

    PubMed

    Chen, Fangjin; Zhuo, Xiaolong; Qin, Tan; Guo, Xiao; Zhang, Chuanmao; Lai, Luhua

    2016-11-24

    Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

  16. 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration

    PubMed Central

    Benner, Christopher; Dowdy, Steven F.; Desai, Arshad; Shiau, Andrew K.

    2016-01-01

    In normal human cells, centrosome loss induced by centrinone—a specific centrosome duplication inhibitor—leads to irreversible, p53-dependent G1 arrest by an unknown mechanism. A genome-wide CRISPR/Cas9 screen for centrinone resistance identified genes encoding the p53-binding protein 53BP1, the deubiquitinase USP28, and the ubiquitin ligase TRIM37. Deletion of TP53BP1, USP28, or TRIM37 prevented p53 elevation in response to centrosome loss but did not affect cytokinesis failure–induced arrest or p53 elevation after doxorubicin-induced DNA damage. Deletion of TP53BP1 and USP28, but not TRIM37, prevented growth arrest in response to prolonged mitotic duration. TRIM37 knockout cells formed ectopic centrosomal-component foci that suppressed mitotic defects associated with centrosome loss. TP53BP1 and USP28 knockouts exhibited compromised proliferation after centrosome removal, suggesting that centrosome-independent proliferation is not conferred solely by the inability to sense centrosome loss. Thus, analysis of centrinone resistance identified a 53BP1-USP28 module as critical for communicating mitotic challenges to the p53 circuit and TRIM37 as an enforcer of the singularity of centrosome assembly. PMID:27432897

  17. MAD2γ, a novel MAD2 isoform, reduces mitotic arrest and is associated with resistance in testicular germ cell tumors

    PubMed Central

    López-Saavedra, Alejandro; Ramírez-Otero, Miguel; Díaz-Chávez, José; Cáceres-Gutiérrez, Rodrigo; Justo-Garrido, Monserrat; Andonegui, Marco A.; Mendoza, Julia; Downie-Ruíz, Ángela; Cortés-González, Carlo; Reynoso, Nancy; Castro-Hernández, Clementina; Domínguez-Gómez, Guadalupe; Santibáñez, Miguel; Fabián-Morales, Eunice; Pruefer, Franz; Luna-Maldonado, Fernando; González-Barrios, Rodrigo; Herrera, Luis A.

    2016-01-01

    ABSTRACT Background: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated. Methods: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index. Results: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells. Conclusions: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs. PMID:27315568

  18. Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma.

    PubMed

    Havas, Aaron P; Rodrigues, Kameron B; Bhakta, Anvi; Demirjian, Joseph A; Hahn, Seongmin; Tran, Jack; Scavello, Margarethakay; Tula-Sanchez, Ana A; Zeng, Yi; Schmelz, Monika; Smith, Catharine L

    2016-12-01

    Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

  19. Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells

    PubMed Central

    1992-01-01

    Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2- related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest. PMID:1532584

  20. The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest.

    PubMed

    Llopis, Alba; Salvador, Noelia; Ercilla, Amaia; Guaita-Esteruelas, Sandra; Barrantes, Ivan del Barco; Gupta, Jalaj; Gaestel, Matthias; Davis, Roger J; Nebreda, Angel R; Agell, Neus

    2012-10-01

    Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.

  1. Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified

    PubMed Central

    Hart, Katherine; Kothari, Anisha; Mackintosh, Samuel G.; Kovak, Matthew R.; Chambers, Timothy C.

    2016-01-01

    Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model. PMID:27738316

  2. Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells

    PubMed Central

    1995-01-01

    Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re- initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events. PMID:7790366

  3. New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer

    PubMed Central

    La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Verrico, Annalisa; Miele, Andrea; Monti, Ludovica; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Ricci, Biancamaria; Soriani, Alessandra; Santoni, Angela; Caraglia, Michele; Porto, Stefania; Pozzo, Eleonora Da; Martini, Claudia; Brancale, Andrea; Marinelli, Luciana; Novellino, Ettore; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Bigogno, Chiara; Dondio, Giulio; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

    2015-01-01

    We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethox-yphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4–7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20–50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer. PMID:26132075

  4. Mitotic arrest of breast cancer MDA-MB-231 cells by a halogenated thieno[3,2-d]pyrimidine.

    PubMed

    Ross, Christina R; Temburnikar, Kartik W; Wilson, Gerald M; Seley-Radtke, Katherine L

    2015-04-15

    Halogenated thieno[3,2-d]pyrimidines exhibit antiproliferative activity against a variety of cancer cell models, such as the mouse lymphocytic leukemia cell line L1210 in which they induce apoptosis independent of cell cycle arrest. Here we assessed these activities on MDA-MB-231 cells, a well-established model of aggressive, metastatic breast cancer. While 2,4-dichloro[3,2-d]pyrimidine was less toxic to MDA-MB-231 cells than previously observed in the L1210 model, flow cytometry analysis showed that MDA-MB-231 cell death involved arrest at the G2/M stage of the cell cycle. Conversely, the introduction of bromine at C7 of the 2,4-dichloro[3,2-d]pyrimidine eliminated cell type-dependent differences in cytotoxicity or cell cycle status. Together, these data indicate that a substituent at C7 can profoundly modify the cytotoxic mechanism of halogenated thieno[3,2-d]pyrimidines in a cell type-specific manner.

  5. 2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

    SciTech Connect

    Hsu, Mei-Hua; Liu, Chin-Yu; Lin, Chiao-Min; Chen, Yen-Jung; Chen, Chun-Jen; Lin, Yu-Fu; Huang, Li-Jiau; Lee, Kuo-Hsiung; Kuo, Sheng-Chu

    2012-03-01

    2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe. Highlights: ► HKL-1 is a potential antimitotic agent against HL-60 cells. ► HKL-1 induces spindle disruption and sustained resulted in mitotic catastrophe. ► CENP-E and aurora B protein expressions significantly reduced. ► Bcl-2 family protein expressions altered and caspase-9/-3 activation. ► HKL-1 is an attractive candidate for possible use as a novel antimitotic agent.

  6. Structure–Biological Function Relationship Extended to Mitotic Arrest-Deficient 2-Like Protein Mad2 Native and Mutants-New Opportunity for Genetic Disorder Control

    PubMed Central

    Avram, Speranta; Milac, Adina; Mernea, Maria; Mihailescu, Dan; Putz, Mihai V.; Buiu, Catalin

    2014-01-01

    Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53–0.65) and fitted correlation r2 (0.82–0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN. PMID:25411801

  7. A novel microtubule inhibitor, MT3-037, causes cancer cell apoptosis by inducing mitotic arrest and interfering with microtubule dynamics

    PubMed Central

    Chang, Ling-Chu; Yu, Yung-Luen; Hsieh, Min-Tsang; Wang, Sheng-Hung; Chou, Ruey-Hwang; Huang, Wei-Chien; Lin, Hui-Yi; Hung, Hsin-Yi; Huang, Li-Jiau; Kuo, Sheng-Chu

    2016-01-01

    We investigated the anticancer potential of a new synthetic compound, 7-(3-fluorophenyl)-4-methylpyrido-[2,3-d]pyrimidin-5(8H)-one (MT3-037). We found that MT3-037 effectively decreased the cancer cell viability by inducing apoptosis. MT3-037 treatments led to cell cycle arrest at M phase, with a marked increase in both expression of cyclin B1 and cyclin-dependent kinase 1 (CDK1) as well as in CDK1 kinase activity. Key proteins that regulate mitotic spindle dynamics, including survivin, Aurora A/B kinases, and polo-like kinase 1 (PLK1), were activated in MT3-037-treated cells. MT3-037-induced apoptosis was accompanied by activation of a pro-apoptotic factor, FADD, and the inactivation of apoptosis inhibitors, Bcl-2 and Bcl-xL, resulting in the cleavage/activation of caspases. The activation of c-Jun N-terminal kinase (JNK) was associated with MT3-037-induced CDK1 and Aurora A/B activation and apoptosis. Immunofluorescence staining of tubulin indicated that MT3-037 altered tubulin networks in cancer cells. Moreover, an in vitro tubulin polymerization assay revealed that MT3-037 inhibited the tubulin polymerization by direct binding to tubulin. Molecular docking studies and binding site completion assays revealed that MT3-037 binds to the colchicine-binding site. Furthermore, MT3-037 significantly inhibited the tumor growth in both MDAMB-468 and Erlotinib-resistant MDA-MB-468 xenograft mouse models. In addition, MT3-037 inhibited the angiogenesis and disrupted the tube formation by human endothelial cells. Our study demonstrates that MT3-037 is a potential tubulin-disrupting agent for antitumor therapy. PMID:27186428

  8. DT-13, a saponin monomer 13 of the Dwarf lilyturf tuber, synergized with vinorelbine to induce mitotic arrest via activation of ERK signaling pathway in NCI-H1299 cells.

    PubMed

    Li, Hongyang; Sun, Li; Li, Hang; Lv, Xiaodan; Semukunzi, Herve; Li, Ruiming; Yu, Jun; Yuan, Shengtao; Lin, Sensen

    2017-03-16

    Vinorelbine (NVB) is a semi-synthetic vinca alkaloid that is approved for the clinical therapy of lung cancer. However, the clinical application of NVB was limited because of the acquisition of resistance and inacceptable toxicity. Therefore, it is of great interest to develop low-cytotoxic drugs that can synergize with NVB. DT-13, a saponin monomer 13 of the Dwarf lilyturf tuber, showed inhibitory effects on tumor metastasis and angiogenesis in the previous studies. Here, we found that DT-13 combined with NVB exhibited synergistic effect to inhibit the cell proliferation in human lung cancer NCI-H1299 cells rather than human embryonic lung fibroblasts WI-38. The combination of DT-13 and NVB significantly inhibited the colony formation, induced cellular and nuclear morphological changes, and triggered cell cycle arrest at mitotic phase. Furthermore, MAPK signaling pathway was activated by the combination treatment, and the activation of ERK was required for the induction of mitotic arrest. Taken together, DT-13 combined with NVB exhibited synergistic anticancer effect in NCI-H1299 cells, and DT-13 may be a candidate agent for adjuvant chemotherapy of NVB in lung cancer.

  9. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

    Turner, Roberta L.; Groitl, Peter; Dobner, Thomas

    2015-01-01

    ABSTRACT Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a

  10. Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

    NASA Technical Reports Server (NTRS)

    Kim, S. H.; Arnold, D.; Lloyd, A.; Roux, S. J.

    2001-01-01

    We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

  11. Trichodermin induces c-Jun N-terminal kinase-dependent apoptosis caused by mitotic arrest and DNA damage in human p53-mutated pancreatic cancer cells and xenografts.

    PubMed

    Chien, Ming-Hsien; Lee, Tzong-Huei; Lee, Wei-Jiunn; Yeh, Yen-Hsiu; Li, Tsai-Kun; Wang, Po-Chuan; Chen, Jih-Jung; Chow, Jyh-Ming; Lin, Yung-Wei; Hsiao, Michael; Wang, Shih-Wei; Hua, Kuo-Tai

    2017-03-01

    Pancreatic cancer is an aggressive malignancy, which generally responds poorly to chemotherapy. In this study, trichodermin, an endophytic fungal metabolite from Nalanthamala psidii, was identified as a potent and selective antitumor agent in human pancreatic cancer. Trichodermin exhibited antiproliferative effects against pancreatic cancer cells, especially p53-mutated cells (MIA PaCa-2 and BxPC-3) rather than normal pancreatic epithelial cells. We found that trichodermin induced caspase-dependent and mitochondrial intrinsic apoptosis. Trichodermin also increased apoptosis through mitotic arrest by activating Cdc2/cyclin B1 complex activity. Moreover, trichodermin promoted the activation of c-Jun N-terminal kinase (JNK), and inhibition of JNK by its inhibitor, shRNA, or siRNA significantly reversed trichodermin-mediated caspase-dependent apoptosis. Trichodermin triggered DNA damage stress to activate p53 function for executing apoptosis in p53-mutated cells. Importantly, we demonstrated that trichodermin with efficacy similar to gemcitabine, profoundly suppressed tumor growth through inducing intratumoral DNA damage and JNK activation in orthotopic pancreatic cancer model. Based on these findings, trichodermin is a potential therapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially the mutant p53 pancreatic cancer.

  12. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway)

    NASA Astrophysics Data System (ADS)

    Ganesh Kumar, C.; Poornachandra, Y.; Chandrasekhar, Cheemalamarri

    2015-11-01

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications.The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2

  13. Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells.

    PubMed

    Zhu, Kuichun; Gerbino, Elvira; Beaupre, Darrin M; Mackley, Paul A; Muro-Cacho, Carlos; Beam, Craig; Hamilton, Andrew D; Lichtenheld, Mathias G; Kerr, William G; Dalton, William; Alsina, Melissa; Sebti, Saïd M

    2005-06-15

    Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G(2)/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients.

  14. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway).

    PubMed

    Kumar, C Ganesh; Poornachandra, Y; Chandrasekhar, Cheemalamarri

    2015-11-28

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications.

  15. Ethyl acetate extract from marine sponge Hyattella cribriformis exhibit potent anticancer activity by promoting tubulin polymerization as evidenced mitotic arrest and induction of apoptosis

    PubMed Central

    Annamalai, Pazhanimuthu; Thayman, Malini; Rajan, Sowmiya; Raman, Lakshmi Sundaram; Ramasubbu, Sankar; Perumal, Pachiappan

    2015-01-01

    Background: Marine sponges are important sources of bioactive compounds. Objective: This study investigated the anticancer properties of Hyattella cribriformis ethyl acetate (EA) fraction in various cancer and normal cell lines. Materials and Methods: anticancer assay was carried out in 15 cell lines to evaluate the anticancer potential of the EA fraction. Impact on cell cycle distribution was determined using flow cytometry. The fraction was investigated for interfering microtubules assembly in both in vitro and cellular assay. Further studies were conducted to determine the fraction induced cell death (apoptosis) using calcein/propidium iodide dual staining, activated caspase-3 and phosphorylation of Bcl-2 protein at Ser70. DNA fragmentation assay was performed to confirm the apoptosis. Results: EA fraction exhibited potent inhibition of cancer cell growth and resulted in 50% growth inhibition (GI50) of 0.27 μg/mL in A673 cell line. Sarcoma (MG-63, Saos-2) and ovarian (SK-OV-3 and OVCAR-3) cancer cell lines also showed superior anticancer activity GI50 of 1.0 μg/mL. Colon and breast cancer cell lines exhibited moderate GI compare other cancer cell lines and normal human lung fibroblast showed GI50 of 15.6 μg/mL. EA fraction showed potent G2/M phase arrest in A673 cell line and induced apoptosis at 48 h exposure. EA fraction promoted microtubule polymerization in tubulin polymerization assay and increased level of polymerized tubulin in the HeLa cells. Fraction induced the activation of caspase-3 and phosphorylation of Bcl-2 anti-apoptotic protein. Fraction induced DNA fragmentation in HeLa cells as evidence of apoptosis. Conclusion: Marine sponge H. cribriformis EA fraction exhibited potent anticancer activity through tubulin polymerization and induction of apoptosis. PMID:25829774

  16. Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage.

    PubMed

    Tao, Weikang; South, Victoria J; Zhang, Yun; Davide, Joseph P; Farrell, Linda; Kohl, Nancy E; Sepp-Lorenzino, Laura; Lobell, Robert B

    2005-07-01

    The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.

  17. Targeting mitotic exit with hyperthermia or APC/C inhibition to increase paclitaxel efficacy.

    PubMed

    Giovinazzi, Serena; Bellapu, Dhruv; Morozov, Viacheslav M; Ishov, Alexander M

    2013-08-15

    Microtubule-poisoning drugs, such as Paclitaxel (or Taxol, PTX), are powerful and commonly used anti-neoplastic agents for the treatment of several malignancies. PTX triggers cell death, mainly through a mitotic arrest following the activation of the spindle assembly checkpoint (SAC). Cells treated with PTX slowly slip from this mitotic block and die by mitotic catastrophe. However, cancer cells can acquire or are intrinsically resistant to this drug, posing one of the main obstacles for PTX clinical effectiveness. In order to override PTX resistance and increase its efficacy, we investigated both the enhancement of mitotic slippage and the block of mitotic exit. To test these opposing strategies, we used physiological hyperthermia (HT) to force exit from PTX-induced mitotic block and the anaphase-promoting complex/cyclosome (APC/C) inhibitor, proTAME, to block mitotic exit. We observed that application of HT on PTX-treated cells forced mitotic slippage, as shown by the rapid decline of cyclin B levels and by microscopy analysis. Similarly, HT induced mitotic exit in cells blocked in mitosis by other antimitotic drugs, such as Nocodazole and the Aurora A inhibitor MLN8054, indicating a common effect of HT on mitotic cells. On the other hand, proTAME prevented mitotic exit of PTX and MLN8054 arrested cells, prolonged mitosis, and induced apoptosis. In addition, we showed that proTAME prevented HT-mediated mitotic exit, indicating that stress-induced APC/C activation is necessary for HT-induced mitotic slippage. Finally, HT significantly increased PTX cytotoxicity, regardless of cancer cells' sensitivity to PTX, and this activity was superior to the combination of PTX with pro-TAME. Our data suggested that forced mitotic exit of cells arrested in mitosis by anti-mitotic drugs, such as PTX, can be a more successful anticancer strategy than blocking mitotic exit by inactivation of the APC/C.

  18. Genome-wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

    PubMed Central

    Díaz-Martínez, Laura A; Karamysheva, Zemfira N; Warrington, Ross; Li, Bing; Wei, Shuguang; Xie, Xian-Jin; Roth, Michael G; Yu, Hongtao

    2014-01-01

    The antimitotic anti-cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint-dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome-wide small interfering RNA (siRNA) screen in taxol-treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro-apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest. PMID:25024437

  19. RPF101, a new capsaicin-like analogue, disrupts the microtubule network accompanied by arrest in the G2/M phase, inducing apoptosis and mitotic catastrophe in the MCF-7 breast cancer cells

    SciTech Connect

    Sá-Júnior, Paulo Luiz de; Pasqualoto, Kerly Fernanda Mesquita; Ferreira, Adilson Kleber; Tavares, Maurício Temotheo; Damião, Mariana Celestina Frojuello Costa Bernstorff; Azevedo, Ricardo Alexandre de; Câmara, Diana Aparecida Dias; Pereira, Alexandre; Madeiro de Souza, Dener; Parise Filho, Roberto

    2013-02-01

    Breast cancer is the world's leading cause of death among women. This situation imposes an urgent development of more selective and less toxic agents. The use of natural molecular fingerprints as sources for new bioactive chemical entities has proven to be a quite promising and efficient method. Capsaicin, which is the primary pungent compound in red peppers, was reported to selectively inhibit the growth of a variety tumor cell lines. Here, we report for the first time a novel synthetic capsaicin-like analogue, RPF101, which presents a high antitumor activity on MCF-7 cell line, inducing arrest of the cell cycle at the G2/M phase through a disruption of the microtubule network. Furthermore, it causes cellular morphologic changes characteristic of apoptosis and a decrease of Δψm. Molecular modeling studies corroborated the biological findings and suggested that RPF101, besides being a more reactive molecule towards its target, may also present a better pharmacokinetic profile than capsaicin. All these findings support the fact that RPF101 is a promising anticancer agent. -- Highlights: ► We report for the first time that RPF101 possesses anticancer properties. ► RPF101 induces apoptosis of human breast cancer cells. ► RPF 101 decreases mitochondrial potential and induces DNA fragmentation.

  20. Mcl-1 dynamics influence mitotic slippage and death in mitosis.

    PubMed

    Sloss, Olivia; Topham, Caroline; Diez, Maria; Taylor, Stephen

    2016-02-02

    Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

  1. PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma

    PubMed Central

    Restall, Ian J; Parolin, Doris A E; Daneshmand, Manijeh; Hanson, Jennifer E L; Simard, Manon A; Fitzpatrick, Megan E; Kumar, Ritesh; Lavictoire, Sylvie J; Lorimer, Ian A J

    2015-01-01

    Cellular senescence is a tumor suppressor mechanism where cells enter a permanent growth arrest following cellular stress. Oncogene-induced senescence (OIS) is induced in non-malignant cells following the expression of an oncogene or inactivation of a tumor suppressor. Previously, we have shown that protein kinase C iota (PKCι) depletion induces cellular senescence in glioblastoma cells in the absence of a detectable DNA damage response. Here we demonstrate that senescent glioblastoma cells exhibit an aberrant centrosome morphology. This was observed in basal levels of senescence, in p21-induced senescence, and in PKCι depletion-induced senescence. In addition, senescent glioblastoma cells are polyploid, Ki-67 negative and arrest at the G1/S checkpoint, as determined by expression of cell cycle regulatory proteins. These markers are all consistent with cells that have undergone mitotic slippage. Failure of the spindle assembly checkpoint to function properly can lead to mitotic slippage, resulting in the premature exit of mitotic cells into the G1 phase of the cell cycle. Although in G1, these cells have the replicated DNA and centrosomal phenotype of a cell that has entered mitosis and failed to divide. Overall, we demonstrate that PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma cells. To our knowledge, this is the first evidence of markers of mitotic slippage directly in senescent cells by co-staining for senescence-associated β-galactosidase and immunofluorescence markers in the same cell population. We suggest that markers of mitotic slippage be assessed in future studies of senescence to determine the extent of mitotic slippage in the induction of cellular senescence. PMID:26208522

  2. PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma.

    PubMed

    Restall, Ian J; Parolin, Doris A E; Daneshmand, Manijeh; Hanson, Jennifer E L; Simard, Manon A; Fitzpatrick, Megan E; Kumar, Ritesh; Lavictoire, Sylvie J; Lorimer, Ian A J

    2015-01-01

    Cellular senescence is a tumor suppressor mechanism where cells enter a permanent growth arrest following cellular stress. Oncogene-induced senescence (OIS) is induced in non-malignant cells following the expression of an oncogene or inactivation of a tumor suppressor. Previously, we have shown that protein kinase C iota (PKCι) depletion induces cellular senescence in glioblastoma cells in the absence of a detectable DNA damage response. Here we demonstrate that senescent glioblastoma cells exhibit an aberrant centrosome morphology. This was observed in basal levels of senescence, in p21-induced senescence, and in PKCι depletion-induced senescence. In addition, senescent glioblastoma cells are polyploid, Ki-67 negative and arrest at the G1/S checkpoint, as determined by expression of cell cycle regulatory proteins. These markers are all consistent with cells that have undergone mitotic slippage. Failure of the spindle assembly checkpoint to function properly can lead to mitotic slippage, resulting in the premature exit of mitotic cells into the G1 phase of the cell cycle. Although in G1, these cells have the replicated DNA and centrosomal phenotype of a cell that has entered mitosis and failed to divide. Overall, we demonstrate that PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma cells. To our knowledge, this is the first evidence of markers of mitotic slippage directly in senescent cells by co-staining for senescence-associated β-galactosidase and immunofluorescence markers in the same cell population. We suggest that markers of mitotic slippage be assessed in future studies of senescence to determine the extent of mitotic slippage in the induction of cellular senescence.

  3. Phosphorylation of XIAP by CDK1–cyclin-B1 controls mitotic cell death

    PubMed Central

    Hou, Ying; Allan, Lindsey A.

    2017-01-01

    ABSTRACT Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1–cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1–cyclin-B1. PMID:27927753

  4. An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2.

    PubMed

    Rundle, Natalie T; Nelson, Jim; Flory, Mark R; Joseph, Jomon; Th'ng, John; Aebersold, Ruedi; Dasso, Mary; Andersen, Raymond J; Roberge, Michel

    2006-08-22

    Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubulin. To identify EKA-interacting proteins, we used a cell-permeable biotinylated form that retains biological activity to isolate binding proteins from living cells. Mass spectrometric analysis identified six EKA-binding proteins, including Ran-binding protein 2, a kinetochore protein whose depletion by small interfering RNA causes a similar mitotic arrest phenotype.

  5. XAB2 functions in mitotic cell cycle progression via transcriptional regulation of CENPE

    PubMed Central

    Hou, Shuai; Li, Na; Zhang, Qian; Li, Hui; Wei, Xinyue; Hao, Tian; Li, Yue; Azam, Sikandar; Liu, Caigang; Cheng, Wei; Jin, Bilian; Liu, Quentin; Li, Man; Lei, Haixin

    2016-01-01

    Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is a multi-functional protein that plays critical role in processes including transcription, transcription-coupled DNA repair, pre-mRNA splicing, homologous recombination and mRNA export. Microarray analysis on gene expression in XAB2 knockdown cells reveals that many genes with significant change in expression function in mitotic cell cycle regulation. Fluorescence-activated cell scanner analysis confirmed XAB2 depletion led to cell arrest in G2/M phase, mostly at prophase or prometaphase. Live cell imaging further disclosed that XAB2 knockdown induced severe mitotic defects including chromosome misalignment and defects in segregation, leading to mitotic arrest, mitotic catastrophe and subsequent cell death. Among top genes down-regulated by XAB2 depletion is mitotic motor protein centrosome-associated protein E (CENPE). Knockdown CENPE showed similar phenotypes to loss of XAB2, but CENPE knockdown followed by XAB2 depletion did not further enhance cell cycle arrest. Luciferase assay on CENPE promoter showed that overexpression of XAB2 increased luciferase activity, whereas XAB2 depletion resulted in striking reduction of luciferase activity. Further mapping revealed a region in CENPE promoter that is required for the transcriptional regulation by XAB2. Moreover, ChIP assay showed that XAB2 interacted with CENPE promoter. Together, these results support a novel function of XAB2 in mitotic cell cycle regulation, which is partially mediated by transcription regulation on CENPE. PMID:27735937

  6. Evidence for regulation of mitotic progression through temporal phosphorylation and dephosphorylation of CK2alpha.

    PubMed

    St-Denis, Nicole A; Derksen, D Richard; Litchfield, David W

    2009-04-01

    Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2alpha, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2alpha phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2alpha is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2alpha (CK2alpha-4D, CK2alpha-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2alpha (CK2alpha-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2alpha requires precise regulation to allow proper mitotic progression.

  7. Induction of mitotic catastrophe by PKC inhibition in Nf1-deficient cells.

    PubMed

    Zhou, Xiaodong; Kim, Sung-Hoon; Shen, Ling; Lee, Hyo-Jung; Chen, Changyan

    2014-01-01

    Mutations of tumor suppressor Nf1 gene deregulate Ras-mediated signaling, which confers the predisposition for developing benign or malignant tumors. Inhibition of protein kinase C (PKC) was shown to be in synergy with aberrant Ras for the induction of apoptosis in various types of cancer cells. However, it has not been investigated whether loss of PKC is lethal for Nf1-deficient cells. In this study, using HMG (3-hydroxy-3-methylgutaryl, a PKC inhibitor), we demonstrate that the inhibition of PKC by HMG treatment triggered a persistently mitotic arrest, resulting in the occurrence of mitotic catastrophe in Nf1-deficient ST8814 cells. However, the introduction of the Nf1 effective domain gene into ST8814 cells abolished this mitotic crisis. In addition, HMG injection significantly attenuated the growth of the xenografted ST8814 tumors. Moreover, Chk1 was phosphorylated, accompanied with the persistent increase of cyclin B1 expression in HMG-treated ST8814 cells. The knockdown of Chk1 by the siRNA prevented the Nf1-deficient cells from undergoing HMG-mediated mitotic arrest as well as mitotic catastrophe. Thus, our data suggested that the suppression of PKC activates the Chk1-mediated mitotic exit checkpoint in Nf1-deficient cells, leading to the induction of apoptosis via mitotic catastrophe. Collectively, the study indicates that targeting PKC may be a potential option for developing new strategies to treat Nf1-deficiency-related diseases.

  8. Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

    SciTech Connect

    Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.; States, J. Christopher

    2008-07-15

    Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.

  9. A simplified Bcl-2 network model reveals quantitative determinants of cell-to-cell variation in sensitivity to anti-mitotic chemotherapeutics

    NASA Astrophysics Data System (ADS)

    Kueh, Hao Yuan; Zhu, Yanting; Shi, Jue

    2016-11-01

    Anti-mitotic drugs constitute a major class of cytotoxic chemotherapeutics used in the clinic, killing cancer cells by inducing prolonged mitotic arrest that activates intrinsic apoptosis. Anti-mitotics-induced apoptosis is known to involve degradation of anti-apoptotic Bcl-2 proteins during mitotic arrest; however, it remains unclear how this mechanism accounts for significant heterogeneity observed in the cell death responses both within and between cancer cell types. To unravel quantitative determinants underlying variability in anti-mitotic drug response, we constructed a single-cell dynamical Bcl-2 network model describing cell death control during mitotic arrest, and constrained the model using experimental data from four representative cancer cell lines. The modeling analysis revealed that, given a variable, slowly accumulating pro-apoptotic signal arising from anti-apoptotic protein degradation, generation of a switch-like apoptotic response requires formation of pro-apoptotic Bak complexes with hundreds of subunits, suggesting a crucial role for high-order cooperativity. Moreover, we found that cell-type variation in susceptibility to drug-induced mitotic death arises primarily from differential expression of the anti-apoptotic proteins Bcl-xL and Mcl-1 relative to Bak. The dependence of anti-mitotic drug response on Bcl-xL and Mcl-1 that we derived from the modeling analysis provides a quantitative measure to predict sensitivity of distinct cancer cells to anti-mitotic drug treatment.

  10. A simplified Bcl-2 network model reveals quantitative determinants of cell-to-cell variation in sensitivity to anti-mitotic chemotherapeutics

    PubMed Central

    Kueh, Hao Yuan; Zhu, Yanting; Shi, Jue

    2016-01-01

    Anti-mitotic drugs constitute a major class of cytotoxic chemotherapeutics used in the clinic, killing cancer cells by inducing prolonged mitotic arrest that activates intrinsic apoptosis. Anti-mitotics-induced apoptosis is known to involve degradation of anti-apoptotic Bcl-2 proteins during mitotic arrest; however, it remains unclear how this mechanism accounts for significant heterogeneity observed in the cell death responses both within and between cancer cell types. To unravel quantitative determinants underlying variability in anti-mitotic drug response, we constructed a single-cell dynamical Bcl-2 network model describing cell death control during mitotic arrest, and constrained the model using experimental data from four representative cancer cell lines. The modeling analysis revealed that, given a variable, slowly accumulating pro-apoptotic signal arising from anti-apoptotic protein degradation, generation of a switch-like apoptotic response requires formation of pro-apoptotic Bak complexes with hundreds of subunits, suggesting a crucial role for high-order cooperativity. Moreover, we found that cell-type variation in susceptibility to drug-induced mitotic death arises primarily from differential expression of the anti-apoptotic proteins Bcl-xL and Mcl-1 relative to Bak. The dependence of anti-mitotic drug response on Bcl-xL and Mcl-1 that we derived from the modeling analysis provides a quantitative measure to predict sensitivity of distinct cancer cells to anti-mitotic drug treatment. PMID:27811996

  11. Heat shock protein inhibitors, 17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis

    SciTech Connect

    Wu Yichen; Yen Wenyen; Lee, T.-C. Yih, L.-H.

    2009-04-15

    Arsenic trioxide (ATO) has recently emerged as a promising therapeutic agent in leukemia because of its ability to induce apoptosis. However, there is no sufficient evidence to support its therapeutic use for other types of cancers. In this study, we investigated if, and how, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), an antagonist of heat shock protein 90 (HSP90), and KNK437, a HSP synthesis inhibitor, potentiated the cytotoxic effect of ATO. Our results showed that cotreatment with ATO and either 17-DMAG or KNK437 significantly increased ATO-induced cell death and apoptosis. siRNA-mediated attenuation of the expression of the inducible isoform of HSP70 (HSP70i) or HSP90{alpha}/{beta} also enhanced ATO-induced apoptosis. In addition, cotreatment with ATO and 17-DMAG or KNK437 significantly increased ATO-induced mitotic arrest and ATO-induced BUBR1 phosphorylation and PDS1 accumulation. Cotreatment also significantly increased the percentage of mitotic cells with abnormal mitotic spindles and promoted metaphase arrest as compared to ATO treatment alone. These results indicated that 17-DMAG or KNK437 may enhance ATO cytotoxicity by potentiating mitotic arrest and mitotic apoptosis possibly through increased activation of the spindle checkpoint.

  12. Cell death by mitotic catastrophe: a molecular definition.

    PubMed

    Castedo, Maria; Perfettini, Jean-Luc; Roumier, Thomas; Andreau, Karine; Medema, Rene; Kroemer, Guido

    2004-04-12

    The current literature is devoid of a clearcut definition of mitotic catastrophe, a type of cell death that occurs during mitosis. Here, we propose that mitotic catastrophe results from a combination of deficient cell-cycle checkpoints (in particular the DNA structure checkpoints and the spindle assembly checkpoint) and cellular damage. Failure to arrest the cell cycle before or at mitosis triggers an attempt of aberrant chromosome segregation, which culminates in the activation of the apoptotic default pathway and cellular demise. Cell death occurring during the metaphase/anaphase transition is characterized by the activation of caspase-2 (which can be activated in response to DNA damage) and/or mitochondrial membrane permeabilization with the release of cell death effectors such as apoptosis-inducing factor and the caspase-9 and-3 activator cytochrome c. Although the morphological aspect of apoptosis may be incomplete, these alterations constitute the biochemical hallmarks of apoptosis. Cells that fail to execute an apoptotic program in response to mitotic failure are likely to divide asymmetrically in the next round of cell division, with the consequent generation of aneuploid cells. This implies that disabling of the apoptotic program may actually favor chromosomal instability, through the suppression of mitotic catastrophe. Mitotic catastrophe thus may be conceived as a molecular device that prevents aneuploidization, which may participate in oncogenesis. Mitotic catastrophe is controlled by numerous molecular players, in particular, cell-cycle-specific kinases (such as the cyclin B1-dependent kinase Cdk1, polo-like kinases and Aurora kinases), cell-cycle checkpoint proteins, survivin, p53, caspases and members of the Bcl-2 family.

  13. About Cardiac Arrest

    MedlinePlus

    ... Thromboembolism Aortic Aneurysm More About Cardiac Arrest Updated:Mar 10,2017 What is cardiac arrest? Cardiac arrest is the abrupt loss of heart function in a person who may or may not have diagnosed heart ...

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

    PubMed Central

    Braunstein, Ilana; Miniowitz, Shirly; Moshe, Yakir; Hershko, Avram

    2007-01-01

    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′-(γ-thio)triphosphate (ATP[γS]) 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. PMID:17360335

  15. Evidence for Regulation of Mitotic Progression through Temporal Phosphorylation and Dephosphorylation of CK2α▿ †

    PubMed Central

    St-Denis, Nicole A.; Derksen, D. Richard; Litchfield, David W.

    2009-01-01

    Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2α, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2α phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2α is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2α (CK2α-4D, CK2α-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2α (CK2α-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2α requires precise regulation to allow proper mitotic progression. PMID:19188443

  16. Micromechanics of human mitotic chromosomes

    NASA Astrophysics Data System (ADS)

    Sun, Mingxuan; Kawamura, Ryo; Marko, John F.

    2011-02-01

    Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

  17. Phosphorylation–dephosphorylation cycle of HP1α governs accurate mitotic progression

    PubMed Central

    Chakraborty, Arindam; Prasanth, Supriya G

    2014-01-01

    Heterochromatin protein 1α (HP1α), a bona fide factor of silent chromatin, is required for establishing as well as maintaining the higher-order chromatin structure in eukaryotes. HP1α is decorated with several post-translational modifications, and many of these are critical for its cellular functions. HP1α is heavily phosphorylated; however, its physiological relevance had remained to be completely understood. We have recently demonstrated that human HP1α is a mitotic target for NDR kinase, and the phosphorylation at the hinge region of HP1α at the G2/M phase of the cell cycle is crucial for mitotic progression and Sgo1 loading at mitotic centromeres (Chakraborty et al., 2014). We now demonstrate that the dephosphorylation of HP1α within its hinge domain occurs during mitosis, specifically soon after prometaphase. In the absence of the hinge-specific HP1α phosphorylation, either as a consequence of depleting NDR1 or in cells expressing a non-phosphorylatable HP1α mutant, the cells arrest in prometaphase with several mitotic defects. In this study we show that NDR1-depleted cells expressing hinge-specific phosphomimetic HP1α mutant rescues the prometaphase arrest but displays defects in mitotic exit, suggesting that the dephosphorylation of HP1α is required for the completion of cytokinesis. Taken together, our results reveal that the phosphorylation–dephosphorylation cycle of HP1α orchestrates accurate progression of cells through mitosis. PMID:24786771

  18. Myosin-10 independently influences mitotic spindle structure and mitotic progression.

    PubMed

    Sandquist, Joshua C; Larson, Matthew E; Hine, Ken J

    2016-06-01

    The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein's spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10's close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10's spindle functions require microtubule binding. In particular, myosin-10's contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding. © 2016 Wiley Periodicals, Inc.

  19. [Cell cycle arrest at M phase induced by vinblastine in MOLT-4 cells].

    PubMed

    Zhong, Yi-Sheng; Pan, Chang-Chuan; Jin, Chang-Nan; Li, Jian-Jun; Xiong, Gong-Peng; Zhang, Jian-Xi; Gong, Jian-Ping

    2009-04-01

    This study was purposed to investigate the biological effect of vinblastine (VLS), usually known as inductor of mitotic arrest, on MOLT-4 of ALL cells and to evaluate its significance. The cell arrest in M phase and/or cell apoptosis were induced by treatment of MOLT-4 cells with 0.05 microg/ml VLS for 0 - 12 hours; the DNA histogram was detected by flow cytometry; the morphological changes of cells were observed by confocal microscopy; the cell cycle distribution, cell apoptosis and morphological changes of cells before and after arrest were analyzed by using arrest increasing rate (AIR), arrest efficiency (AE), apoptosis rate (AR) and morphologic parameters respectively. The results indicated that the cell arrest did not accompanied by significant increase of apoptosis rate; the DNA histogram of cell arrest showed dynamic change of cell cycle in time-dependent manner; the arrest efficiency could be quantified. The cell arrest at M phase was accompanied by cell stack in S phase, the cell proliferation rate dropped after cell arrest occurred. The cells arrested at M phase possessed of characteristic morphologic features in cell mitosis. It is concluded that the vinblastine can solely induce arrest of MOLT-4 cells at M phase. This study provides experimental basis for further investigating the relation of cell cycle arrest to apoptosis, mechanism of checkpoint and development of new anticancer drugs.

  20. Mechanisms of Mitotic Spindle Assembly

    PubMed Central

    Petry, Sabine

    2016-01-01

    Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ~200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes. PMID:27145846

  1. A LCMT1-PME-1 methylation equilibrium controls mitotic spindle size.

    PubMed

    Xia, Xiaoyu; Gholkar, Ankur; Senese, Silvia; Torres, Jorge Z

    2015-01-01

    Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to the regulation of cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to mitotic arrest, spindle assembly checkpoint activation, defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.

  2. Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines

    PubMed Central

    Bowman, Doug; Shinde, Vaishali; Lasky, Kerri; Shi, Judy; Vos, Tricia; Stringer, Bradley; Amidon, Ben; D'Amore, Natalie; Hyer, Marc L.

    2014-01-01

    Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens. PMID:25365521

  3. Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression

    SciTech Connect

    Takahashi, Akinori; Kikuguchi, Chisato; Morita, Masahiro; Shimodaira, Tetsuhiro; Tokai-Nishizumi, Noriko; Yokoyama, Kazumasa; Ohsugi, Miho; Suzuki, Toru; Yamamoto, Tadashi

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer CNOT3 depletion increases the mitotic index. Black-Right-Pointing-Pointer CNOT3 inhibits the expression of MAD1. Black-Right-Pointing-Pointer CNOT3 destabilizes the MAD1 mRNA. Black-Right-Pointing-Pointer MAD1 knockdown attenuates the CNOT3 depletion-induced mitotic arrest. -- Abstract: The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint, suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.

  4. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

    PubMed Central

    1996-01-01

    M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity. PMID:8567717

  5. Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint

    PubMed Central

    Salmela, Anna-Leena; Pouwels, Jeroen; Varis, Asta; Kukkonen, Anu M.; Toivonen, Pauliina; Halonen, Pasi K.; Perälä, Merja; Kallioniemi, Olli; Gorbsky, Gary J.; Kallio, Marko J.

    2009-01-01

    Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2–160 μg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound. PMID:19395653

  6. Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

    PubMed Central

    McCoy, Rajiv C.; Demko, Zachary P.; Ryan, Allison; Banjevic, Milena; Hill, Matthew; Sigurjonsson, Styrmir; Rabinowitz, Matthew; Petrov, Dmitri A.

    2015-01-01

    Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight

  7. Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

    PubMed

    Lehr, Hans-Anton; Rochat, Candice; Schaper, Cornelia; Nobile, Antoine; Shanouda, Sherien; Vijgen, Sandrine; Gauthier, Arnaud; Obermann, Ellen; Leuba, Susana; Schmidt, Marcus; C, Curzio Ruegg; Delaloye, Jean-Francois; Simiantonaki, Nectaria; Schaefer, Stephan C

    2013-03-01

    Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

  8. Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde

    SciTech Connect

    Zhang Bo; Huang Bo; Guan Hua; Zhang Shimeng; Xu Qinzhi; He Xingpeng; Liu Xiaodan; Wang Yu; Shang Zengfu; Zhou Pingkun

    2011-05-01

    Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.

  9. Microelasticity of Single Mitotic Chromosomes

    NASA Astrophysics Data System (ADS)

    Poirier, Michael; Eroglu, Sertac; Chatenay, Didier; Marko, John F.; Hirano, Tatsuya

    2000-03-01

    The force-extension behavior of mitotic chromosomes from the newt TVI tumor cell line was studied using micropipette manipulation and force measuring techniques. Reversible, linear elastic response was observed for extensions up to 5 times the native length; the force required to double chromosome length was 1 nanonewton (nN). For further elongations, the linear response teminates at a force plateau of 15 nN and at an extension of 20x. Beyond this extension, the chromosome breaks at elongations between 20x and 70x. These results will be compared to the similar behavior of mitotic chromosomes from explanted newt cells (Poirier, Eroglu, Chatenay and Marko, Mol. Biol. Cell, in press). Also, the effect of biochemical modifications on the elasticity was studied. Ethidium Bromide, which binds to DNA, induces up to a 10 times increase in the Young's modulus. Anti-XCAP-E, which binds to a putative chromosome folding protein, induces up to a 2 times increase in the Young's modulus. Preliminary results on the dynamical relaxation of chromosomes will also be presented. Support of this research through a Biomedical Engineering Research Grant from The Whitaker Foundation is gratefully acknowledged.

  10. Chromatin shapes the mitotic spindle.

    PubMed

    Dinarina, Ana; Pugieux, Céline; Corral, Maria Mora; Loose, Martin; Spatz, Joachim; Karsenti, Eric; Nédélec, François

    2009-08-07

    In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

  11. Inhibition of mitotic-specific histone phophorylation by sodium arsenite

    SciTech Connect

    Cobo, J.M.; Valdez, J.G.; Gurley, L.R.

    1994-10-01

    Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10{mu}M sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetric manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific HI and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase HI Phosphorylation affected. The phosphorylation of HI was inhibited only in mitosis, reducing HI phosphorylation to 38.1% of control levels, which was the level of interphase HI phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibite cell proliferation by interfering with the cyclin B/p34{sup cdc2} histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed by our laboratory that HI and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference of this process by sodium arsenite may lead to structurally damaged chromosomes resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.

  12. Prolonged oestrogen treatment does not correlate with a sustained increase in anterior pituitary mitotic index in ovariectomized Wistar rats.

    PubMed

    Nolan, L A; Levy, A

    2009-03-01

    Oestrogen is a powerful mitogen that is believed to exert a continuous, dose-dependent trophic stimulus at the anterior pituitary. This persistent mitotic effect contrasts with corticosterone and testosterone, changes in the levels of which induce only transient, self-limiting fluctuations in pituitary mitotic activity. To further define the putative long-term trophic effects of oestrogen, we have accurately analysed the effects of 7 and 28 days oestrogen treatment on anterior pituitary mitotic activity in ovariectomized 10-week-old Wistar rats using both bromodeoxyuridine (BrdU) and timed colchicine-induced mitotic arrest. An oestrogen dose-dependent increase in mitotic index was seen 7 days after the start of treatment as expected, representing an acceleration in gross mitotic activity from 1.7%/day in ovariectomized animals in the absence of any oestrogen replacement to 3.7%/day in the presence of a pharmacological dose of oestrogen (50 mcg/rat per day: approximately 230 mcg/kg per day). Despite continued exposure to high-dose oestrogen and persistence of the increase in pituitary wet weight, the increase in mitotic index was unexpectedly not sustained. After 28 days of high-dose oestrogen treatment, anterior pituitary mitotic index and BrdU-labelling index were not significantly different from baseline. Although a powerful pituitary mitogen in the short term, responsible, presumably, for increased trophic variability in oestrus cycling females, these data indicate that in keeping with other trophic stimuli to the pituitary and in contrast to a much established dogma, the mitotic response to longer-term high-dose oestrogen exposure is transient and is not the driver of persistent pituitary growth, at least in female Wistar rats.

  13. Regulation of Mitotic Exit in Saccharomyces cerevisiae.

    PubMed

    Baro, Bàrbara; Queralt, Ethel; Monje-Casas, Fernando

    2017-01-01

    The Mitotic Exit Network (MEN) is an essential signaling pathway, closely related to the Hippo pathway in mammals, which promotes mitotic exit and initiates cytokinesis in the budding yeast Saccharomyces cerevisiae. Here, we summarize the current knowledge about the MEN components and their regulation.

  14. Mitotic chromosome condensation in vertebrates

    SciTech Connect

    Vagnarelli, Paola

    2012-07-15

    Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes of

  15. Cell biology of mitotic recombination.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2015-03-02

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  16. Cell Biology of Mitotic Recombination

    PubMed Central

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

  17. AIP regulates stability of Aurora-A at early mitotic phase coordinately with GSK-3beta.

    PubMed

    Fumoto, K; Lee, P-C; Saya, H; Kikuchi, A

    2008-07-24

    Glycogen synthase kinase-3 (GSK-3beta) regulates microtubule dynamics and cellular polarity through phosphorylating various microtubule associating proteins and plus-end tracking proteins. Although it was also reported that GSK-3beta is inactivated by protein kinase B at the spindle poles, functions and targets of GSK-3beta in the mitotic phase are unknown. Here, we identified Aurora-A-interacting protein (AIP), a negative regulator of Aurora-A, as a binding partner of GSK-3beta. AIP was colocalized with Aurora-A and GSK-3beta to the spindle poles in metaphase, and its depletion in cells stabilized and activated Aurora-A in early mitotic phase and caused mitotic cell arrest. Treatment of the cells with a GSK-3beta inhibitor reduced the protein level of Aurora-A and this reduction was suppressed by AIP knockdown. AIP was phosphorylated by GSK-3beta, and an AIP mutant in which the GSK-3beta phosphorylation site was mutated could bind and downregulate Aurora-A more efficiently. These results suggest that GSK-3beta modulates the early mitotic Aurora-A level through binding and phosphorylating AIP.

  18. The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

    SciTech Connect

    Salmela, Anna-Leena; Pouwels, Jeroen; Kukkonen-Macchi, Anu; Waris, Sinikka; Toivonen, Pauliina; Jaakkola, Kimmo; Maeki-Jouppila, Jenni; Kallio, Lila; Kallio, Marko J.

    2012-03-10

    The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3 Prime ,5-dihydroxy-4 Prime ,6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.

  19. Profiling DNA damage response following mitotic perturbations

    PubMed Central

    S. Pedersen, Ronni; Karemore, Gopal; Gudjonsson, Thorkell; Rask, Maj-Britt; Neumann, Beate; Hériché, Jean-Karim; Pepperkok, Rainer; Ellenberg, Jan; Gerlich, Daniel W.; Lukas, Jiri; Lukas, Claudia

    2016-01-01

    Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed phenotypes. To demonstrate the potential of this resource, we show that DNA breakage after cytokinesis failure is preceded by replication stress, which mounts during consecutive cell cycles and coincides with decreased proliferation. Together, our results provide a resource to gauge the magnitude and dynamics of DNA breakage associated with mitotic aberrations and suggest that replication stress might limit propagation of cells with abnormal karyotypes. PMID:27976684

  20. p31comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

    PubMed Central

    Teichner, Adar; Eytan, Esther; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Dumin, Elena; Gromis, Jonathan; Hershko, Avram

    2011-01-01

    Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31comet in this process. It is known that p31comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC. PMID:21300909

  1. Moderate intensity static magnetic fields affect mitotic spindles and increase the antitumor efficacy of 5-FU and Taxol.

    PubMed

    Luo, Yan; Ji, Xinmiao; Liu, Juanjuan; Li, Zhiyuan; Wang, Wenchao; Chen, Wei; Wang, Junfeng; Liu, Qingsong; Zhang, Xin

    2016-06-01

    Microtubules are the fundamental components in mitotic spindle, which plays essential roles in cell division. It was well known that purified microtubules could be affected by static magnetic fields (SMFs) in vitro because of the diamagnetic anisotropy of tubulin. However, whether these effects lead to cell division defects was unknown. Here we find that 1T SMFs induce abnormal mitotic spindles and increase mitotic index. Synchronization experiments show that SMFs delay cell exit from mitosis and cause mitotic arrest. These mimic the cellular effects of a microtubule-targeting drug Paclitaxel (Taxol), which is frequently used in combination with 5-Fluorouracil (5-FU) and Cisplatin in cancer treatment. Using four different human cancer cell lines, HeLa, HCT116, CNE-2Z and MCF7, we find that SMFs increase the antitumor efficacy of 5-FU or 5-FU/Taxol, but not Cisplatin, which indicates that the SMF-induced combinational effects with chemodrugs are drug-specific. Our study not only reveals the effect of SMFs on microtubules to cause abnormal mitotic spindles and delay cells exit from mitosis, but also implies the potential applications of SMFs in combination with chemotherapy drugs 5-FU or 5-FU/Taxol, but not with Cisplatin in cancer treatment.

  2. Drug-induced premature chromosome condensation (PCC) protocols: cytogenetic approaches in mitotic chromosome and interphase chromatin.

    PubMed

    Gotoh, Eisuke

    2015-01-01

    Chromosome analysis is a fundamental technique which is used in wide areas of cytogenetic study including karyotyping species, hereditary diseases diagnosis, or chromosome biology study. Chromosomes are usually prepared from mitotic cells arrested by colcemid block protocol. However, obtaining mitotic chromosomes is often hampered under several circumstances. As a result, cytogenetic analysis will be sometimes difficult or even impossible in such cases. Premature chromosome condensation (PCC) (see Note 1) is an alternative method that has proved to be a unique and useful way in chromosome analysis. Former, PCC has been achieved following cell fusion method (cell-fusion PCC) mediated either by fusogenic viruses (e.g., Sendai virus) or cell fusion chemicals (e.g., polyethylene glycol), but the cell fusion PCC has several drawbacks. The novel drug-induced PCC using protein phosphatase inhibitors was introduced about 20 years ago. This method is much simpler and easier even than the conventional mitotic chromosome preparation protocol use with colcemid block and furthermore obtained PCC index (equivalent to mitotic index for metaphase chromosome) is usually much higher than colcemid block method. Moreover, this method allows the interphase chromatin to be condensed to visualize like mitotic chromosomes. Therefore drug-induced PCC has opened the way for chromosome analysis not only in metaphase chromosomes but also in interphase chromatin. The drug-induced PCC has thus proven the usefulness in cytogenetics and other cell biology fields. For this second edition version, updated modifications/changes are supplemented in Subheadings 2, 3, and 4, and a new section describing the application of PCC in chromosome science fields is added with citation of updated references.

  3. Epigenetic countermarks in mitotic chromosome condensation.

    PubMed

    van Wely, Karel H M; Mora Gallardo, Carmen; Vann, Kendra R; Kutateladze, Tatiana G

    2017-01-03

    Mitosis in metazoans is characterized by abundant phosphorylation of histone H3 and involves the recruitment of condensin complexes to chromatin. The relationship between the 2 phenomena and their respective contributions to chromosome condensation in vivo remain poorly understood. Recent studies have shown that H3T3 phosphorylation decreases binding of histone readers to methylated H3K4 in vitro and is essential to displace the corresponding proteins from mitotic chromatin in vivo. Together with previous observations, these data provide further evidence for a role of mitotic histone H3 phosphorylation in blocking transcriptional programs or preserving the 'memory' PTMs. Mitotic protein exclusion can also have a role in depopulating the chromatin template for subsequent condensin loading. H3 phosphorylation thus serves as an integral step in the condensation of chromosome arms.

  4. The mitotic spindle and actin tails.

    PubMed

    Karsenti, Eric; Nédélec, François

    2004-04-01

    To segregate their chromosomes, eukaryotic cells rely on a dynamic structure made of microtubules: the mitotic spindle. This structure can form in cells lacking centrosomes, because their chromosomes also nucleate microtubules. This second assembly pathway is observed even in some cells that naturally have centrosomes, for example when the centrosomes are ablated by laser surgery. Recent results have started to address the complementary question of whether centrosome-nucleated microtubules alone could sustain the formation of a functional mitotic spindle. We wonder in this respect whether lower eukaryotes such as yeasts are different from higher eukaryotes such as vertebrates.

  5. THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

    PubMed Central

    Mazia, Daniel; Mitchison, J. M.; Medina, Heitor; Harris, Patricia

    1961-01-01

    A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus. PMID:13768661

  6. Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin

    SciTech Connect

    Komura, Jun-ichiro; Ikehata, Hironobu; Mori, Toshio; Ono, Tetsuya

    2012-03-10

    During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.

  7. Centrin: Another target of monastrol, an inhibitor of mitotic spindle

    NASA Astrophysics Data System (ADS)

    Duan, Lian; Wang, Tong-Qing; Bian, Wei; Liu, Wen; Sun, Yue; Yang, Bin-Sheng

    2015-02-01

    Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibiting antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compounds 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.

  8. Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

    PubMed Central

    Magidson, Valentin; He, Jie; Ault, Jeffrey G.; O’Connell, Christopher B.; Yang, Nachen; Tikhonenko, Irina; McEwen, Bruce F.

    2016-01-01

    Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression. PMID:26833787

  9. Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes.

    PubMed

    Kaisari, Sharon; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Teichner, Adar; Hershko, Avram

    2017-01-31

    The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31(comet) Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.

  10. Inhibition of the mitotic exit network in response to damaged telomeres.

    PubMed

    Valerio-Santiago, Mauricio; de Los Santos-Velázquez, Ana Isabel; Monje-Casas, Fernando

    2013-01-01

    When chromosomal DNA is damaged, progression through the cell cycle is halted to provide the cells with time to repair the genetic material before it is distributed between the mother and daughter cells. In Saccharomyces cerevisiae, this cell cycle arrest occurs at the G2/M transition. However, it is also necessary to restrain exit from mitosis by maintaining Bfa1-Bub2, the inhibitor of the Mitotic Exit Network (MEN), in an active state. While the role of Bfa1 and Bub2 in the inhibition of mitotic exit when the spindle is not properly aligned and the spindle position checkpoint is activated has been extensively studied, the mechanism by which these proteins prevent MEN function after DNA damage is still unclear. Here, we propose that the inhibition of the MEN is specifically required when telomeres are damaged but it is not necessary to face all types of chromosomal DNA damage, which is in agreement with previous data in mammals suggesting the existence of a putative telomere-specific DNA damage response that inhibits mitotic exit. Furthermore, we demonstrate that the mechanism of MEN inhibition when telomeres are damaged relies on the Rad53-dependent inhibition of Bfa1 phosphorylation by the Polo-like kinase Cdc5, establishing a new key role of this kinase in regulating cell cycle progression.

  11. The novel combination of chlorpromazine and pentamidine exerts synergistic antiproliferative effects through dual mitotic action.

    PubMed

    Lee, Margaret S; Johansen, Lisa; Zhang, Yanzhen; Wilson, Amy; Keegan, Mitchell; Avery, William; Elliott, Peter; Borisy, Alexis A; Keith, Curtis T

    2007-12-01

    Combination therapy has proven successful in treating a wide variety of aggressive human cancers. Historically, combination treatments have been discovered through serendipity or lengthy trials using known anticancer agents with similar indications. We have used combination high-throughput screening to discover the unexpected synergistic combination of an antiparasitic agent, pentamidine, and a phenothiazine antipsychotic, chlorpromazine. This combination, CRx-026, inhibits the growth of tumor cell lines in vivo more effectively than either pentamidine or chlorpromazine alone. Here, we report that CRx-026 exerts its antiproliferative effect through synergistic dual mitotic action. Chlorpromazine is a potent and specific inhibitor of the mitotic kinesin KSP/Eg5 and inhibits tumor cell proliferation through mitotic arrest and accumulation of monopolar spindles. Pentamidine treatment results in chromosomal segregation defects and delayed progression through mitosis, consistent with inhibition of the phosphatase of regenerating liver family of phosphatases. We also show that CRx-026 synergizes in vitro and in vivo with the microtubule-binding agents paclitaxel and vinorelbine. These data support a model where dual action of pentamidine and chlorpromazine in mitosis results in synergistic antitumor effects and show the importance of systematic screening for combinations of targeted agents.

  12. Rapid measurement of mitotic spindle orientation in cultured mammalian cells

    PubMed Central

    Decarreau, Justin; Driver, Jonathan; Asbury, Charles; Wordeman, Linda

    2014-01-01

    Summary Factors that influence the orientation of the mitotic spindle are important for the maintenance of stem cell populations and in cancer development. However, screening for these factors requires rapid quantification of alterations of the angle of the mitotic spindle in cultured cell lines. Here we describe a method to image mitotic cells and rapidly score the angle of the mitotic spindle using a simple MATLAB application to analyze a stack of Z-images. PMID:24633791

  13. SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS

    PubMed Central

    Bibring, Thomas; Baxandall, Jane

    1971-01-01

    Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein. PMID:5543404

  14. Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

    PubMed Central

    Kimura, M; Yoshioka, T; Saio, M; Banno, Y; Nagaoka, H; Okano, Y

    2013-01-01

    Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization

  15. Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin.

    PubMed

    Komura, Jun-ichiro; Ikehata, Hironobu; Mori, Toshio; Ono, Tetsuya

    2012-03-10

    During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression.

  16. Mutation in the Bimd Gene of Aspergillus Nidulans Confers a Conditional Mitotic Block and Sensitivity to DNA Damaging Agents

    PubMed Central

    Denison, S. H.; Kafer, E.; May, G. S.

    1993-01-01

    Mutation in the bimD gene of Aspergillus nidulans results in a mitotic block in anaphase characterized by a defective mitosis. Mutation in bimD also confers, at temperatures permissive for the mitotic arrest phenotype, an increased sensitivity to DNA damaging agents, including methyl methanesulfonate and ultraviolet light. In order to better understand the relationship between DNA damage and mitotic progression, we cloned the bimD gene from Aspergillus. A cosmid containing the bimD gene was identified among pools of cosmids by cotransformation with the nutritional selective pyrG gene of a strain carrying the recessive, temperature-sensitive lethal bimD6 mutation. The bimD gene encodes a predicted polypeptide of 166,000 daltons in mass and contains amino acid sequence motifs similar to those found in some DNA-binding transcription factors. These sequences include a basic domain followed by a leucine zipper, which together are called a bZIP motif, and a carboxyl-terminal domain enriched in acidic amino acids. Overexpression of the wild-type bimD protein resulted in an arrest of the nuclear division cycle that was reversible and determined to be in either the G(1) or S phase of the cell cycle. Our data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis. PMID:8375649

  17. Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation*

    PubMed Central

    Hernández-Ortega, Sara; Bru, Samuel; Ricco, Natalia; Ramírez, Sara; Casals, Núria; Jiménez, Javier; Isasa, Marta; Crosas, Bernat; Clotet, Josep

    2013-01-01

    Progression through the G1 phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G1 cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G1 cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G1 cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G1. PMID:23264631

  18. Pb-inhibited mitotic activity in onion roots involves DNA damage and disruption of oxidative metabolism.

    PubMed

    Kaur, Gurpreet; Singh, Harminder Pal; Batish, Daizy Rani; Kohli, Ravinder Kumar

    2014-09-01

    Plant responses to abiotic stress significantly affect the development of cells, tissues and organs. However, no studies correlating Pb-induced mitotic inhibition and DNA damage and the alterations in redox homeostasis during root division per se were found in the literature. Therefore, an experiment was conducted to evaluate the impact of Pb on mitotic activity and the associated changes in the oxidative metabolism in onion roots. The cytotoxic effect of Pb on cell division was assessed in the root meristems of Allium cepa (onion). The mitotic index (MI) was calculated and chromosomal abnormalities were sought. Pb-treatment induced a dose-dependent decrease in MI in the onion root tips and caused mitotic abnormalities such as distorted metaphase, fragments, sticky chromosomes, laggards, vagrant chromosomes and bridges. Single Cell Gel Electrophoresis was also performed to evaluate Pb induced genotoxicity. It was accompanied by altered oxidative metabolism in the onion root tips suggesting the interference of Pb with the redox homeostasis during cell division. There was a higher accumulation of malondialdehyde, conjugated dienes and hydrogen peroxide, and a significant increase in the activities of superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases in Pb-treated onion roots, whereas catalases activity exhibited a decreasing pattern upon Pb exposure. The study concludes that Pb-induced cytotoxicity and genotoxicity in the onion roots is mediated through ROS and is also tightly linked to the cell cycle. The exposure to higher concentrations arrested cell cycle leading to cell death, whereas different repair responses are generated at lower concentrations, thereby allowing the cell to complete the cell cycle.

  19. CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.

    PubMed

    Kashima, Lisa; Idogawa, Masashi; Mita, Hiroaki; Shitashige, Miki; Yamada, Tesshi; Ogi, Kazuhiro; Suzuki, Hiromu; Toyota, Minoru; Ariga, Hiroyoshi; Sasaki, Yasushi; Tokino, Takashi

    2012-04-13

    The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

  20. Registry of Unexplained Cardiac Arrest

    ClinicalTrials.gov

    2016-05-16

    Cardiac Arrest; Long QT Syndrome; Brugada Syndrome; Catecholaminergi Polymorphic Ventricular Tachycardia; Idiopathic VentricularFibrillation; Early Repolarization Syndrome; Arrhythmogenic Right Ventricular Cardiomyopathy

  1. Suppression of spindly delays mitotic exit and exacerbates cell death response of cancer cells treated with low doses of paclitaxel.

    PubMed

    Silva, Patrícia M A; Ribeiro, Nilza; Lima, Raquel T; Andrade, Cláudia; Diogo, Vânia; Teixeira, Joana; Florindo, Cláudia; Tavares, Álvaro; Vasconcelos, M Helena; Bousbaa, Hassan

    2017-02-27

    Microtubule-targeting agents (MTAs) are used extensively for the treatment of diverse types of cancer. They block cancer cells in mitosis through the activation of the spindle assembly checkpoint (SAC), the surveillance mechanism that ensures accurate chromosome segregation at the onset of anaphase. However, the cytotoxic activity of MTAs is limited by premature mitotic exit (mitotic slippage) due to SAC silencing. Here we have explored the dual role of the protein Spindly in chromosome attachments and SAC silencing to analyze the consequences of its depletion on the viability of tumor cells treated with clinically relevant doses of paclitaxel. As expected, siRNA-mediated Spindly suppression induced chromosome misalignment and accumulation of cells in mitosis. Remarkably, these cells were more sensitive to low-doses of paclitaxel. Sensitization was due to an increase in the length of mitotic arrest and high frequency of multinucleated cells, both correlated with an exacerbated post-mitotic cell death response as determined by cell fate profiling. Thus, by affecting both SAC silencing and chromosome attachment, Spindly targeting offers a double-edged sword that potentiates tumor cell killing by clinically relevant doses of paclitaxel, providing a rationale for combination chemotherapy against cancer.

  2. Novel DNA damage checkpoint in mitosis: Mitotic DNA damage induces re-replication without cell division in various cancer cells.

    PubMed

    Hyun, Sun-Yi; Rosen, Eliot M; Jang, Young-Joo

    2012-07-06

    DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair.

  3. Measuring mitotic spindle dynamics in budding yeast

    NASA Astrophysics Data System (ADS)

    Plumb, Kemp

    In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

  4. Mitotic Spindle Positioning in Breast Cancer

    DTIC Science & Technology

    2009-10-01

    5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Tirnauer, Jennifer S. M.D. 5d. PROJECT NUMBER Email: tirnauer@uchc.edu 5e. TASK...SUPPLEMENTARY NOTES 14. ABSTRACT The purpose of this project was to determine whether mitotic spindle position differs in benign versus malignant...postdoc working on the project has left, I want to re-visit the experiments with MCF-10A cells using serum free media. 15. SUBJECT TERMS breast

  5. Automatic microscopy for mitotic cell location.

    NASA Technical Reports Server (NTRS)

    Herron, J.; Ranshaw, R.; Castle, J.; Wald, N.

    1972-01-01

    Advances are reported in the development of an automatic microscope with which to locate hematologic or other cells in mitosis for subsequent chromosome analysis. The system under development is designed to perform the functions of: slide scanning to locate metaphase cells; conversion of images of selected cells into binary form; and on-line computer analysis of the digitized image for significant cytogenetic data. Cell detection criteria are evaluated using a test sample of 100 mitotic cells and 100 artifacts.

  6. Mitotic spindle studied using picosecond laser scissors

    NASA Astrophysics Data System (ADS)

    Baker, N. M.; Botvinick, E. L.; Shi, Linda; Berns, M. B.; Wu, George

    2006-08-01

    In previous studies we have shown that the second harmonic 532 nm, from a picosecond frequency doubled Nd:YAG laser, can cleanly and selectively disrupt spindle fiber microtubules in live cells (Botvinick et al 2004, Biophys. J. 87:4303-4212). In the present study we have ablated different locations and amounts of the metaphase mitotic spindle, and followed the cells in order to observe the fate of the irradiated spindle and the ability of the cell to continue through mitosis. Cells of the rat kangaroo line (PTK2) were stably transfected by ECFP-tubulin and, using fluorescent microscopy and the automated RoboLase microscope, (Botvinick and Berns, 2005, Micros. Res. Tech. 68:65-74) brightly fluorescent individual cells in metaphase were irradiated with 0.2447 nJ/micropulse corresponding to an irradiance of 1.4496*10^7 J/(ps*cm^2) . Upon irradiation the exposed part of the mitotic spindle immediately lost fluorescence and the following events were observed in the cells over time: (1) immediate contraction of the spindle pole towards the cut, (2) recovery of connection between pole and cut microtubule, (3) completion of mitosis. This system should be very useful in studying internal cellular dynamics of the mitotic spindle.

  7. Chemically diverse microtubule stabilizing agents initiate distinct mitotic defects and dysregulated expression of key mitotic kinases.

    PubMed

    Rohena, Cristina C; Peng, Jiangnan; Johnson, Tyler A; Crews, Phillip; Mooberry, Susan L

    2013-04-15

    Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds.

  8. Chemically Diverse Microtubule Stabilizing Agents Initiate Distinct Mitotic Defects and Dysregulated Expression of Key Mitotic Kinases

    PubMed Central

    Rohena, Cristina C.; Peng, Jiangnan; Johnson, Tyler A.; Crews, Phillip; Mooberry, Susan L.

    2013-01-01

    Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent than paclitaxel or laulimalide, which is consistent with the distinct defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer. This suggests a common mechanism of aster formation. However, taccalonolide AJ also causes pericentrin accumulation on every spindle aster. The presence of pericentrin at every spindle aster initiated by taccalonolide AJ might facilitate the maintenance and stability of the highly focused asters formed by this stabilizer. Laulimalide and paclitaxel cause completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Delineating how diverse microtubule stabilizers affect mitotic signaling pathways could identify key proteins involved in modulating sensitivity and resistance to the antimitotic actions of these compounds. PMID:23399639

  9. The Nuclear Matrix Protein Megator Regulates Stem Cell Asymmetric Division through the Mitotic Checkpoint Complex in Drosophila Testes.

    PubMed

    Liu, Ying; Singh, Shree Ram; Zeng, Xiankun; Zhao, Jiangsha; Hou, Steven X

    2015-12-01

    In adult Drosophila testis, asymmetric division of germline stem cells (GSCs) is specified by an oriented spindle and cortically localized adenomatous coli tumor suppressor homolog 2 (Apc2). However, the molecular mechanism underlying these events remains unclear. Here we identified Megator (Mtor), a nuclear matrix protein, which regulates GSC maintenance and asymmetric division through the spindle assembly checkpoint (SAC) complex. Loss of Mtor function results in Apc2 mis-localization, incorrect centrosome orientation, defective mitotic spindle formation, and abnormal chromosome segregation that lead to the eventual GSC loss. Expression of mitotic arrest-deficient-2 (Mad2) and monopolar spindle 1 (Mps1) of the SAC complex effectively rescued the GSC loss phenotype associated with loss of Mtor function. Collectively our results define a new role of the nuclear matrix-SAC axis in regulating stem cell maintenance and asymmetric division.

  10. ECPR for Refractory Out-Of-Hospital Cardiac Arrest

    ClinicalTrials.gov

    2017-02-22

    Cardiac Arrest; Heart Arrest; Sudden Cardiac Arrest; Cardiopulmonary Arrest; Death, Sudden, Cardiac; Cardiopulmonary Resuscitation; CPR; Extracorporeal Cardiopulmonary Resuscitation; Extracorporeal Membrane Oxygenation

  11. LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs.

    PubMed

    Po'uha, S T; Shum, M S Y; Goebel, A; Bernard, O; Kavallaris, M

    2010-01-28

    LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH-EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.

  12. Distal ulnar growth arrest.

    PubMed

    Nelson, O A; Buchanan, J R; Harrison, C S

    1984-03-01

    Four cases of arrest of distal ulnar physeal growth occurring in children ages 7 to 13 years had follow-up for 2 to 10 years. Each patient developed bowing of the radial diaphysis, ulnar translation of the distal radial epiphysis, and increased ulnar angulation of the distal radiocarpal joint surface. Growth discrepancies were documented in both the ulna (range 2.2 to 3.9 cm) and to a lesser extent the radius (range 0.2 to 1.6 cm) when compared to the opposite forearm in each patient. The progression of deformity appeared to be greatest during adolescence. Radial deviation and pronation were limited to varying degrees in each case. No patient had significant pain or functional impairment, but the cosmetic appearance was always displeasing. Indications for surgical treatment include increasing ulnar angulation of the distal radial articular surface, progressive loss of motion, and displeasing cosmetic appearance.

  13. [Thrombolysis in cardiac arrest].

    PubMed

    Ruiz Bailén, M; Rucabado Aguilar, L; Morante Valle, A; Castillo Rivera, A

    2006-03-01

    Both acute myocardial infarction and pulmonary thromboembolism are responsible for a great number of cardiac arrests. Both present high rates of mortality. Thrombolysis has proved to be an effective treatment for acute myocardial infarction and pulmonary thromboembolism with shock. It would be worth considering whether thrombolysis could be effective and safe during or after cardiopulmonary resuscitation (CPR). Unfortunately, too few clinical studies presenting sufficient scientific data exist in order to respond adequately to this question. However, most studies they show that thrombolysis applied during and after CPR is a therapeutic option that is not associated with greater risk of serious hemorrhaging and could possibly have beneficial effects. On the other hand, experimental data exists which show that thrombolytics can attenuate neurological damage produced after CPR. Nevertheless, clinical trials would be necessary in order to adequately establish the effectiveness and safety of thrombolysis in patients who require CPR.

  14. Deep hypothermic circulatory arrest

    PubMed Central

    Ziganshin, Bulat A.

    2013-01-01

    Effective cerebral protection remains the principle concern during aortic arch surgery. Hypothermic circulatory arrest (HCA) is entrenched as the primary neuroprotection mechanism since the 70s, as it slows injury-inducing pathways by limiting cerebral metabolism. However, increases in HCA duration has been associated with poorer neurological outcomes, necessitating the adjunctive use of antegrade (ACP) and retrograde cerebral perfusion (RCP). ACP has superseded RCP as the preferred perfusion strategy as it most closely mimic physiological perfusion, although there exists uncertainty regarding several technical details, such as unilateral versus bilateral perfusion, flow rate and temperature, perfusion site, undue trauma to head vessels, and risks of embolization. Nevertheless, we believe that the convenience, simplicity and effectiveness of straight DHCA justifies its use in the majority of elective and emergency cases. The following perspective offers a historical and clinical comparison of the DHCA with other techniques of cerebral protection. PMID:23977599

  15. Cell cycle arrest and activation of development in marine invertebrate deuterostomes.

    PubMed

    Costache, Vlad; McDougall, Alex; Dumollard, Rémi

    2014-08-01

    Like most metazoans, eggs of echinoderms and tunicates (marine deuterostomes, there is no data for the cephalochordates) arrest awaiting fertilization due to the activity of the Mos/MEK/MAPK cascade and are released from this cell cycle arrest by sperm-triggered Ca2+ signals. Invertebrate deuterostome eggs display mainly three distinct types of cell cycle arrest before fertilization mediated by potentially different cytostatic factors (CSF): one CSF causes arrest during meiotic metaphase I (MI-CSF in tunicates and some starfishes), another CSF likely causes arrest during meiotic metaphase II (amphioxus), and yet another form of CSF causes arrest to occur after meiotic exit during G1 of the first mitotic cycle (G1-CSF). In tunicates and echinoderms these different CSF activities have been shown to rely on the Mos//MAPK pathway for establishment and on Ca2+ signals for their inactivation. Despite these molecular similarities, release of MI-CSF arrest is caused by APC/C activation (to destroy cyclin B) whereas release from G1-CSF is caused by stimulating S phase and the synthesis of cyclins. Further research is needed to understand how both the Mos//MAPK cascade and Ca2+ achieve these tasks in different marine invertebrate deuterostomes. Another conserved feature of eggs is that protein synthesis of specific mRNAs is necessary to proceed through oocyte maturation and to maintain CSF-induced cell cycle arrest. Then activation of development at fertilization is accompanied by an increase in the rate of protein synthesis but the mechanisms involved are still largely unknown in most of the marine deuterostomes. How the sperm-triggered Ca2+ signals cause an increase in protein synthesis has been studied mainly in sea urchin eggs. Here we review these conserved features of eggs (arrest, activation and protein synthesis) focusing on the non-vertebrate deuterostomes.

  16. The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

    PubMed Central

    Sivakumar, Sushama; Daum, John R.; Tipton, Aaron R.; Rankin, Susannah; Gorbsky, Gary J.

    2014-01-01

    The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit. PMID:24403607

  17. Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells.

    PubMed

    Stan, Silvia D; Zeng, Yan; Singh, Shivendra V

    2008-01-01

    Withaferin A (WA) is derived from the medicinal plant Withania somnifera that has been safely used for centuries in the Indian Ayurvedic medicine for treatment of various ailments. We now demonstrate that WA treatment causes G2 and mitotic arrest in human breast cancer cells. Treatment of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) cell lines with WA resulted in a concentration- and time-dependent increase in G2-M fraction, which correlated with a decrease in levels of cyclin-dependent kinase 1 (Cdk1), cell division cycle 25C (Cdc25C) and/or Cdc25B proteins, leading to accumulation of Tyrosine15 phosphorylated (inactive) Cdk1. Ectopic expression of Cdc25C conferred partial yet significant protection against WA-mediated G2-M phase cell cycle arrest in MDA-MB-231 cells. The WA-treated MDA-MB-231 and MCF-7 cells were also arrested in mitosis as judged by fluorescence microscopy and analysis of Ser10 phosphorylated histone H3. Mitotic arrest resulting from exposure to WA was accompanied by an increase in the protein level of anaphase promoting complex/cyclosome substrate securin. In conclusion, the results of this study suggest that G2-M phase cell cycle arrest may be an important mechanism in antiproliferative effect of WA against human breast cancer cells.

  18. Acquired Upper Extremity Growth Arrest.

    PubMed

    Gauger, Erich M; Casnovsky, Lauren L; Gauger, Erica J; Bohn, Deborah C; Van Heest, Ann E

    2016-09-29

    This study reviewed the clinical history and management of acquired growth arrest in the upper extremity in pediatric patients. The records of all patients presenting from 1996 to 2012 with radiographically proven acquired growth arrest were reviewed. Records were examined to determine the etiology and site of growth arrest, management, and complications. Patients with tumors or hereditary etiology were excluded. A total of 44 patients (24 boys and 20 girls) with 51 physeal arrests who presented at a mean age of 10.6 years (range, 0.8-18.2 years) were included in the study. The distal radius was the most common site (n=24), followed by the distal humerus (n=8), metacarpal (n=6), distal ulna (n=5), proximal humerus (n=4), radial head (n=3), and olecranon (n=1). Growth arrest was secondary to trauma (n=22), infection (n=11), idiopathy (n=6), inflammation (n=2), compartment syndrome (n=2), and avascular necrosis (n=1). Twenty-six patients (59%) underwent surgical intervention to address deformity caused by the physeal arrest. Operative procedures included ipsilateral unaffected bone epiphysiodesis (n=21), shortening osteotomy (n=10), lengthening osteotomy (n=8), excision of physeal bar or bone fragment (n=2), angular correction osteotomy (n=1), and creation of single bone forearm (n=1). Four complications occurred; 3 of these required additional procedures. Acquired upper extremity growth arrest usually is caused by trauma or infection, and the most frequent site is the distal radius. Growth disturbances due to premature arrest can be treated effectively with epiphysiodesis or osteotomy. In this series, the specific site of anatomic growth arrest was the primary factor in determining treatment. [Orthopedics. 201x; xx(x):xx-xx.].

  19. The Golgi mitotic checkpoint is controlled by BARS-dependent fission of the Golgi ribbon into separate stacks in G2.

    PubMed

    Colanzi, Antonino; Hidalgo Carcedo, Cristina; Persico, Angela; Cericola, Claudia; Turacchio, Gabriele; Bonazzi, Matteo; Luini, Alberto; Corda, Daniela

    2007-05-16

    The Golgi ribbon is a complex structure of many stacks interconnected by tubules that undergo fragmentation during mitosis through a multistage process that allows correct Golgi inheritance. The fissioning protein CtBP1-S/BARS (BARS) is essential for this, and is itself required for mitotic entry: a block in Golgi fragmentation results in cell-cycle arrest in G2, defining the 'Golgi mitotic checkpoint'. Here, we clarify the precise stage of Golgi fragmentation required for mitotic entry and the role of BARS in this process. Thus, during G2, the Golgi ribbon is converted into isolated stacks by fission of interstack connecting tubules. This requires BARS and is sufficient for G2/M transition. Cells without a Golgi ribbon are independent of BARS for Golgi fragmentation and mitotic entrance. Remarkably, fibroblasts from BARS-knockout embryos have their Golgi complex divided into isolated stacks at all cell-cycle stages, bypassing the need for BARS for Golgi fragmentation. This identifies the precise stage of Golgi fragmentation and the role of BARS in the Golgi mitotic checkpoint, setting the stage for molecular analysis of this process.

  20. High throughput screening of natural products for anti-mitotic effects in MDA-MB-231 human breast carcinoma cells

    PubMed Central

    Mazzio, E; Badisa, R; Mack, N; Deiab, S; Soliman, KFA

    2013-01-01

    Some of the most effective anti-mitotic microtubule-binding agents, such as paclitaxel (Taxus brevifolia) were originally discovered through robust NCI botanical screenings. In this study, a high-through microarray format was utilized to screen 897 aqueous extracts of commonly used natural products (0.00015–0.5 mg/ml) relative to paclitaxel for anti-mitotic effects (independent of toxicity) on proliferation of MDA-MB-231 cells. The data obtained showed that less than 1.34 % tested showed inhibitory growth (IG50) properties <0.0183 mg/ml. The most potent anti-mitotics (independent of toxicity) were Mandrake root (Podophyllum peltatum), Truja Twigs (Thuja occidentalis), Colorado desert mistletoe (Phoradendron flavescens), Tou Gu Cao Speranskia Herb (Speranskia tuberculata), Bentonite Clay, Bunge Root (Pulsatilla chinensis), Brucea Fruit (Brucea javanica), Madder Root (Rubia tinctorum), Gallnut of Chinese Sumac (Melaphis chinensis), Elecampane Root (Inula Helenium), Yuan Zhi Root (Polygala tenuifolia), Pagoda Tree Fruit (Melia Toosendan), Stone Root (Collinsonia Canadensis) and others such as American Witchhazel, Arjun and Bladderwrack. The strongest tumoricidal herbs identified from amongst the subset evaluated for anti-mitotic properties were wild yam (Dioscorea villosa), beth-root (Trillium Pendulum) and alkanet-root (Lithospermum canescens). Additional data was obtained on a lesser-recognized herb: (Speranskia tuberculata) which showed growth inhibition on BT-474 (human ductal breast carcinoma) and Ishikawa (human endometrial adenocarcinoma) cells with ability to block replicative DNA synthesis leading to G2 arrest in MDA-MB-231 cells. In conclusion, these findings present relative potency of natural anti-mitotic resources effective against human breast carcinoma MDA-MB-231 cell division. PMID:24105850

  1. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

    PubMed Central

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G.; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S.

    2015-01-01

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling. PMID:25883264

  2. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.

    PubMed

    Shuda, Masahiro; Velásquez, Celestino; Cheng, Erdong; Cordek, Daniel G; Kwun, Hyun Jin; Chang, Yuan; Moore, Patrick S

    2015-05-12

    Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

  3. [Arrest of maturation in spermatogenesis].

    PubMed

    Francavilla, S; Bellocci, M; Martini, M; Bruno, B; Moscardelli, S; Fabbrini, A; Properzi, G

    1982-07-30

    The ultrastructural aspects of the germinal epithelium of 10 infertile men affected by maturative arrest of spermatogenesis were studied. We noted an increased number of malformed germinal cells. Marginal nuclear vescicles were present in spermatogonia of patients affected by spermatogonial arrest. The few spermatid present in the germinal epithelium of the patients affected by a spermatidic arrest presented changes of the nuclear condensation, the acrosome, and the tail. The Sertoli cells presented an immature aspect of the nucleus and changes of the "mantle". A possible correlation between the Sertoli cells changes and the altered spermatogenesis was proposed.

  4. Meiotic and mitotic recombination in meiosis.

    PubMed

    Kohl, Kathryn P; Sekelsky, Jeff

    2013-06-01

    Meiotic crossovers facilitate the segregation of homologous chromosomes and increase genetic diversity. The formation of meiotic crossovers was previously posited to occur via two pathways, with the relative use of each pathway varying between organisms; however, this paradigm could not explain all crossovers, and many of the key proteins involved were unidentified. Recent studies that identify some of these proteins reinforce and expand the model of two meiotic crossover pathways. The results provide novel insights into the evolutionary origins of the pathways, suggesting that one is similar to a mitotic DNA repair pathway and the other evolved to incorporate special features unique to meiosis.

  5. Mitotic recombination of chromosome 17 in astrocytomas

    SciTech Connect

    James, C.D.; Carlbom, E.; Nordenskjold, M.; Collins, V.P.; Cavenee, W.K. )

    1989-04-01

    Allelic combinations at seven loci on human chromosome 17 defined by restriction fragment length polymorphisms were determined in tumor and normal tissues from 35 patients with gliomas. Loss of constitutional heterozygosity at one or more of these loci was observed in 8 of the 24 tumors displaying astrocytic differentiation and in the single primitive neuroectodermal tumor examined. The astrocytomas showing these losses included examples of each adult malignancy grade of the disease, including glioblastoma (malignancy grade IV), and seven of them demonstrated concurrent maintenance of heterozygosity for at least one chromosome 17 locus. Determination of allele dosage together with the genotypic data indicated that the tumor chromosomes 17 were derived by mitotic recombination in 7 of the 9 cases with shared homozygosity of the region 17p11.2-ptr in all cases. In contrast, tumors of oligodendrocytic, ependymal, or mixed cellular differentiation did not exhibit loss of alleles at any of the loci examined. These data suggest that the somatic attainment of homozygosity for loci on chromosome 17p is frequently associated with the oncogenesis of central nervous system tumors, particularly those showing solely astrocytic differentiation, and that mitotic recombination mapping is a useful approach towards the subregional localization of a locus whose rearrangement is involved in this disease.

  6. Divergence of mitotic strategies in fission yeasts

    PubMed Central

    Gu, Ying; Yam, Candice; Oliferenko, Snezhana

    2012-01-01

    The aim of mitosis is to produce two daughter nuclei, each containing a chromosome complement identical to that of the mother nucleus. This can be accomplished through a variety of strategies, with “open” and “closed” modes of mitosis positioned at the opposite ends of the spectrum and a range of intermediate patterns in between. In the “closed” mitosis, the nuclear envelope remains intact throughout the nuclear division. In the “open” division type, the envelope of the original nucleus breaks down early in mitosis and reassembles around the segregated daughter genomes. In any case, the nuclear membrane has to remodel to accommodate the mitotic spindle assembly, chromosome segregation and formation of the daughter nuclei. We have recently shown that within the fission yeast clade, the mitotic control of the nuclear surface area may determine the choice between the nuclear envelope breakdown and a fully “closed” division. Here we discuss our data and argue that comparative cell biology studies using two fission yeast species, Schizosaccharomyces pombe and Schizosaccharomyces japonicus, could provide unprecedented insights into physiology and evolution of mitosis. PMID:22572960

  7. Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions.

    PubMed

    Meijer, L; Pondaven, P

    1988-01-01

    Fertilized sea urchin eggs undergo a series of rapid and synchronized mitotic divisions. Extracts were made at various times throughout the first three mitotic divisions and assayed for phosphorylating activity toward histone H1. Histone H1 kinase (HH1K) undergoes a transient activation (8- to 10-fold increase) 20 min before each cleavage. The amplitude of the HH1K peak strongly depends on the synchrony of the egg population. Concomitant cytological observations show that the time-course of HH1K correlates with the time-course of nuclear envelope breakdown and of metaphase. This correlation is observed at each cell division cycle. HH1K from each of the three first mitoses show identical time- and concentration-dependence curves as well as identical dose-inhibition curves with 6-dimethylaminopurine and quercetin, suggesting that the same (group of) kinase(s) is (are) activated before each cleavage. Ionophore A23187 does not trigger, but inhibits, HH1K activation; however, partial activation of the eggs with ammonia at pH 9.0 (but not at pH 8.0) triggers the transient HH1K activation. Appearance of the HH1K cycle requires protein synthesis since it is completely abolished in emetine-treated eggs. Although cytochalasin B blocks egg cleavage, it does not inhibit HH1K activation nor nuclear divisions. A prolonged HH1K activation cycle is observed in eggs arrested in metaphase with colchicine or nocodazole. Despite the existence of a cycle in cAMP concentration during mitosis, forskolin, an activator of adenylate cyclase, does not modify the time-course of HH1K activation and of cell division. The cycling HH1K is independent of calcium-calmodulin, calcium-phospholipids, or cyclic AMP. It clearly resembles the mammalian "growth-associated histone kinase." The relationship between the transient activation of HH1K and the intracellular mitotic factors driving the cell cycle is discussed.

  8. The moyamoya disease susceptibility variant RNF213 R4810K (rs112735431) induces genomic instability by mitotic abnormality

    SciTech Connect

    Hitomi, Toshiaki; Habu, Toshiyuki; Kobayashi, Hatasu; Okuda, Hiroko; Harada, Kouji H.; Osafune, Kenji; Taura, Daisuke; Sone, Masakatsu; Asaka, Isao; Ameku, Tomonaga; Watanabe, Akira; Kasahara, Tomoko; Sudo, Tomomi; Shiota, Fumihiko; Hashikata, Hirokuni; Takagi, Yasushi; Morito, Daisuke; Miyamoto, Susumu; Nakao, Kazuwa; Koizumi, Akio

    2013-10-04

    Highlights: •Overexpression of RNF213 R4810K inhibited cell proliferation. •Overexpression of RNF213 R4810K had the time of mitosis 4-fold and mitotic failure. •R4810K formed a complex with MAD2 more readily than wild-type. •iPSECs from the MMD patients had elevated mitotic failure compared from the control. •RNF213 R4810K induced mitotic abnormality and increased risk of aneuploidy. -- Abstract: Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n = 3, 61.0 ± 8.2%) compared with wild-type subjects (n = 6, 13.1 ± 7.7%; p < 0.01). Aneuploidy was observed more frequently in fibroblasts (p < 0.01) and induced pluripotent stem cells (iPSCs) (p < 0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p < 0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p < 0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.

  9. A three-dimensional approach to mitotic chromosome structure: evidence for a complex hierarchical organization

    PubMed Central

    1987-01-01

    We describe findings on the architecture of Drosophila melanogaster mitotic chromosomes, made using a three-dimensional-oriented structural approach. Using high-voltage and conventional transmission electron microscopy combined with axial tomography and digital contrast- enhancement techniques, we have for the first time visualized significant structural detail within minimally perturbed mitotic chromosomes. Chromosomes prepared by several different preparative procedures showed a consistent size hierarchy of discrete chromatin structural domains with cross-sectional diameters of 120, 240, 400-500, and 800-1,000 A. In fully condensed, metaphase-arrested chromosomes, there is evidence for even larger-scale structural organization in the range of 1,300-3,000-A size. The observed intrachromosomal arrangements of these higher-order structural domains show that both the radial loop and sequential helical coiling models of chromosome structure are over- simplifications of the true situation. Finally, our results suggest that the pathway of chromatin condensation through mitosis consists of concurrent changes occurring at several levels of chromatin organization, rather than a strictly sequential folding process. PMID:3112167

  10. NITROUS OXIDE: EFFECTS ON THE MITOTIC APPARATUS AND CHROMOSOME MOVEMENT IN HELA CELLS

    PubMed Central

    Brinkley, B. R.; Rao, Potu N.

    1973-01-01

    When HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy. When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G1 synchronously. Ultrastructural studies of N2O-blocked cells revealed a bipolar spindle with centriole pairs at each pole. Both chromosomal and interpolar (pole-to-pole) microtubules were also present. Thus, N2O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly. However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement. The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis. These ultrastructural studies indicated that N2O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents. Like Colcemid, however, prolonged arrest in mitosis with N2O led to an increased incidence of multipolar spindles. PMID:4726309

  11. Absence of a conventional spindle mitotic checkpoint in the binucleated single-celled parasite Giardia intestinalis.

    PubMed

    Markova, Kristyna; Uzlikova, Magdalena; Tumova, Pavla; Jirakova, Klara; Hagen, Guy; Kulda, Jaroslav; Nohynkova, Eva

    2016-10-01

    The spindle assembly checkpoint (SAC) joins the machinery of chromosome-to-spindle microtubule attachment with that of the cell cycle to prevent missegregation of chromosomes during mitosis. Although a functioning SAC has been verified in a limited number of organisms, it is regarded as an evolutionarily conserved safeguard mechanism. In this report, we focus on the existence of the SAC in a single-celled parasitic eukaryote, Giardia intestinalis. Giardia belongs to Excavata, a large and diverse supergroup of unicellular eukaryotes in which SAC control has been nearly unexplored. We show that Giardia cells with absent or defective mitotic spindles due to the inhibitory effects of microtubule poisons do not arrest in mitosis; instead, they divide without any delay, enter the subsequent cell cycle and even reduplicate DNA before dying. We identified a limited repertoire of kinetochore and SAC components in the Giardia genome, indicating that this parasite is ill equipped to halt mitosis before the onset of anaphase via SAC control of chromosome-spindle microtubule attachment. Finally, based on overexpression, we show that Giardia Mad2, a core SAC protein in other eukaryotes, localizes along intracytoplasmic portions of caudal flagellar axonemes, but never within nuclei, even in mitotic cells with blocked spindles, where the SAC should be active. These findings are consistent with the absence of a conventional SAC, known from yeast and metazoans, in the parasitic protist Giardia.

  12. Juvenile Arrests, 1998. Juvenile Justice Bulletin.

    ERIC Educational Resources Information Center

    Snyder, Howard N.

    This report provides a summary and analysis of national and state juvenile arrest data in the United States. In 1998, law enforcement agencies made an estimated 2.6 million arrests of persons under age 18. Federal Bureau of Investigations statistics indicate that juveniles account for 18% of all arrests, and 17% of all violent crime arrests in…

  13. Statistical correlations of crime with arrests

    NASA Astrophysics Data System (ADS)

    Kuelling, Albert C.

    1997-01-01

    Regression analysis shows that the overall crime rate correlates with the overall arrest rate. Violent crime only weakly correlates with the violent arrest rate, but strongly correlates with the property arrest rate. Contrary to common impressions, increasing arrest rates do not significantly increase loading on incarceration facilities.

  14. Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

    PubMed

    Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

    2014-01-01

    Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological

  15. Curcumin affects components of the chromosomal passenger complex and induces mitotic catastrophe in apoptosis-resistant Bcr-Abl-expressing cells.

    PubMed

    Wolanin, Kamila; Magalska, Adriana; Mosieniak, Grazyna; Klinger, Rut; McKenna, Sharon; Vejda, Susanne; Sikora, Ewa; Piwocka, Katarzyna

    2006-07-01

    The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G(2)-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells-morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid

  16. Two different mitotic checkpoint inhibitors of the anaphase-promoting complex/cyclosome antagonize the action of the activator Cdc20

    PubMed Central

    Eytan, Esther; Braunstein, Ilana; Ganoth, Dvora; Teichner, Adar; Hittle, James C.; Yen, Tim J.; Hershko, Avram

    2008-01-01

    The mitotic checkpoint system ensures the fidelity of chromosome segregation by preventing the completion of mitosis in the presence of any misaligned chromosome. When activated, it blocks the initiation of anaphase by inhibiting the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Little is known about the biochemical mechanisms by which this system inhibits APC/C, except for the existence of a mitotic checkpoint complex (MCC) inhibitor of APC/C composed of the APC/C activator Cdc20 associated with the checkpoint proteins Mad2, BubR1, and Bub3. We have been studying the mechanisms of the mitotic checkpoint system in extracts that reproduce its downstream events. We found that inhibitory factors are associated with APC/C in the checkpoint-arrested state, which can be recovered from immunoprecipitates. Only a part of the inhibitory activity was caused by MCC [Braunstein I, Miniowitz S, Moshe Y, Hershko A (2007) Proc Natl Acad Sci USA 104:4870–4875]. Here, we show that during exit from checkpoint, rapid disassembly of MCC takes place while APC/C is still inactive. This observation suggested the possible involvement of multiple factors in the regulation of APC/C by the mitotic checkpoint. We have separated a previously unknown inhibitor of APC/C from MCC. This inhibitor, called mitotic checkpoint factor 2 (MCF2), is associated with APC/C only in the checkpoint-arrested state. The inhibition of APC/C by both MCF2 and MCC was decreased at high concentrations of Cdc20. We propose that both MCF2 and MCC inhibit APC/C by antagonizing Cdc20, possibly by interaction with the Cdc20-binding site of APC/C. PMID:18591651

  17. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

    PubMed

    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  18. Radiation-induced senescence-like terminal growth arrest in thyroid cells.

    PubMed

    Podtcheko, Alexei; Namba, Hiroyuki; Saenko, Vladimir; Ohtsuru, Akira; Starenki, Dmitriy; Meirmanov, Serik; Polona, Iryna; Rogounovitch, Tatiana; Yamashita, Shunichi

    2005-04-01

    Premature senescence may play an important role as an acute, drug-, or ionizing radiation (IR)-inducible growth arrest program along with interphase apoptosis and mitotic catastrophe. The aim of the study was to evaluate whether IR can induce senescence-like phenotype (SLP) associated with terminal growth arrest in the thyroid cells, and if so, to evaluate impact of terminal growth arrest associated with SLP in intrinsic radiosensitivity of various thyroid carcinomas. The induction of SLP in thyroid cells were identified by: (1) senescence associated beta-galactosidase (SA-beta-Gal) staining method, (2) dual-flow cytometric analysis of cell proliferation and side light scatter using vital staining with PKH-2 fluorescent dye, (3) double labeling for 5-bromodeoxyuridine and SA- beta-Gal, (4) Staining for SA-beta-Gal with consequent antithyroglobulin immunohistochemistry. IR induced SLP associated with terminal growth arrest in four thyroid cancer cells lines and in primary thyrocytes in time- and dose-dependent manner. Analysis of relationship between induction of SLP and radiosensitivity revealed a trend in which more radioresistant cell lines strongly tended to show lower specific SLP yields (r = -0.93, p = 0.068). We find out that SA-beta-Gal staining is detectable in irradiated ARO xenotransplants, but not in control tumors. We, therefore, conclude that induction of SLP with terminal growth arrest contribute to the elimination of clonogenic populations after IR.

  19. Dephosphorylation and subcellular compartment change of the mitotic Bloom's syndrome DNA helicase in response to ionizing radiation.

    PubMed

    Dutertre, Stéphanie; Sekhri, Redha; Tintignac, Lionel A; Onclercq-Delic, Rosine; Chatton, Bruno; Jaulin, Christian; Amor-Guéret, Mounira

    2002-02-22

    Bloom's syndrome is a rare human autosomal recessive disorder that combines a marked genetic instability and an increased risk of developing all types of cancers and which results from mutations in both copies of the BLM gene encoding a RecQ 3'-5' DNA helicase. We recently showed that BLM is phosphorylated and excluded from the nuclear matrix during mitosis. We now show that the phosphorylated mitotic BLM protein is associated with a 3'-5' DNA helicase activity and interacts with topoisomerase III alpha. We demonstrate that in mitosis-arrested cells, ionizing radiation and roscovitine treatment both result in the reversion of BLM phosphorylation, suggesting that BLM could be dephosphorylated through the inhibition of cdc2 kinase. This was supported further by our data showing that cdc2 kinase activity is inhibited in gamma-irradiated mitotic cells. Finally we show that after ionizing radiation, BLM is not involved in the establishment of the mitotic DNA damage checkpoint but is subjected to a subcellular compartment change. These findings lead us to propose that BLM may be phosphorylated during mitosis, probably through the cdc2 pathway, to form a pool of rapidly available active protein. Inhibition of cdc2 kinase after ionizing radiation would lead to BLM dephosphorylation and possibly to BLM recruitment to some specific sites for repair.

  20. Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis*

    PubMed Central

    Zhu, Lijuan; Wang, Zhikai; Wang, Wenwen; Wang, Chunli; Hua, Shasha; Su, Zeqi; Brako, Larry; Garcia-Barrio, Minerva; Ye, Mingliang; Wei, Xuan; Zou, Hanfa; Ding, Xia; Liu, Lifang; Liu, Xing; Yao, Xuebiao

    2015-01-01

    Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation. PMID:26378239

  1. Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

    PubMed Central

    Min, Mingwei; Mevissen, Tycho E. T.; De Luca, Maria; Komander, David; Lindon, Catherine

    2015-01-01

    The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner. PMID:26446837

  2. Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis.

    PubMed

    Zhu, Lijuan; Wang, Zhikai; Wang, Wenwen; Wang, Chunli; Hua, Shasha; Su, Zeqi; Brako, Larry; Garcia-Barrio, Minerva; Ye, Mingliang; Wei, Xuan; Zou, Hanfa; Ding, Xia; Liu, Lifang; Liu, Xing; Yao, Xuebiao

    2015-11-06

    Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.

  3. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

    SciTech Connect

    Yang, Feng; Camp, David G.; Gritsenko, Marina A.; Luo, Quanzhou; Kelly, Ryan T.; Clauss, Therese RW; Brinkley, William R.; Smith, Richard D.; Stenoien, David L.

    2007-11-16

    The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins including SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.

  4. Mitotic entry: Non-genetic heterogeneity exposes the requirement for Plk1.

    PubMed

    Aspinall, Claire F; Zheleva, Daniella; Tighe, Anthony; Taylor, Stephen S

    2015-11-03

    The quest to develop novel antimitotic chemotherapy agents has led to the generation of several small molecule inhibitors targeting Plk1, a protein kinase required for multiple aspects of cell division. Previous studies have shown that upon exposure to Plk1 inhibitors, cells enter mitosis, delay briefly in prophase and then arrest in mitosis due to an inability to undergo centrosome separation. Here, we show that four different classes of Plk1 inhibitor block mitotic entry in several cancer cell lines and non-transformed RPE-1 cells. The proportion of cells that arrest in G2 is cell line and concentration dependent, and is subject to non-genetic heterogeneity. Following inhibitor washout, the G2 block is alleviated and cells enter mitosis but then fail to complete cell division indicating that most Plk1 inhibitors are not fully reversible. An exception is CYC140844; in contrast to five other inhibitors examined here, this novel Plk1 inhibitor is fully reversible. We discuss the implications for developing Plk1 inhibitors as chemotherapy agents and research tools.

  5. THE CHEMOTHERAPY OF CARDIAC ARREST.

    PubMed

    MINUCK, M

    1965-01-02

    Direct-air ventilation, external cardiac compression, and external defibrillation are established techniques for patients who unexpectedly develop cardiac arrest. The proper use of drugs can increase the incidence of successful resuscitation. Intracardiac adrenaline (epinephrine) acts as a powerful stimulant during cardiac standstill and, in addition, converts fine ventricular fibrillation to a coarser type, more responsive to electrical defibrillation. Routine use of intravenous sodium bicarbonate is recommended to combat the severe metabolic acidosis accompanying cardiac arrest. Lidocaine is particularly useful when ventricular fibrillation or ventricular tachycardia tends to recur. Analeptics are contraindicated, since they invariably increase oxygen requirements of already hypoxic cerebral tissues. The following acrostic is a useful mnemonic for recalling the details of the management of cardiac arrest in their proper order: A (Airway), B (Breathing), C (Circulation), D (Diagnosis of underlying cause), E (Epinephrine), F (Fibrillation), G (Glucose intravenously), pH (Sodium bicarbonate), I (Intensive care).

  6. Torin1-mediated TOR kinase inhibition reduces Wee1 levels and advances mitotic commitment in fission yeast and HeLa cells.

    PubMed

    Atkin, Jane; Halova, Lenka; Ferguson, Jennifer; Hitchin, James R; Lichawska-Cieslar, Agata; Jordan, Allan M; Pines, Jonathon; Wellbrock, Claudia; Petersen, Janni

    2014-03-15

    The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.

  7. The Spo12 protein of Saccharomyces cerevisiae: a regulator of mitotic exit whose cell cycle-dependent degradation is mediated by the anaphase-promoting complex.

    PubMed Central

    Shah, R; Jensen, S; Frenz, L M; Johnson, A L; Johnston, L H

    2001-01-01

    The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis. PMID:11729145

  8. Analysis of Crack Arrest Toughness.

    DTIC Science & Technology

    1988-01-15

    vload(m) vp tn(m) Vertical Source Load (kN) on wedge HY80 Finite Element 0.0122 0.0099 3.81x10 -4 144 Steel Calculations Experiment 0.0122 --- 3.74x10-4...curve, are bona fide measures of the fracture arrest capability of tough ductile steels . The second is that the J-values represent the crack driving...fibrous mode of crack extension. (b) A new test method for studying fast fracture and arrest in tough steels . (c) Measurements of fast fracture and crack

  9. Activation of JNK triggers release of Brd4 from mitotic chromosomes and mediates protection from drug-induced mitotic stress.

    PubMed

    Nishiyama, Akira; Dey, Anup; Tamura, Tomohiko; Ko, Minoru; Ozato, Keiko

    2012-01-01

    Some anti-cancer drugs, including those that alter microtubule dynamics target mitotic cells and induce apoptosis in some cell types. However, such drugs elicit protective responses in other cell types allowing cells to escape from drug-induced mitotic inhibition. Cells with a faulty protective mechanism undergo defective mitosis, leading to genome instability. Brd4 is a double bromodomain protein that remains on chromosomes during mitosis. However, Brd4 is released from mitotic chromosomes when cells are exposed to anti-mitotic drugs including nocodazole. Neither the mechanisms, nor the biological significance of drug-induced Brd4 release has been fully understood. We found that deletion of the internal C-terminal region abolished nocodazole induced Brd4 release from mouse P19 cells. Furthermore, cells expressing truncated Brd4, unable to dissociate from chromosomes were blocked from mitotic progression and failed to complete cell division. We also found that pharmacological and peptide inhibitors of the c-jun-N-terminal kinases (JNK) pathway, but not inhibitors of other MAP kinases, prevented release of Brd4 from chromosomes. The JNK inhibitor that blocked Brd4 release also blocked mitotic progression. Further supporting the role of JNK in Brd4 release, JNK2-/- embryonic fibroblasts were defective in Brd4 release and sustained greater inhibition of cell growth after nocodazole treatment. In sum, activation of JNK pathway triggers release of Brd4 from chromosomes upon nocodazole treatment, which mediates a protective response designed to minimize drug-induced mitotic stress.

  10. Significant decrease of ADP release rate underlies the potent activity of dimethylenastron to inhibit mitotic kinesin Eg5 and cancer cell proliferation

    SciTech Connect

    Sun, Linlin; Sun, Xiaodong; Xie, Songbo; Yu, Haiyang; Zhong, Diansheng

    2014-05-09

    Highlights: • DIMEN displays higher anti-proliferative activity than enastron. • DIMEN induced mitotic arrest and apoptosis more significantly than enastron. • DIMEN blocked the conformational change of ADP-binding pocket more effectively. • DIMEN hindered ADP release more potently than enastron. - Abstract: Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreatic and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.

  11. Cardiac arrest during dipyridamole imaging

    SciTech Connect

    Blumenthal, M.S.; McCauley, C.S.

    1988-05-01

    A case of cardiac arrest and subsequent acute myocardial infarction occurring during thallium-201 imaging with oral dipyridamole augmentation is presented. Previous reports emphasizing the safety of this procedure are briefly reviewed and a recommendation for close hemodynamic and arrhythmia monitoring during the study is made. Large doses of oral dipyridamole may be contraindicated in patients with unstable angina.

  12. DUI Arrests and Academic Attrition

    ERIC Educational Resources Information Center

    Thompson, Kevin M.; Richardson, Katie

    2008-01-01

    A sobering 2002 study reported that over 2 million college students drove under the influence of alcohol (DUI) in 1999. Among those driving while intoxicated, approximately 1.7% or roughly 34,000 students reported being arrested on DUI charges. Regrettably, a significant proportion of the 1,400 college student deaths and 500,000 injuries are…

  13. Gentle arrester for moving bodies

    NASA Technical Reports Server (NTRS)

    Hull, R. A.

    1981-01-01

    Wire cable absorbs energy at constant rate with reduced shock and rebounding. Cable typically elongates to 90 percent of its potential, but is surrounded by braided sheath to absorb remaining energy should it break prematurely. Applications of arrester include passenger restraint in air and land vehicles, parachute risers, and ground snatch by aircraft. Possible cable material is type 302 stainless steel.

  14. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    PubMed

    Shim, Hee Jin; Lee, Eun-Mi; Nguyen, Long Duy; Shim, Jaekyung; Song, Young-Han

    2014-01-01

    Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

  15. Sudden Cardiac Arrest (SCA) Risk Assessment

    MedlinePlus

    ... HRS Find a Specialist Share Twitter Facebook SCA Risk Assessment Sudden Cardiac Arrest (SCA) occurs abruptly and without ... people of all ages and health conditions. Start Risk Assessment The Sudden Cardiac Arrest (SCA) Risk Assessment Tool ...

  16. Cardiac Arrest: MedlinePlus Health Topic

    MedlinePlus

    ... dying from a second SCA. NIH: National Heart, Lung, and Blood Institute Start Here About Cardiac Arrest (American Heart ... Society) What Is Sudden Cardiac Arrest? (National Heart, Lung, and Blood Institute) Latest News How Devices in Public Places ...

  17. Cardiac arrest: resuscitation and reperfusion.

    PubMed

    Patil, Kaustubha D; Halperin, Henry R; Becker, Lance B

    2015-06-05

    The modern treatment of cardiac arrest is an increasingly complex medical procedure with a rapidly changing array of therapeutic approaches designed to restore life to victims of sudden death. The 2 primary goals of providing artificial circulation and defibrillation to halt ventricular fibrillation remain of paramount importance for saving lives. They have undergone significant improvements in technology and dissemination into the community subsequent to their establishment 60 years ago. The evolution of artificial circulation includes efforts to optimize manual cardiopulmonary resuscitation, external mechanical cardiopulmonary resuscitation devices designed to augment circulation, and may soon advance further into the rapid deployment of specially designed internal emergency cardiopulmonary bypass devices. The development of defibrillation technologies has progressed from bulky internal defibrillators paddles applied directly to the heart, to manually controlled external defibrillators, to automatic external defibrillators that can now be obtained over-the-counter for widespread use in the community or home. But the modern treatment of cardiac arrest now involves more than merely providing circulation and defibrillation. As suggested by a 3-phase model of treatment, newer approaches targeting patients who have had a more prolonged cardiac arrest include treatment of the metabolic phase of cardiac arrest with therapeutic hypothermia, agents to treat or prevent reperfusion injury, new strategies specifically focused on pulseless electric activity, which is the presenting rhythm in at least one third of cardiac arrests, and aggressive post resuscitation care. There are discoveries at the cellular and molecular level about ischemia and reperfusion pathobiology that may be translated into future new therapies. On the near horizon is the combination of advanced cardiopulmonary bypass plus a cocktail of multiple agents targeted at restoration of normal metabolism and

  18. Immunochemical studies of 22S protein from isolated mitotic apparatus.

    PubMed

    Bibring, T; Baxandall, J

    1969-05-01

    Evidence is presented that the "22S protein" of mitotic apparatus isolated from sea urchin eggs is not microtubule protein. An antibody preparation active against 22S protein is described, and immunochemical studies of the distribution of 22S protein in various cellular fractions and among morphological features of mitotic apparatus are reported. The protein is ubiquitous in the metaphase egg fractions that were tested but is not found in sperm flagella. It is immunologically distinct from proposed microtubule protein isolated from mitotic apparatus by the method of Sakai, and from proposed microtubule protein obtained after extraction with mild acid. It exists in nontubule material of isolated mitotic apparatus but is not detectable in microtubules.

  19. IMMUNOCHEMICAL STUDIES OF 22S PROTEIN FROM ISOLATED MITOTIC APPARATUS

    PubMed Central

    Bibring, Thomas; Baxandall, Jane

    1969-01-01

    Evidence is presented that the "22S protein" of mitotic apparatus isolated from sea urchin eggs is not microtubule protein. An antibody preparation active against 22S protein is described, and immunochemical studies of the distribution of 22S protein in various cellular fractions and among morphological features of mitotic apparatus are reported. The protein is ubiquitous in the metaphase egg fractions that were tested but is not found in sperm flagella. It is immunologically distinct from proposed microtubule protein isolated from mitotic apparatus by the method of Sakai, and from proposed microtubule protein obtained after extraction with mild acid. It exists in nontubule material of isolated mitotic apparatus but is not detectable in microtubules. PMID:4977446

  20. Micromechanical study of mitotic chromosome structure

    NASA Astrophysics Data System (ADS)

    Marko, John

    2011-03-01

    Our group has developed micromanipulation techniques for study of the highly compacted mitotic form of chromosome found in eukaryote cells during cell division. Each metaphase chromosome contains two duplicate centimeter-long DNA molecules, folded up by proteins into cylindrical structures several microns in length. Native chromosomes display linear and reversible stretching behavior over a wide range of extensions (up to 5x native length for amphibian chromosomes), described by a Young modulus of about 300 Pa. Studies using DNA-cutting and protein-cutting enzymes have revealed that metaphase chromosomes behave as a network of chromatin fibers held together by protein-based isolated crosslinks. Our results are not consistent with the more classical model of loops of chromatin attached to a protein-based structural organizer or ``scaffold". In short, our experiments indicate that metaphase chromosomes can be considered to be ``gels" of chromatin; the stretching modulus of a whole chromosome is consistent with stretching of the chromatin fibers contained within it. Experiments using topoisomerases suggest that topological constraints may play an appreciable role in confining chromatin in the metaphase chromosome. Finally, recent experiments on human chromosomes will be reviewed, including results of experiments where chromosome-folding proteins are specifically depleted using siRNA methods. Supported by NSF-MCB-1022117, DMR-0715099, PHY-0852130, DMR-0520513, NCI 1U54CA143869-01 (NU-PS-OC), and the American Heart Association.

  1. SEROLOGICAL SIMILARITY OF FLAGELLAR AND MITOTIC MICROTUBULES

    PubMed Central

    Fulton, Chandler; Kane, R. E.; Stephens, R. E.

    1971-01-01

    An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins. PMID:4106543

  2. Mitotic apparatus: the selective extraction of protein with mild acid.

    PubMed

    Bibring, T; Baxandall, J

    1968-07-26

    The treatment of isolated mitotic apparatus with mild (pH 3) hydrochloric acid results in the extraction of less than 10 percent of its protein, accompanied by the selective morphological disappearance of the microtubules. The same extraction can be shown to dissolve outer doublet microtubules from sperm flagella. A protein with points of similarity to the flagellar microtubule protein is the major component of the extract from mitotic apparatus.

  3. 14 CFR 1203b.103 - Arrest authority.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Arrest authority. 1203b.103 Section 1203b.103 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION SECURITY PROGRAMS; ARREST AUTHORITY AND USE OF FORCE BY NASA SECURITY FORCE PERSONNEL § 1203b.103 Arrest authority. (a) NASA...

  4. 25 CFR 11.301 - Arrests.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Arrests. 11.301 Section 11.301 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAW AND ORDER COURTS OF INDIAN OFFENSES AND LAW AND ORDER CODE Criminal Procedure § 11.301 Arrests. (a) Arrest is the taking of a person into police custody in order...

  5. 10 CFR 1049.4 - Arrest authority.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Arrest authority. 1049.4 Section 1049.4 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS OF THE STRATEGIC PETROLEUM RESERVE § 1049.4 Arrest authority. (a) Under the Act, the authority of...

  6. 10 CFR 1049.4 - Arrest authority.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 4 2013-01-01 2013-01-01 false Arrest authority. 1049.4 Section 1049.4 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS OF THE STRATEGIC PETROLEUM RESERVE § 1049.4 Arrest authority. (a) Under the Act, the authority of...

  7. 33 CFR 154.822 - Detonation arresters, flame arresters, and flame screens.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Detonation arresters, flame arresters, and flame screens. 154.822 Section 154.822 Navigation and Navigable Waters COAST GUARD... BULK Vapor Control Systems § 154.822 Detonation arresters, flame arresters, and flame screens. (a)...

  8. 33 CFR 154.822 - Detonation arresters, flame arresters, and flame screens.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Detonation arresters, flame arresters, and flame screens. 154.822 Section 154.822 Navigation and Navigable Waters COAST GUARD... BULK Vapor Control Systems § 154.822 Detonation arresters, flame arresters, and flame screens. (a)...

  9. Micromechanical-biochemical studies of mitotic chromosome elasticity and structure

    NASA Astrophysics Data System (ADS)

    Poirier, Michael Guy

    The structure of mitotic chromosomes was studied by combining micromechanical force measurements with microfluidic biochemical exposures. Our method is to use glass micropipettes attached to either end of a single chromosome to do mechanical experiments in the extracellular buffer. A third pipette can be used to locally 'spray' reactants so as to carry out dynamical mechanical-chemical experiments. The following elastic properties of mitotic chromosomes are found: Young's modulus, Y = 300 Pa; Poisson ratio, sigma = 0.1; Bending rigidity, B = 1 x 10 -22 J·m; Internal viscosity, eta' = 100 kg/m·sec; Volume fraction, ϕ = 0.7; Extensions of less than 3 times the relaxed length are linear and reversible; Extensions beyond 30 fold exhibit a force plateau at 15 nN and convert the chromosome to a disperse ghost-like state with little change in chromatin structure; Mitotic chromosomes are relatively isotropic; dsDNA cuts of at least every 3 kb cause the a mitotic chromosomes to fall apart; dsDNA cuts less frequently than every 50 kb do not affect mitotic chromosome structure. These results lead to the conclusion that mitotic chromosomes are a network crosslinked every 50 kb between which chromatin is fold by chromatin folding proteins, which are likely to be condensins.

  10. Timeless links replication termination to mitotic kinase activation.

    PubMed

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-05-06

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  11. Mad2 and Mad3 Cooperate to Arrest Budding Yeast in Mitosis

    PubMed Central

    Lau, Derek T. C.; Murray, Andrew W.

    2012-01-01

    Summary Background The spindle checkpoint ensures accurate chromosome transmission by delaying chromosome segregation until all chromosomes are correctly aligned on the mitotic spindle. The checkpoint is activated by kinetochores that are not attached to microtubules or are attached but not under tension and arrests cells at metaphase by inhibiting the anaphase-promoting complex (APC) and its co-activator Cdc20. Despite numerous studies, we still do not understand how the checkpoint proteins coordinate with each other to inhibit APCCdc20 activity. Results To ask how the checkpoint components induce metaphase arrest, we constructed fusions of checkpoint proteins and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic possible protein interactions during checkpoint activation. We found that expression of a Mad2-Mad3 protein fusion or non-covalently linked Mad2 and Mad3, but not the overexpression of the two separate proteins, induces metaphase arrest that is independent of functional kinetochores or other checkpoint proteins. We further showed that artificially tethering Mad2 to Cdc20 also arrests cells in metaphase independently of other checkpoint components. Conclusion Our results suggest that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, which is sufficient to inhibit APC activity and is the most downstream event in spindle checkpoint activation. PMID:22209528

  12. Anti-mitotic potential of 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620/5-FU

    SciTech Connect

    Kim, Nam Hyun; Kim, Su-Nam; Oh, Joa Sub; Lee, Seokjoon; Kim, Yong Kee

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer DBC exerts antiproliferative potential against 5FU-resistant human gastric cancer cells. Black-Right-Pointing-Pointer This effect is mediated by destabilization of microtubules and subsequent mitotic arrest. Black-Right-Pointing-Pointer DBC enhances apoptosis via caspase activation and downregulation of antiapoptotic genes. -- Abstract: In this study, we investigate an anti-mitotic potential of the novel synthetic coumarin-based compound, 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin, in 5-fluorouracil-resistant human gastric cancer cell line SNU-620-5FU and its parental cell SNU-620. It exerts the anti-proliferative effects with similar potencies against both cancer cells, which is mediated by destabilization of microtubules and subsequent mitotic arrest. Furthermore, this compound enhances caspase-dependent apoptotic cell death via decreased expression of anti-apoptotic genes. Taken together, our data strongly support anti-mitotic potential of 7-diethylamino-3(2 Prime -benzoxazolyl)-coumarin against drug-resistant cancer cells which will prompt us to further develop as a novel microtubule inhibitor for drug-resistant cancer chemotherapy.

  13. Out-of-hospital Cardiac Arrest (OHCA) Biomarkers

    ClinicalTrials.gov

    2017-04-07

    Neurological Outcome; Cardiac Arrest; Out-Of-Hospital Cardiac Arrest; Brain Anoxia Ischemia; Hypoxia, Brain; Hypoxia-Ischemia, Brain; Cardiac Arrest With Successful Resuscitation; Cardiac Arrest, Out-Of-Hospital; Brain Injuries

  14. Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway.

    PubMed

    Nakayama, Yuji; Igarashi, Asae; Kikuchi, Ikue; Obata, Yuuki; Fukumoto, Yasunori; Yamaguchi, Naoto

    2009-09-10

    Polyploid cells result in aneuploidy through aberrant chromosome segregation, possibly leading to tumorigenesis. Although polyploid cells are induced through over-replication by a variety of agents, including DNA-damaging drugs, the mechanisms that induce polyploidy have been hitherto unknown. Here, we show that treatment with bleomycin, a glycopeptide anticancer drug, induces over-replication at low cytotoxic doses. During bleomycin-induced over-replication, mitotic entry is inhibited through tyrosine phosphorylation of CDK1 along the ATM/ATR pathway in the early phase of treatment. Bleomycin-induced over-replication is inhibited by the inhibitors of the ATM/ATR pathway through abrogation of bleomycin-induced G2 arrest, and the ATM/ATR inhibitors promote cell death instead of over-replication. Following the phosphorylation of CDK1, the level of cyclin B1 is decreased in the late phase of treatment. Time-lapse imaging of clone cells that express a live cell marker of endogenous cyclin B1 revealed that cyclin B1 is degraded in G2-arrested cells upon bleomycin treatment. Our findings lead to a model of how the ATM/ATR pathway acts as a molecular switch for regulating cell fates, flipping between cell death via progress into mitosis, and over-replication via sustained G2 arrest upon DNA damage, where cyclin B1 degradation is an important factor for inducing over-replication.

  15. Dynamic Positioning of Mitotic Spindles in Yeast:

    PubMed Central

    Yeh, Elaine; Yang, Charlie; Chin, Elaine; Maddox, Paul; Salmon, E. D.; Lew, Daniel J.; Bloom, Kerry

    2000-01-01

    In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being “pushed” by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus “pulling” the nucleus toward the bud neck. Failure of “pulling” is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud. PMID:11071919

  16. Assessment of Mitotic Activity in Pituitary Adenomas and Carcinomas.

    PubMed

    Thapar, Kamal; Yamada, Yukio; Scheithauer, Bernd; Kovacs, Kalman; Yamada, Shozo; Stefaneanu, Lucia

    1996-01-01

    Assessment of mitotic activity represents one of the oldest and most routinely used histopathologic methods of evaluating the biological aggressiveness of human tumors. In the case of pituitary tumors, however, the relevance of this approach as a means of gauging tumor behavior remains ill-defined. In this article, the relationship between the mitotic index and biological aggressiveness of pituitary tumors was evaluated in a series of 54 pituitary adenomas and 6 primary pituitary carcinomas. All tumors were fully classified by immunohistochemistry and electron microscopy; adenomas were further stratified on the basis of their invasion status, the latter being defined as gross, operatively, or radiologically apparent infiltration of dura or bone. Mitotic figures were present in 11 tumors, 10 being either invasive adenomas or pituitary carcinomas. A significant association between the presence of mitotic figures and tumor behavior was noted, as evidenced by progressive increments in the proportion of cases expressing mitotic figures in the categories of noninvasive adenoma, invasive adenoma, and pituitary carcinoma (3.9, 21.4, and 66.7%, respectively; Fisher's exact test, two-tailed, p < 0.001). The mitotic index, however, appeared to be a less informative parameter, being extremely low in all cases (mean = 0.016% +/- 0.005 [+/- SEMI). Although the mean mitotic index in pituitary carcinomas (0.09% +/- 0.035) was significantly higher than the mean mitotic index of either noninvasive adenomas (0.002% +/- 0.002) or invasive adenomas (0.013% +/- 0.005), no practical threshold value capable of distinguishing these three groups was evident. Comparison of the mitotic index with Ki-67 derived growth fractions in these tumors revealed a significant but weak linear correlation (r = 0.41, p < 0.01). These data suggest that when, mitotic figures are present, they do provide some indication of the behavior and invasive potential of pituitary tumors. For routine diagnostic

  17. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  18. Prognostic differences of World Health Organization-assessed mitotic activity index and mitotic impression by quick scanning in invasive ductal breast cancer patients younger than 55 years.

    PubMed

    Skaland, Ivar; van Diest, Paul J; Janssen, Emiel A M; Gudlaugsson, Einar; Baak, Jan P A

    2008-04-01

    The proliferation marker mitotic activity index is the strongest prognostic indicator in lymph node-negative breast cancer. The World Health Organization (WHO) 2003-defined procedure for determining WHO-mitotic activity index is often replaced by a quick scan mitotic impression. We evaluated the prognostic consequences of this practice in 433 T(1-3)N(0)M(0) lymph node-negative invasive ductal type breast cancers with long-term follow-up (median, 112 months; range, 12-187 months). Twenty-seven percent of the studied cases developed distant metastases, and 25% died of disease. Agreement between WHO-mitotic activity index (0-5 = 1, 6-10 = 2, >10 = 3) and mitotic impression (1, 2, 3) categories was 66% (kappa = 0.41), including 85% for category 1, 26% for category 2, and 52% for category 3. The WHO-mitotic activity index was a much stronger prognosticator than the mitotic impression, and the 10-year survival rates of the same categories (eg, mitotic activity index and mitotic impression category both 2) differed greatly. When grade was assessed by combining WHO-mitotic activity index or mitotic impression with the same values for tubular formation and nuclear atypia, grades disagreed in 18% of the cases. Deviation from the formal WHO-mitotic activity index assessment guidelines in breast cancer often results in erroneous prognosis estimations with therapeutic consequences and may explain why the prognostic value of proliferative activity in breast cancer is not always confirmed.

  19. A dynamic mode of mitotic bookmarking by transcription factors

    PubMed Central

    Teves, Sheila S; An, Luye; Hansen, Anders S; Xie, Liangqi; Darzacq, Xavier; Tjian, Robert

    2016-01-01

    During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking. DOI: http://dx.doi.org/10.7554/eLife.22280.001 PMID:27855781

  20. A Brief History of Research on Mitotic Mechanisms

    PubMed Central

    McIntosh, J. Richard; Hays, Thomas

    2016-01-01

    This chapter describes in summary form some of the most important research on chromosome segregation, from the discovery and naming of mitosis in the nineteenth century until around 1990. It gives both historical and scientific background for the nine chapters that follow, each of which provides an up-to-date review of a specific aspect of mitotic mechanism. Here, we trace the fruits of each new technology that allowed a deeper understanding of mitosis and its underlying mechanisms. We describe how light microscopy, including phase, polarization, and fluorescence optics, provided descriptive information about mitotic events and also enabled important experimentation on mitotic functions, such as the dynamics of spindle fibers and the forces generated for chromosome movement. We describe studies by electron microscopy, including quantitative work with serial section reconstructions. We review early results from spindle biochemistry and genetics, coupled to molecular biology, as these methods allowed scholars to identify key molecular components of mitotic mechanisms. We also review hypotheses about mitotic mechanisms whose testing led to a deeper understanding of this fundamental biological event. Our goal is to provide modern scientists with an appreciation of the work that has laid the foundations for their current work and interests. PMID:28009830

  1. Loops determine the mechanical properties of mitotic chromosomes

    NASA Astrophysics Data System (ADS)

    Zhang, Yang; Heermann, Dieter W.

    2013-03-01

    In mitosis, chromosomes undergo a condensation into highly compacted, rod-like objects. Many models have been put forward for the higher-order organization of mitotic chromosomes including radial loop and hierarchical folding models. Additionally, mechanical properties of mitotic chromosomes under different conditions were measured. However, the internal organization of mitotic chromosomes still remains unclear. Here we present a polymer model for mitotic chromosomes and show how chromatin loops play a major role for their mechanical properties. The key assumption of the model is the ability of the chromatin fibre to dynamically form loops with the help of binding proteins. Our results show that looping leads to a tight compaction and significantly increases the bending rigidity of chromosomes. Moreover, our qualitative prediction of the force elongation behaviour is close to experimental findings. This indicates that the internal structure of mitotic chromosomes is based on self-organization of the chromatin fibre. We also demonstrate how number and size of loops have a strong influence on the mechanical properties. We suggest that changes in the mechanical characteristics of chromosomes can be explained by an altered internal loop structure. YZ gratefully appreciates funding by the German National Academic Foundation (Studienstiftung des deutschen Volkes) and support by the Heidelberg Graduate School for Mathematical and Computational Methods in the Sciences (HGS MathComp).

  2. Mechanical control of mitotic progression in single animal cells.

    PubMed

    Cattin, Cedric J; Düggelin, Marcel; Martinez-Martin, David; Gerber, Christoph; Müller, Daniel J; Stewart, Martin P

    2015-09-08

    Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback-controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50-100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement.

  3. Mitotic Exit Control as an Evolved Complex System

    SciTech Connect

    Bosl, W; Li, R

    2005-04-25

    The exit from mitosis is the last critical decision a cell has to make during a division cycle. A complex regulatory system has evolved to evaluate the success of mitotic events and control this decision. Whereas outstanding genetic work in yeast has led to rapid discovery of a large number of interacting genes involved in the control of mitotic exit, it has also become increasingly difficult to comprehend the logic and mechanistic features embedded in the complex molecular network. Our view is that this difficulty stems in part from the attempt to explain mitotic exit control using concepts from traditional top-down engineering design, and that exciting new results from evolutionary engineering design applied to networks and electronic circuits may lend better insights. We focus on four particularly intriguing features of the mitotic exit control system: the two-stepped release of Cdc14; the self-activating nature of Tem1 GTPase; the spatial sensor associated with the spindle pole body; and the extensive redundancy in the mitotic exit network. We attempt to examine these design features from the perspective of evolutionary design and complex system engineering.

  4. Mitotic Chromosome Loss in a Disomic Haploid of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Campbell, D. A.; Fogel, S.; Lusnak, K.

    1975-01-01

    Experiments designed to characterize the incidence of mitotic chromosome loss in a yeast disomic haploid were performed. The selective methods employed utilize the non-mating property of strains disomic for linkage group III and heterozygous at the mating type locus. The principal findings are: (1) The frequency of spontaneous chromosome loss in the disome is of the order 10-4 per cell; this value approximates the frequency in the same population of spontaneous mitotic exchange resulting in homozygosity at the mating type locus. (2) The recovered diploids are pure clones, and thus represent unique events in the disomic haploid. (3) Of the euploid chromosomes recovered after events leading to chromosome loss, approximately 90% retain the parental marker configuration expected from segregation alone; however, the remainder are recombinant for marker genes, and are the result of mitotic exchanges in the disome, especially in regions near the centromere. The recombinant proportion significantly exceeds that expected if chromosome loss and mitotic exchange in the disome were independent events. The data are consistent with a model proposing mitotic nondisjunction as the event responsible for chromosome loss in the disomic haploid. PMID:1092597

  5. Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells.

    PubMed

    Solc, Petr; Schultz, Richard M; Motlik, Jan

    2010-09-01

    Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic

  6. Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression.

    PubMed

    Matus, David Q; Lohmer, Lauren L; Kelley, Laura C; Schindler, Adam J; Kohrman, Abraham Q; Barkoulas, Michalis; Zhang, Wan; Chi, Qiuyi; Sherwood, David R

    2015-10-26

    Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia, F-actin-rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together, these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1-arrested and actively cycling cells may be needed to halt metastatic cancer.

  7. The nuclear localization signal of mitotic kinesin-like protein Mklp-1: effect on Mklp-1 function during cytokinesis.

    PubMed

    Liu, Xiaoqi; Erikson, Raymond L

    2007-02-23

    The mitotic kinesin-like protein (Mklp-1) localizes in the nucleus during interphase due to the presence of nuclear localization signal(s) [NLS(s)] within its sequence. Here, we mapped two NLSs to be 899SRKRRSST906 and 949KRKKP953 in the tail domain of Mklp-1, and showed that ectopic expression of a mutant Mklp-1 without the NLSs leads to cell cycle arrest at cytokinesis, indicating that the NLSs are necessary for Mklp-1 to execute its normal function during cell division. Furthermore, mutation of two serine residues in the first NLS to aspartic acid, which mimics phosphorylation, attenuated its nuclear localization function, suggesting that the function of this NLS might be regulated by phosphorylation.

  8. Shaping mitotic chromosomes: From classical concepts to molecular mechanisms

    PubMed Central

    Kschonsak, Marc; Haering, Christian H

    2015-01-01

    How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss – in light of these recent insights – the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles. PMID:25988527

  9. Shaping mitotic chromosomes: From classical concepts to molecular mechanisms.

    PubMed

    Kschonsak, Marc; Haering, Christian H

    2015-07-01

    How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss - in light of these recent insights - the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles.

  10. Plk2 regulates mitotic spindle orientation and mammary gland development.

    PubMed

    Villegas, Elizabeth; Kabotyanski, Elena B; Shore, Amy N; Creighton, Chad J; Westbrook, Thomas F; Rosen, Jeffrey M

    2014-04-01

    Disruptions in polarity and mitotic spindle orientation contribute to the progression and evolution of tumorigenesis. However, little is known about the molecular mechanisms regulating these processes in vivo. Here, we demonstrate that Polo-like kinase 2 (Plk2) regulates mitotic spindle orientation in the mammary gland and that this might account for its suggested role as a tumor suppressor. Plk2 is highly expressed in the mammary gland and is required for proper mammary gland development. Loss of Plk2 leads to increased mammary epithelial cell proliferation and ductal hyperbranching. Additionally, a novel role for Plk2 in regulating the orientation of the mitotic spindle and maintaining proper cell polarity in the ductal epithelium was discovered. In support of a tumor suppressor function for Plk2, loss of Plk2 increased the formation of lesions in multiparous glands. Collectively, these results demonstrate a novel role for Plk2 in regulating mammary gland development.

  11. Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

    PubMed

    Ley, K D

    1975-07-01

    Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.

  12. Evidence for arrested bone formation during spaceflight

    NASA Technical Reports Server (NTRS)

    Turner, R. T.; Bobyn, J. D.; Duvall, P.; Morey, E. R.; Baylink, D. J.; Spector, M.

    1982-01-01

    Addressing the question of whether the bone formed in space is unusual, the morphology of bone made at the tibial diaphysis of rats before, during, and after spaceflight is studied. Evidence of arrest lines in the bone formed in space is reported suggesting that bone formation ceases along portions of the periosteum during spaceflight. Visualized by microradiography, the arrest lines are shown to be less mineralized than the surrounding bone matrix. When viewed by scanning electron microscopy, it is seen that bone fractures more readily at the site of an arrest line. These observations are seen as suggesting that arrest lines are a zone of weakness and that their formation may result in decreased bone strength in spite of normalization of bone formation after flight. The occurrence, location, and morphology of arrest lines are seen as suggesting that they are a visible result of the phenomenon of arrested bone formation.

  13. CaMKKβ-AMPKα2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells

    PubMed Central

    Lee, In Jeong; Lee, Chang-Woo; Lee, Jae-Ho

    2015-01-01

    Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKαThr172 signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKαThr172 signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα Thr172 signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase. PMID:25590814

  14. Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3.

    PubMed

    Toni, Lee S; Padilla, Pamela A

    2016-02-01

    Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.

  15. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

    PubMed Central

    Brandeis, M; Hunt, T

    1996-01-01

    We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells. Images PMID:8895573

  16. Novel Mad2-targeting miR-493-3p controls mitotic fidelity and cancer cells’ sensitivity to paclitaxel

    PubMed Central

    Mäki-Jouppila, Jenni; Chen, Ping; Elgaaen, Bente Vilming; Straume, Anne Hege; Huhtinen, Kaisa; Cárpen, Olli; Lønning, Per Eystein; Davidson, Ben; Hautaniemi, Sampsa; Kallio, Marko J.

    2016-01-01

    The molecular pathways that contribute to the proliferation and drug response of cancer cells are highly complex and currently insufficiently characterized. We have identified a previously unknown microRNA-based mechanism that provides cancer cells means to stimulate tumorigenesis via increased genomic instability and, at the same time, evade the action of clinically utilized microtubule drugs. We demonstrate miR-493-3p to be a novel negative regulator of mitotic arrest deficient-2 (MAD2), an essential component of the spindle assembly checkpoint that monitors the fidelity of chromosome segregation. The microRNA targets the 3′ UTR of Mad2 mRNA thereby preventing translation of the Mad2 protein. In cancer cells, overexpression of miR-493-3p induced a premature mitotic exit that led to increased frequency of aneuploidy and cellular senescence in the progeny cells. Importantly, excess of the miR-493-3p conferred resistance of cancer cells to microtubule drugs. In human neoplasms, miR-493-3p and Mad2 expression alterations correlated with advanced ovarian cancer forms and high miR-493-3p levels were associated with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially in the paclitaxel therapy arm. Our results suggest that intratumoral profiling of miR-493-3p and Mad2 levels can have diagnostic value in predicting the efficacy of taxane chemotherapy. PMID:26943585

  17. Force and the spindle: Mechanical cues in mitotic spindle orientation

    PubMed Central

    Nestor-Bergmann, Alexander; Goddard, Georgina; Woolner, Sarah

    2014-01-01

    The mechanical environment of a cell has a profound effect on its behaviour, from dictating cell shape to driving the transcription of specific genes. Recent studies have demonstrated that mechanical forces play a key role in orienting the mitotic spindle, and therefore cell division, in both single cells and tissues. Whilst the molecular machinery that mediates the link between external force and the mitotic spindle remains largely unknown, it is becoming increasingly clear that this is a widely used mechanism which could prove vital for coordinating cell division orientation across tissues in a variety of contexts. PMID:25080021

  18. Simulated Cardiopulmonary Arrests in a Hospital Setting.

    ERIC Educational Resources Information Center

    Mishkin, Barbara H.; And Others

    1982-01-01

    Describes a simulated interdisciplinary role rehearsal for cardiopulmonary arrest to prepare nurses to function effectively. Includes needs analysis, program components, and responses of program participants. (Author)

  19. Application of lightning arrester on transmission lines

    NASA Astrophysics Data System (ADS)

    Matsubara, K.

    1990-05-01

    A lightning arrester on transmission lines is expected to reduce lightning incidents; however, the problems of the application guide to be established such as the method of application, the treating capacity for lightning energy, and the coordination to an arc horn remain as they are. The method of application for a lightning arrester on transmission lines and its effect are examined from the viewpoint of the generating incident on transmission lines by lightning and the operating viewpoint of a lightning arrester for a 77kV system. The double circuit setup of an arrester is the best method to reduce the lightning incidents and the single circuit setup can reduce the incident rate by 40 percent preventing the flashover of the circuit on which the arrester is not set up. For installing an arrester on a short section, the flashover point can transfer to the next steel tower where the arrester is not set up, only when lightning strikes the last steel tower. Inflowing current into an arrester from a direct lightning strike is considerably large and the energy at that time becomes large but this energy can be reduced by the operation of the arrester set up on another steel tower.

  20. Mitotic Spindle Disruption by Alternating Electric Fields Leads to Improper Chromosome Segregation and Mitotic Catastrophe in Cancer Cells

    PubMed Central

    Giladi, Moshe; Schneiderman, Rosa S; Voloshin, Tali; Porat, Yaara; Munster, Mijal; Blat, Roni; Sherbo, Shay; Bomzon, Zeev; Urman, Noa; Itzhaki, Aviran; Cahal, Shay; Shteingauz, Anna; Chaudhry, Aafia; Kirson, Eilon D; Weinberg, Uri; Palti, Yoram

    2015-01-01

    Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields. TTFields are a unique anti-mitotic treatment modality delivered in a continuous, noninvasive manner to the region of a tumor. It was previously postulated that by exerting directional forces on highly polar intracellular elements during mitosis, TTFields could disrupt the normal assembly of spindle microtubules. However there is limited evidence directly linking TTFields to an effect on microtubules. Here we report that TTFields decrease the ratio between polymerized and total tubulin, and prevent proper mitotic spindle assembly. The aberrant mitotic events induced by TTFields lead to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells. The effect of TTFields on cell viability and clonogenic survival substantially depends upon the cell division rate. We show that by extending the duration of exposure to TTFields, slowly dividing cells can be affected to a similar extent as rapidly dividing cells. PMID:26658786

  1. Arresting relaxation in Pickering Emulsions

    NASA Astrophysics Data System (ADS)

    Atherton, Tim; Burke, Chris

    2015-03-01

    Pickering emulsions consist of droplets of one fluid dispersed in a host fluid and stabilized by colloidal particles absorbed at the fluid-fluid interface. Everyday materials such as crude oil and food products like salad dressing are examples of these materials. Particles can stabilize non spherical droplet shapes in these emulsions through the following sequence: first, an isolated droplet is deformed, e.g. by an electric field, increasing the surface area above the equilibrium value; additional particles are then adsorbed to the interface reducing the surface tension. The droplet is then allowed to relax toward a sphere. If more particles were adsorbed than can be accommodated by the surface area of the spherical ground state, relaxation of the droplet is arrested at some non-spherical shape. Because the energetic cost of removing adsorbed colloids exceeds the interfacial driving force, these configurations can remain stable over long timescales. In this presentation, we present a computational study of the ordering present in anisotropic droplets produced through the mechanism of arrested relaxation and discuss the interplay between the geometry of the droplet, the dynamical process that produced it, and the structure of the defects observed.

  2. Biz1, a Zinc Finger Protein Required for Plant Invasion by Ustilago maydis, Regulates the Levels of a Mitotic Cyclin[W

    PubMed Central

    Flor-Parra, Ignacio; Vranes, Miroslav; Kämper, Jörg; Pérez-Martín, José

    2006-01-01

    Plant invasion by pathogenic fungi involves regulated growth and highly organized fungal morphological changes. For instance, when the smut fungus Ustilago maydis infects maize (Zea mays), its dikaryotic infective filament is cell cycle arrested, and appressoria are differentiated prior to plant penetration. Once the filament enters the plant, the cell cycle block is released and fungal cells begin proliferation, suggesting a tight interaction between plant invasion and the cell cycle and morphogenesis control systems. We describe a novel factor, Biz1 (b-dependent zinc finger protein), which has two Cys2His2 zinc finger domains and nuclear localization, suggesting a transcriptional regulatory function. The deletion of biz1 shows no detectable phenotypic alterations during axenic growth. However, mutant cells show a severe reduction in appressoria formation and plant penetration, and those hyphae that invade the plant arrest their pathogenic development directly after plant penetration. biz1 is induced via the b-mating–type locus, the key control instance for pathogenic development. The gene is expressed at high levels throughout pathogenic development, which induces a G2 cell cycle arrest that is a direct consequence of the downregulation of the mitotic cyclin Clb1. Our data support a model in which Biz1 is involved in cell cycle arrest preceding plant penetration as well as in the induction of appressoria. PMID:16905655

  3. LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

    PubMed Central

    Chiyoda, Tatsuyuki; Sugiyama, Naoyuki; Shimizu, Takatsune; Naoe, Hideaki; Kobayashi, Yusuke; Ishizawa, Jo; Arima, Yoshimi; Tsuda, Hiroshi; Ito, Masaaki; Kaibuchi, Kozo; Aoki, Daisuke; Ishihama, Yasushi

    2012-01-01

    In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase–targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage–induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint. PMID:22641346

  4. The DEAD-box RNA helicase Vasa functions in embryonic mitotic progression in the sea urchin.

    PubMed

    Yajima, Mamiko; Wessel, Gary M

    2011-06-01

    Vasa is a broadly conserved ATP-dependent RNA helicase that functions in the germ line of organisms from cnidarians to mammals. Curiously, Vasa is also present in the somatic cells of many animals and functions as a regulator of multipotent cells. Here, we report a mitotic function of Vasa revealed in the sea urchin embryo. We found that Vasa protein is present in all blastomeres of the early embryo and that its abundance oscillates with the cell cycle. Vasa associates with the spindle and the separating sister chromatids at metaphase, and then quickly disappears after telophase. Inhibition of Vasa protein synthesis interferes with proper chromosome segregation, arrests cells at M-phase, and delays overall cell cycle progression. Cdk activity is necessary for the proper localization of Vasa, implying that Vasa is involved in the cyclin-dependent cell cycle network, and Vasa is required for the efficient translation of cyclinB mRNA. Our results suggest an evolutionarily conserved role of Vasa that is independent of its function in germ line determination.

  5. Glycophthalocyanines as photosensitizers for triggering mitotic catastrophe and apoptosis in cancer cells.

    PubMed

    Soares, Ana R M; Neves, Maria G P M S; Tomé, Augusto C; Iglesias-de la Cruz, M Carmen; Zamarrón, Alicia; Carrasco, Elisa; González, Salvador; Cavaleiro, José A S; Torres, Tomás; Guldi, Dirk M; Juarranz, Angeles

    2012-04-16

    Photodynamic therapy (PDT) is a treatment modality for different forms of cancer based on the combination of light, molecular oxygen, and a photosensitizer (PS) compound. When activated by light, the PS generates reactive oxygen species leading to tumor destruction. Phthalocyanines are compounds that have already shown to be efficient PSs for PDT. Several examples of carbohydrate substituted phthalocyanines have been reported, assuming that the presence of carbohydrate moieties could improve their tumor selectivity. This work describes the photoeffects of symmetric and asymmetric phthalocyanines with D-galactose (so-called GPh1, GPh2, and GPh3) on HeLa carcinoma cells and their involvement in cell death. Photophysical properties and in vitro photodynamic activities for the compounds considered revealed that the asymmetric glycophthalocyanine GPh3 is very efficient and selective, producing higher photocytotoxicity on cancer cells than in nonmalignat HaCaT. The cell toxiticy after PDT treatment was dependent upon light exposure level and GPh3 concentration. GPh3 causes cell cycle arrest at the metaphase stage leading to multiple spindle poles, mitotic catastrophe, followed by apoptosis in cancer cells. These effects were partially negated by the pancaspase inhibitor Z-VAD-FMK. Together, these results indicate that GPh3 is an excellent candidate drug for PDT, able to induce selective tumor cell death.

  6. A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

    PubMed Central

    DeMoe, Joanna H.; Santaguida, Stefano; Daum, John R.; Musacchio, Andrea; Gorbsky, Gary J.

    2008-01-01

    In mitosis the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate (1). The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Since complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide a therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel. PMID:19190331

  7. Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C.

    PubMed

    Ganapathy, Suthakar; Fagman, Johan B; Shen, Ling; Yu, Tianqi; Zhou, Xiaodong; Dai, Wei; Makriyannis, Alexandros; Chen, Changyan

    2016-12-20

    Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

  8. Regulation of Aurora-A kinase on the mitotic spindle.

    PubMed

    Kufer, Thomas A; Nigg, Erich A; Silljé, Herman H W

    2003-12-01

    The error-free segregation of duplicated chromosomes during cell division is essential for the maintenance of an intact genome. This process is brought about by a highly dynamic bipolar array of microtubules, the mitotic spindle. The formation and function of the mitotic spindle during M-phase of the cell cycle is regulated by protein phosphorylation, involving multiple protein kinases and phosphatases. Prominent among the enzymes implicated in spindle assembly is the serine/threonine-specific protein kinase Aurora-A. In several common human tumors, Aurora-A is overexpressed, and deregulation of this kinase was shown to result in mitotic defects and aneuploidy. Moreover, recent genetic evidence directly links the human Aurora-A gene to cancer susceptibility. Several of the physiological substrates of Aurora-A presumably await identification, but recent studies are beginning to shed light on the regulation of this critical mitotic kinase. Here, we review these findings with particular emphasis on the role of TPX2, a prominent spindle component implicated in a Ran-GTP-mediated spindle assembly pathway.

  9. Cortical neurons gradually attain a post-mitotic state.

    PubMed

    Anda, Froylan Calderon de; Madabhushi, Ram; Rei, Damien; Meng, Jia; Gräff, Johannes; Durak, Omer; Meletis, Konstantinos; Richter, Melanie; Schwanke, Birgit; Mungenast, Alison; Tsai, Li-Huei

    2016-09-01

    Once generated, neurons are thought to permanently exit the cell cycle and become irreversibly differentiated. However, neither the precise point at which this post-mitotic state is attained nor the extent of its irreversibility is clearly defined. Here we report that newly born neurons from the upper layers of the mouse cortex, despite initiating axon and dendrite elongation, continue to drive gene expression from the neural progenitor tubulin α1 promoter (Tα1p). These observations suggest an ambiguous post-mitotic neuronal state. Whole transcriptome analysis of sorted upper cortical neurons further revealed that neurons continue to express genes related to cell cycle progression long after mitotic exit until at least post-natal day 3 (P3). These genes are however down-regulated thereafter, associated with a concomitant up-regulation of tumor suppressors at P5. Interestingly, newly born neurons located in the cortical plate (CP) at embryonic day 18-19 (E18-E19) and P3 challenged with calcium influx are found in S/G2/M phases of the cell cycle, and still able to undergo division at E18-E19 but not at P3. At P5 however, calcium influx becomes neurotoxic and leads instead to neuronal loss. Our data delineate an unexpected flexibility of cell cycle control in early born neurons, and describe how neurons transit to a post-mitotic state.

  10. GSK3 Regulates Mitotic Chromosomal Alignment through CRMP4

    PubMed Central

    Ong Tone, Stephan; Dayanandan, Bama

    2010-01-01

    Background Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis. Methodology and Principal Findings Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton. Conclusion and Significance Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulating chromosomal alignment and mitotic progression through its effect on spindle microtubules. PMID:21179545

  11. Cytotoxic effects of cylindrospermopsin in mitotic and non-mitotic Vicia faba cells.

    PubMed

    Garda, Tamás; Riba, Milán; Vasas, Gábor; Beyer, Dániel; M-Hamvas, Márta; Hajdu, Gréta; Tándor, Ildikó; Máthé, Csaba

    2015-02-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin known as a eukaryotic protein synthesis inhibitor. We aimed to study its effects on growth, stress responses and mitosis of a eukaryotic model, Vicia faba (broad bean). Growth responses depended on exposure time (3 or 6d), cyanotoxin concentration, culture conditions (dark or continuous light) and V. faba cultivar ("Standard" or "ARC Egypt Cross"). At 6d of exposure, CYN had a transient stimulatory effect on root system growth, roots being possibly capable of detoxification. The toxin induced nucleus fragmentation, blebbing and chromosomal breaks indicating double stranded DNA breaks and programmed cell death. Root necrotic tissue was observed at 0.1-20 μg mL(-1) CYN that probably impeded toxin uptake into vascular tissue. Growth and cell death processes observed were general stress responses. In lateral root tip meristems, lower CYN concentrations (0.01-0.1 μg mL(-1)) induced the stimulation of mitosis and distinct mitotic phases, irrespective of culture conditions or the cultivar used. Higher cyanotoxin concentrations inhibited mitosis. Short-term exposure of hydroxylurea-synchronized roots to 5 μg mL(-1) CYN induced delay of mitosis that might have been related to a delay of de novo protein synthesis. CYN induced the formation of double, split and asymmetric preprophase bands (PPBs), in parallel with the alteration of cell division planes, related to the interference of cyanotoxin with protein synthesis, thus it was a plant- and CYN specific alteration.

  12. Juvenile Arrests, 2000. Juvenile Justice Bulletin.

    ERIC Educational Resources Information Center

    Snyder, Howard N.

    This bulletin examines the national and state juvenile arrest rate in 2000 using data reported annually by local law enforcement agencies nationwide to the FBI's Uniform Crime Reporting program. Results indicate that the murder rate in 2000 was the lowest since 1965; juvenile arrests for violence in 2000 were the lowest since 1988; few juveniles…

  13. Juvenile Arrests 1996. Juvenile Justice Bulletin.

    ERIC Educational Resources Information Center

    Snyder, Howard N.

    In 1996, law enforcement agencies in the United States made an estimated 2.9 million arrests of persons under the age of 18. According to Federal Bureau of Investigation (FBI) figures, juveniles accounted for 19% of all arrests and 19% of all violent crime in 1996. The substantial growth in juvenile crime that began in the late 1980s peaked in…

  14. The Arrest Records of Rosa Parks.

    ERIC Educational Resources Information Center

    Bredhoff, Stacey; Schamel, Wynell; Potter, Lee Ann

    1999-01-01

    Provides background information on the arrest of Rosa Parks and the effects this event had on the Civil Rights Movement. Offers a collection of teaching activities in which the students examine the arrest records of Rosa Parks and explains that these activities are designed to accompany a unit on racial segregation. (CMK)

  15. 10 CFR 1047.4 - Arrest authority.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Arrest authority. 1047.4 Section 1047.4 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS... of the Atomic Energy Act: (i) Felonies: (A) Section 222. Violation of Specific Sections—42...

  16. 10 CFR 1047.4 - Arrest authority.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Arrest authority. 1047.4 Section 1047.4 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS... of the Atomic Energy Act: (i) Felonies: (A) Section 222. Violation of Specific Sections—42...

  17. 10 CFR 1047.4 - Arrest authority.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 4 2013-01-01 2013-01-01 false Arrest authority. 1047.4 Section 1047.4 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) LIMITED ARREST AUTHORITY AND USE OF FORCE BY PROTECTIVE FORCE OFFICERS... of the Atomic Energy Act: (i) Felonies: (A) Section 222. Violation of Specific Sections—42...

  18. 36 CFR 222.76 - Arrest.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 36 Parks, Forests, and Public Property 2 2014-07-01 2014-07-01 false Arrest. 222.76 Section 222.76 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE RANGE MANAGEMENT Management of Wild Free-Roaming Horses and Burros § 222.76 Arrest. Any employee designated by the...

  19. 36 CFR 222.36 - Arrest.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 2 2011-07-01 2011-07-01 false Arrest. 222.36 Section 222.36 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE RANGE MANAGEMENT Management of Wild Free-Roaming Horses and Burros § 222.36 Arrest. Any employee designated by the...

  20. 36 CFR 222.36 - Arrest.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 36 Parks, Forests, and Public Property 2 2012-07-01 2012-07-01 false Arrest. 222.36 Section 222.36 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE RANGE MANAGEMENT Management of Wild Free-Roaming Horses and Burros § 222.36 Arrest. Any employee designated by the...

  1. 36 CFR 222.36 - Arrest.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Arrest. 222.36 Section 222.36 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE RANGE MANAGEMENT Management of Wild Free-Roaming Horses and Burros § 222.36 Arrest. Any employee designated by the...

  2. 36 CFR 222.76 - Arrest.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 36 Parks, Forests, and Public Property 2 2013-07-01 2013-07-01 false Arrest. 222.76 Section 222.76 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF AGRICULTURE RANGE MANAGEMENT Management of Wild Free-Roaming Horses and Burros § 222.76 Arrest. Any employee designated by the...

  3. Psychopathology in Women Arrested for Domestic Violence

    ERIC Educational Resources Information Center

    Stuart, Gregory L.; Moore, Todd M.; Gordon, Kristina Coop; Ramsey, Susan E.; Kahler, Christopher W.

    2006-01-01

    This study examined the prevalence of psychopathology among women arrested for violence and whether the experience of intimate partner violence (IPV) was associated with Axis I psychopathology. Women who were arrested for domestic violence perpetration and court referred to violence intervention programs (N=103) completed measures of IPV…

  4. Mechanism of APC/CCDC20 activation by mitotic phosphorylation

    PubMed Central

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2016-01-01

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  5. Physeal arrest of the distal radius.

    PubMed

    Abzug, Joshua M; Little, Kevin; Kozin, Scott H

    2014-06-01

    Fractures of the distal radius are among the most common pediatric fractures. Although most of these fractures heal without complication, some result in partial or complete physeal arrest. The risk of physeal arrest can be reduced by avoiding known risk factors during fracture management, including multiple attempts at fracture reduction. Athletes may place substantial compressive and shear forces across the distal radial physes, making them prone to growth arrest. Timely recognition of physeal arrest can allow for more predictable procedures to be performed, such as distal ulnar epiphysiodesis. In cases of partial arrest, physeal bar excision with interposition grafting can be performed. Once ulnar abutment is present, more invasive procedures may be required, including ulnar shortening osteotomy or radial lengthening.

  6. Crack arrest in thick section steel plate

    NASA Astrophysics Data System (ADS)

    Smith, E.

    1983-03-01

    Crack arrest in thick section steel plate is considered in relation to the conditions for crack arrest in a nuclear reactor pressure vessel, when this is subjected to thermal stresses resulting from a hypothetical loss of coolant accident. The results of a theoretical analysis, based primarily on recent developments in quasi-static crack propagation theory, provide further support for the view that the arrest toughness KIa is essentially a material property. However, since the theoretical results also suggest that KIa is reduced by neutron irradiation, and because there is, as yet, no conclusive experimental data on the effect of neutron irradiation on KIa, it is proposed that with highly irradiated steel, instead of using a KIa crack arrest criterion, it is better to use a more conservative criterion, based on the concept that arrest occurs within the vessel at a position where the temperature exceeds that temperature above which the cleavage fracture mode is unable to operate.

  7. Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome

    PubMed Central

    Moshe, Yakir; Boulaire, Jérôme; Pagano, Michele; Hershko, Avram

    2004-01-01

    Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1–Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein β-TrCP. As with other substrates of SCFβ-TrCP, the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCFβ-TrCP. Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis. PMID:15148369

  8. [Cardiopulmonary resuscitation in cardiac arrest following trauma].

    PubMed

    Leidel, B A; Kanz, K-G

    2016-11-01

    For decades, survival rates of cardiac arrest following trauma were reported between 0 and 2 %. Since 2005, survival rates have increased with a wide range up to 39 % and good neurological recovery in every second person injured for unknown reasons. Especially in children, high survival rates with good neurologic outcomes are published. Resuscitation following traumatic cardiac arrest differs significantly from nontraumatic causes. Paramount is treatment of reversible causes, which include massive bleeding, hypoxia, tension pneumothorax, and pericardial tamponade. Treatment of reversible causes should be simultaneous. Chest compression is inferior following traumatic cardiac arrest and should never delay treatment of reversible causes of the traumatic cardiac arrest. In massive bleeding, bleeding control has priority. Damage control resuscitation with permissive hypotension, aggressive coagulation therapy, and damage control surgery represent the pillars of initial treatment. Cardiac arrest due to hypoxia should be resolved by airway management and ventilation. Tension pneumothorax should be decompressed by finger thoracostomy, pericardial tamponade by resuscitative thoracotomy. In addition, resuscitative thoracotomy allows direct and indirect bleeding control. Untreated impact brain apnea may rapidly lead to cardiac arrest and requires quick opening of the airway and effective oxygenation. Established algorithms for treatment of cardiac arrest following trauma enable a safe, structured, and effective management.

  9. Extracorporeal Membrane Oxygenation for Refractory Cardiac Arrest

    PubMed Central

    Conrad, Steven A; Rycus, Peter T

    2017-01-01

    Extracorporeal cardiopulmonary resuscitation (ECPR) is the use of rapid deployment venoarterial (VA) extracorporeal membrane oxygenation to support systemic circulation and vital organ perfusion in patients in refractory cardiac arrest not responding to conventional cardiopulmonary resuscitation (CPR). Although prospective controlled studies are lacking, observational studies suggest improved outcomes compared with conventional CPR when ECPR is instituted within 30–60 min following cardiac arrest. Adult and pediatric patients with witnessed in-hospital and out-of-hospital cardiac arrest and good quality CPR, failure of at least 15 min of conventional resuscitation, and a potentially reversible cause for arrest are candidates. Percutaneous cannulation where feasible is rapid and can be performed by nonsurgeons (emergency physicians, intensivists, cardiologists, and interventional radiologists). Modern extracorporeal systems are easy to prime and manage and are technically easy to manage with proper training and experience. ECPR can be deployed in the emergency department for out-of-hospital arrest or in various inpatient units for in-hospital arrest. ECPR should be considered for patients with refractory cardiac arrest in hospitals with an existing extracorporeal life support program, able to provide rapid deployment of support, and with resources to provide postresuscitation evaluation and management. PMID:28074817

  10. Extracorporeal membrane oxygenation for refractory cardiac arrest.

    PubMed

    Conrad, Steven A; Rycus, Peter T

    2017-01-01

    Extracorporeal cardiopulmonary resuscitation (ECPR) is the use of rapid deployment venoarterial (VA) extracorporeal membrane oxygenation to support systemic circulation and vital organ perfusion in patients in refractory cardiac arrest not responding to conventional cardiopulmonary resuscitation (CPR). Although prospective controlled studies are lacking, observational studies suggest improved outcomes compared with conventional CPR when ECPR is instituted within 30-60 min following cardiac arrest. Adult and pediatric patients with witnessed in-hospital and out-of-hospital cardiac arrest and good quality CPR, failure of at least 15 min of conventional resuscitation, and a potentially reversible cause for arrest are candidates. Percutaneous cannulation where feasible is rapid and can be performed by nonsurgeons (emergency physicians, intensivists, cardiologists, and interventional radiologists). Modern extracorporeal systems are easy to prime and manage and are technically easy to manage with proper training and experience. ECPR can be deployed in the emergency department for out-of-hospital arrest or in various inpatient units for in-hospital arrest. ECPR should be considered for patients with refractory cardiac arrest in hospitals with an existing extracorporeal life support program, able to provide rapid deployment of support, and with resources to provide postresuscitation evaluation and management.

  11. Theory of dynamic arrest in colloidal mixtures

    NASA Astrophysics Data System (ADS)

    Juárez-Maldonado, R.; Medina-Noyola, M.

    2008-05-01

    We present a first-principles theory of dynamic arrest in colloidal mixtures based on the multicomponent self-consistent generalized Langevin equation theory of colloid dynamics [M. A. Chávez-Rojo and M. Medina-Noyola, Phys. Rev. E 72, 031107 (2005); M. A. Chávez-Rojo and M. Medina-Noyola, Phys. Rev. E76, 039902 (2007)]. We illustrate its application with a description of dynamic arrest in two simple model colloidal mixtures: namely, hard-sphere and repulsive Yukawa binary mixtures. Our results include observation of the two patterns of dynamic arrest, one in which both species become simultaneously arrested and the other involving the sequential arrest of the two species. The latter case gives rise to mixed states in which one species is arrested while the other species remains mobile. We also derive the (”bifurcation” or fixed-point”) equations for the nonergodic parameters of the system, which takes the surprisingly simple form of a system of coupled equations for the localization length of the particles of each species. The solution of this system of equations indicates unambiguously which species is arrested (finite localization length) and which species remains ergodic (infinite localization length). As a result, we are able to draw the entire ergodic-nonergodic phase diagram of the binary hard-sphere mixture.

  12. Irreversible translation arrest in the reperfused brain

    PubMed Central

    DeGracia, Donald J; Hu, Bingren R

    2012-01-01

    Irreversible translation arrest occurs in reperfused neurons that will die by delayed neuronal death. It is now recognized that suppression of protein synthesis is a general response of eukaryotic cells to exogenous stressors. Indeed, stress-induced translation arrest can be viewed as a component of cell stress responses, and consists of initiation, maintenance, and termination phases that work in concert with stress-induced transcriptional mechanisms. Within this framework, we review translation arrest in reperfused neurons. This framework provides a basis to recognize that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is the initiator of translation arrest, and a key marker indicating activation of neuronal stress responses. However, eIF2 alpha phosphorylation is reversible. Other phases of stress-induced translation arrest appear to contribute to irreversible translation arrest specifically in ischemic vulnerable neuron populations. We detail two lines of evidence supporting this view. First, ischemia, as a stress stimulus, induces irreversible co-translational protein misfolding and aggregation after 4 to 6 h of reperfusion, trapping protein synthesis machinery into functionally inactive protein aggregates. Second, ischemia and reperfusion leads to modifications of stress granules (SGs) that sequester functionally inactive 48S preinitiation complexes to maintain translation arrest. At later reperfusion durations, these mechanisms may converge such that SGs become sequestered in protein aggregates. These mechanisms result in elimination of functionally active ribosomes and preclude recovery of protein synthesis in selectively vulnerable neurons. Thus, recognizing translation arrest as a component of endogenous cellular stress response pathways will aid in making sense of the complexities of postischemic translation arrest. PMID:16926841

  13. Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

    PubMed

    Lee, Min Ho; Cho, Yoonjung; Jung, Byung Chul; Kim, Sung Hoon; Kang, Yeo Wool; Pan, Cheol-Ho; Rhee, Ki-Jong; Kim, Yoon Suk

    2015-08-14

    Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth.

  14. 53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis

    PubMed Central

    Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

    2016-01-01

    Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency. DOI: http://dx.doi.org/10.7554/eLife.16270.001 PMID:27371829

  15. Sudden death during arrest and phencyclidine intoxication.

    PubMed

    Pestaner, Joseph P; Southall, Pamela E

    2003-06-01

    Deaths of individuals being arrested are important and complex medicolegal cases. Conclusions regarding the cause and manner of death for such cases must take into account multiple factors that may have played a role, as well as anticipate the forensic issues that will arise. In this article, we review the deaths of 2 individuals in which phencyclidine intoxication was a factor that contributed to death during arrest. Most cases of sudden death during arrest have involved cocaine intoxication; because phencyclidine's pharmacologic properties are quite different from those of cocaine, these cases allow for comparisons to those factors that may have greater importance.

  16. Cardiac Arrest: Obstetric CPR/ACLS.

    PubMed

    Cobb, Benjamin; Lipman, Steven

    2017-01-10

    In contrast with other high-resource countries, maternal mortality has seen an increase in the United States. Caring for pregnant women in cardiac arrest may prove uniquely challenging given the rarity of the event coupled by the physiological changes of pregnancy. Optimization of resuscitative efforts warrants special attention as described in the 2015 American Heart Association's "Scientific Statement on Maternal Cardiac Arrest." Current recommendations address a variety of topics ranging from the basic components of chest compressions and airway management to some of the logistical complexities and operational challenges involved in maternal cardiac arrest.

  17. Innovations in polymer arrester moisture sealing testing

    SciTech Connect

    Bennett, J.A.; Mackevich, J.P.; Mosso, R.J.

    1994-12-31

    The vast majority of porcelain distribution arrester failures are the result of moisture ingress. Standards lag technology and do not currently address the unique design aspects of polymer arresters. Traditional sealing test methods cannot be run on polymer arresters because of lack of internal air space. A novel design test is proposed which involves sensitive interfacial leakage current measurements as the diagnostic. Samples are thermally cycled in water to produce thermal excursions and aging, while encouraging water ingress, should the sealing system be compromised. The proposed test is a modification of a protocol established for polymer insulators, which has been correlated to field service.

  18. Innovation in polymer arrester moisture sealing testing

    SciTech Connect

    Bennett, J.A.; Mackevich, J.P.; Mosso, R.J.

    1995-01-01

    The vast majority of porcelain distribution arrester failures are the result of moisture ingress. Standards lag technology and do not currently address the unique design aspects of polymer arresters. Traditional sealing test methods cannot be run on polymer arresters because of lack of internal air space. A novel design test is proposed which involves sensitive interfacial leakage current measurements as the diagnostic. Samples are thermally cycled in water to produce thermal excursions and aging, while encouraging water ingress, should the sealing system be compromised. The proposed test is a modification of a protocol established for polymer insulators, which has been correlated to field service.

  19. Chromosome Damage and Early Developmental Arrest Caused by the Rex Element of Drosophila Melanogaster

    PubMed Central

    Robbins, L. G.; Pimpinelli, S.

    1994-01-01

    Rex (Ribosomal exchange) is a genetically identified repeated element within the ribosomal DNA (rDNA) of Drosophila melanogaster. Rex has a semidominant maternal effect that promotes exchange between and within rDNA arrays in the first few embryonic mitoses. Several of Rex's genetic properties suggest that its primary effect is rDNA-specific chromosome breakage that is resolved by recombination. We report here that rDNA crossovers are only a small, surviving minority of Rex-induced events. Cytology of embryos produced by Rex-homozygous females reveals obvious chromosome damage in at least a quarter of the embryos within the first three mitotic divisions. More than half of the embryos produced by Rex females die, and the developmental arrest is among the earliest reported for any maternal-effect lethal. The striking lethal phenotype suggests that embryos with early chromosome damage could be particularly fruitful subjects for analysis of the cell biology of early embryos. PMID:7828823

  20. Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

    PubMed Central

    Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F.; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L.; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B.; Murray, Brion W.

    2015-01-01

    Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment

  1. Preventing and arresting coronary atherosclerosis.

    PubMed

    Roberts, W C

    1995-09-01

    The good news about coronary atherosclerosis is that it takes an awful lot of plaque before symptoms of myocardial ischemia occur. The bad news is that despite the need for large quantities of plaque for symptoms to occur, nevertheless nearly half of us in the United States eventually have the necessary quantity. Atherosclerosis is infrequently hereditary in origin. Most of us get atherosclerosis because we consume too much fat, cholesterol, and calories. The consequence is an elevated ( > 150 mg/dl) serum total cholesterol level, and the higher the number is above 150, the greater is the quantity of plaque deposited in our arteries. If the serum total cholesterol level can be prevented from rising to more than 150 mg/dl, plaques are not laid down; if elevated levels are lowered to 150 mg/dl, further plaque does not form, and parts of those present may vanish. A fruit-vegetarian-starch diet is necessary as a rule to achieve the 150 mg/dl level in most adults. Lipid-lowering drugs are required in the patients with familial hypercholesterolemia and in most patients with atherosclerotic events. The best news about atherosclerosis is that it can be prevented in those without the hereditary form, and it can be arrested by lowering elevated serum total (and LDL) cholesterol to the 150 mg/dl level.

  2. Improving Survival after Cardiac Arrest.

    PubMed

    Bjørshol, Conrad Arnfinn; Søreide, Eldar

    2017-02-01

    Each year, approximately half a million people suffer out-of-hospital cardiac arrest (CA) in Europe: The majority die. Survival after CA varies greatly between regions and countries. The authors give an overview of the important elements necessary to promote improved survival after CA as a function of the chain of survival and formula for survival concepts. The chain of survival incorporates bystanders (who identify warning symptoms, call the emergency dispatch center, initiate cardiopulmonary resuscitation [CPR]), dispatchers (who identify CA, and instruct and reassure the caller), first responders (who provide high-quality CPR, early defibrillation), paramedics and other prehospital care providers (who continue high-quality CPR, and provide timely defibrillation and advanced life support, transport to CA center), and hospitals (targeted temperature management, percutaneous coronary intervention, delayed prognostication). The formula for survival concept consists of (1) medical science (international guidelines), (2) educational efficiency (e.g., low-dose, high-frequency training for lay people, first responders, and professionals; and (3) local implementation of all factors in the chain of survival and formula for survival. Survival rates after CA can be advanced through the improvement of the different factors in both the chain of survival and the formula for survival. Importantly, the neurologic outcome in the majority of CA survivors has continued to improve.

  3. Mitotic Spindle Assembly in Land Plants: Molecules and Mechanisms

    PubMed Central

    Yamada, Moé; Goshima, Gohta

    2017-01-01

    In textbooks, the mitotic spindles of plants are often described separately from those of animals. How do they differ at the molecular and mechanistic levels? In this chapter, we first outline the process of mitotic spindle assembly in animals and land plants. We next discuss the conservation of spindle assembly factors based on database searches. Searches of >100 animal spindle assembly factors showed that the genes involved in this process are well conserved in plants, with the exception of two major missing elements: centrosomal components and subunits/regulators of the cytoplasmic dynein complex. We then describe the spindle and phragmoplast assembly mechanisms based on the data obtained from robust gene loss-of-function analyses using RNA interference (RNAi) or mutant plants. Finally, we discuss future research prospects of plant spindles. PMID:28125061

  4. Brownian dynamics simulation of fission yeast mitotic spindle formation

    NASA Astrophysics Data System (ADS)

    Edelmaier, Christopher

    2014-03-01

    The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

  5. Analysis of the Functionality of the Mitotic Checkpoints.

    PubMed

    Fraschini, Roberta

    2017-01-01

    During cell division the main goal of the cell is to produce two daughter cells with the same genome as the mother, i.e., maintain its genetic stability. Since this issue is essential to preserve the cell ability to proliferate properly, all eukaryotic cells have developed several pathways, called mitotic checkpoints, that regulate mitotic entry, progression, and exit in response to different cellular signals. Given the evolutive conservation of mechanisms and proteins involved in the cell cycle control from yeast to humans, the budding yeast S. cerevisiae has been very helpful to gain insight in these complex regulations. Here, we describe how the checkpoint can be activated and which cellular phenotypes can be used as markers of checkpoint activation.

  6. A comprehensive model to predict mitotic division in budding yeasts

    PubMed Central

    Sutradhar, Sabyasachi; Yadav, Vikas; Sridhar, Shreyas; Sreekumar, Lakshmi; Bhattacharyya, Dibyendu; Ghosh, Santanu Kumar; Paul, Raja; Sanyal, Kaustuv

    2015-01-01

    High-fidelity chromosome segregation during cell division depends on a series of concerted interdependent interactions. Using a systems biology approach, we built a robust minimal computational model to comprehend mitotic events in dividing budding yeasts of two major phyla: Ascomycota and Basidiomycota. This model accurately reproduces experimental observations related to spindle alignment, nuclear migration, and microtubule (MT) dynamics during cell division in these yeasts. The model converges to the conclusion that biased nucleation of cytoplasmic microtubules (cMTs) is essential for directional nuclear migration. Two distinct pathways, based on the population of cMTs and cortical dyneins, differentiate nuclear migration and spindle orientation in these two phyla. In addition, the model accurately predicts the contribution of specific classes of MTs in chromosome segregation. Thus we present a model that offers a wider applicability to simulate the effects of perturbation of an event on the concerted process of the mitotic cell division. PMID:26310442

  7. G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound

    PubMed Central

    Cabrera, Maia; Gomez, Natalia; Remes Lenicov, Federico; Echeverría, Emiliana; Shayo, Carina; Moglioni, Albertina; Fernández, Natalia; Davio, Carlos

    2015-01-01

    Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4’-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies. PMID:26360247

  8. Mitotic exit: Determining the PP2A dephosphorylation program.

    PubMed

    Pereira, Gislene; Schiebel, Elmar

    2016-08-29

    In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55's dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis.

  9. Mitotic exit: Determining the PP2A dephosphorylation program

    PubMed Central

    2016-01-01

    In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55’s dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis. PMID:27551057

  10. Mitotic activity in dorsal epidermis of Rana pipiens.

    NASA Technical Reports Server (NTRS)

    Garcia-Arce, H.; Mizell, S.

    1972-01-01

    Study of statistically significant rhythms of mitotic division in dorsal epidermis of frogs, Rana pipiens, exposed to a 12:12 light:dark environment for 14 days. The results include the findings that (1) male animals have a primary period of 22 hr in summer and 18 hr in winter, (2) female animals have an 18 hr period, and (3) parapinealectomy and blinding abolish the rhythm.

  11. Cyto-3D-print to attach mitotic cells.

    PubMed

    Castroagudin, Michelle R; Zhai, Yujia; Li, Zhi; Marnell, Michael G; Glavy, Joseph S

    2016-08-01

    The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal.

  12. Pattern formation in stochastic systems: Magnetized billiards and mitotic spindles

    NASA Astrophysics Data System (ADS)

    Schaffner, Stuart C.

    Physical systems that exhibit chaotic behavior or are subject to thermal noise are treated as random processes, especially if the state of the system cannot be measured precisely. Here we examine two such systems. The first is a single electron confined to a wedge-shaped section of a disk, called a billiard, in the presence of a uniform transverse magnetic field. The system exhibits a mixture of chaotic and nonchaotic behavior at different values of the magnetic field strength. If the size of the billiard is on the order of micrometers, as in a quantum dot, both quantum and classical analyses are necessary. The second system is a collection of stiff fibers, called microtubules, suspended in a fluid called the cytoplasm, and lying over chromosomes in a cell. The cytoplasm supplies molecular motors and fuel for the motors. The chromosomes supply motor attachment points. The combination causes the microtubules to self-assemble into a coherent structure called the mitotic spindle. This structure is vital to cell division in plants and animals. Elements of the mitotic spindle have sizes ranging from nanometers to micrometers, and all are subject to considerable thermal agitation. Mitotic spindle self-assembly occurs despite the randomizing effect of this thermal motion. We studied both systems by constructing physical models described by mathematical equations. From these we were able to perform computer simulations. For the billiard problem, we made innovative use of geometric symmetries. These symmetries allowed us to construct efficient representations of both classical and quantum systems. We found a new region of integrable trajectories for a magnetic field above that required to produce completely chaotic orbits. For the mitotic spindle, we were the first to demonstrate spindle self-assembly in a model that matches conditions reported by experimental biologists. Our simulations have shed significant light on which of the many elements in this complex system are

  13. A roller coaster ride with the mitotic cyclins.

    PubMed

    Fung, Tsz Kan; Poon, Randy Y C

    2005-06-01

    Cyclins are discovered as proteins that accumulate progressively through interphase and disappear abruptly at mitosis during each cell cycle. In mammalian cells, cyclin A accumulates from late G1 phase and is destroyed before metaphase, and cyclin B is destroyed slightly later at anaphase. The abundance of the mitotic cyclins is mainly regulated at the levels of transcription and proteolysis. Transcription is stimulated and repressed by several transcription factors, including B-MYB, E2F, FOXM1, and NF-Y. Elements in the promoter, including CCRE/CDE and CHR, are in part responsible for the cell cycle oscillation of transcription. Destruction of the mitotic cyclins is carried out by the ubiquitin ligases APC/C(CDC20) and APC/C(CDH1). Central to our knowledge is the understanding of how APC/C is turned on from anaphase to early G1 phase, and turned off from late G1 till the spindle-assembly checkpoint is deactivated in metaphase. Reciprocal actions of cyclin-dependent kinases (CDKs) on APC/C, as well as on the SCF complexes ensure that the mitotic cyclins are destroyed only at the proper time.

  14. Influence of centriole number on mitotic spindle length and symmetry

    PubMed Central

    Keller, Lani C.; Wemmer, Kimberly A.; Marshall, Wallace F.

    2010-01-01

    The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assembly in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions. PMID:20540087

  15. Distinct Kinesin-14 mitotic mechanisms in spindle bipolarity.

    PubMed

    Simeonov, Dimitre R; Kenny, Katelyn; Seo, Lan; Moyer, Amanda; Allen, Jessica; Paluh, Janet L

    2009-11-01

    Kinesin-like proteins are integral to formation and function of a conserved mitotic spindle apparatus that directs chromosome segregation and precedes cell division. Ubiquitous to the mechanism of spindle assembly and stability are balanced Kinesin-5 promoting and Kinesin-14 opposing forces. Distinct Kinesin-14 roles in bipolarity in eukaryotes have not been shown, but are suggested by gamma-tubulin-based pole interactions that affect establishment and by microtubule cross-linking and sliding that maintain bipolarity and spindle length. Distinct roles also imply specialized functional domains. By cross-species analysis of compatible mechanisms in establishing mitotic bipolarity we demonstrate that Kinesin-14 human HSET (HsHSET) functionally replaces Schizosaccharomyces pombe Pkl1 and its action is similarly blocked by mutation in a Kinesin-14 binding site on gamma-tubulin. Drosophila DmNcd localizes preferentially to bundled interpolar microtubules in fission yeast and does not replace SpPkl1. Analysis of twenty-six Kinesin-14 derivatives, including Tail, Stalk or Neck-Motor chimeras, for spindle localization, spindle assembly and mitotic progression defined critical domains. The Tail of SpPkl1 contains functional elements enabling its role in spindle assembly that are distinct from but transferable to DmNcd, whereas HsHSET function utilizes both Tail and Stalk features. Our analysis is the first to demonstrate distinct mechanisms between SpPkl1 and DmNcd, and reveal that HsHSET shares functional overlap in spindle pole mechanisms.

  16. On the molecular mechanisms of mitotic kinase activation

    PubMed Central

    Bayliss, Richard; Fry, Andrew; Haq, Tamanna; Yeoh, Sharon

    2012-01-01

    During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients. PMID:23226601

  17. Toward a systems-level view of mitotic checkpoints.

    PubMed

    Ibrahim, Bashar

    2015-03-01

    Reproduction and natural selection are the key elements of life. In order to reproduce, the genetic material must be doubled, separated and placed into two new daughter cells, each containing a complete set of chromosomes and organelles. In mitosis, transition from one process to the next is guided by intricate surveillance mechanisms, known as the mitotic checkpoints. Dis-regulation of cell division through checkpoint malfunction can lead to developmental defects and contribute to the development or progression of tumors. This review approaches two important mitotic checkpoints, the spindle assembly checkpoint (SAC) and the spindle position checkpoint (SPOC). The highly conserved spindle assembly checkpoint (SAC) controls the onset of anaphase by preventing premature segregation of the sister chromatids of the duplicated genome, to the spindle poles. In contrast, the spindle position checkpoint (SPOC), in the budding yeast Saccharomyces cerevisiae, ensures that during asymmetric cell division mitotic exit does not occur until the spindle is properly aligned with the cell polarity axis. Although there are no known homologs, there is indication that functionally similar checkpoints exist also in animal cells. This review can be regarded as an "executable model", which could be easily translated into various quantitative concrete models like Petri nets, ODEs, PDEs, or stochastic particle simulations. It can also function as a base for developing quantitative models explaining the interplay of the various components and proteins controlling mitosis.

  18. On the molecular mechanisms of mitotic kinase activation.

    PubMed

    Bayliss, Richard; Fry, Andrew; Haq, Tamanna; Yeoh, Sharon

    2012-11-01

    During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein-protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.

  19. Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis.

    PubMed Central

    Ahmad, K; Golic, K G

    1999-01-01

    Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence. PMID:10049921

  20. Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes.

    PubMed

    Zhen, Chao Yu; Duc, Huy Nguyen; Kokotovic, Marko; Phiel, Christopher J; Ren, Xiaojun

    2014-11-15

    Polycomb group (PcG) proteins are epigenetic transcriptional factors that repress key developmental regulators and maintain cellular identity through mitosis via a poorly understood mechanism. Using quantitative live-cell imaging in mouse ES cells and tumor cells, we demonstrate that, although Polycomb repressive complex (PRC) 1 proteins (Cbx-family proteins, Ring1b, Mel18, and Phc1) exhibit variable capacities of association with mitotic chromosomes, Cbx2 overwhelmingly binds to mitotic chromosomes. The recruitment of Cbx2 to mitotic chromosomes is independent of PRC1 or PRC2, and Cbx2 is needed to recruit PRC1 complex to mitotic chromosomes. Quantitative fluorescence recovery after photobleaching analysis indicates that PRC1 proteins rapidly exchange at interphasic chromatin. On entry into mitosis, Cbx2, Ring1b, Mel18, and Phc1 proteins become immobilized at mitotic chromosomes, whereas other Cbx-family proteins dynamically bind to mitotic chromosomes. Depletion of PRC1 or PRC2 protein has no effect on the immobilization of Cbx2 on mitotic chromosomes. We find that the N-terminus of Cbx2 is needed for its recruitment to mitotic chromosomes, whereas the C-terminus is required for its immobilization. Thus these results provide fundamental insights into the molecular mechanisms of epigenetic inheritance.

  1. Composite Pressure Vessel Including Crack Arresting Barrier

    NASA Technical Reports Server (NTRS)

    DeLay, Thomas K. (Inventor)

    2013-01-01

    A pressure vessel includes a ported fitting having an annular flange formed on an end thereof and a tank that envelopes the annular flange. A crack arresting barrier is bonded to and forming a lining of the tank within the outer surface thereof. The crack arresting barrier includes a cured resin having a post-curing ductility rating of at least approximately 60% through the cured resin, and further includes randomly-oriented fibers positioned in and throughout the cured resin.

  2. Distribution Surge Arrester Failures due to Winter Lightning and Measurement of Energy Absorption Capability of Arresters

    NASA Astrophysics Data System (ADS)

    Sugimoto, Hitoshi; Shimasaki, Katsuhiko; Kado, Hiroyuki

    Surge arresters and distribution equipments with zinc-oxide elements are used for lightning protection of overhead power distribution lines in Japan. However, these surge arresters are sometimes damaged by direct lightning strokes, especially in winter. Increasing of surge arrester failures in winter is attributed to a very large electric charge of winter lightning than that of summer lightning. For improvement of surge arresters, we have measured the energy absorption capability of surge arresters using a half cycle of alternating current with a frequency of 50Hz for simulating a winter lightning current. The mean values of arrester failure energy increased in proportion to the volume of zinc-oxide element, however the values of arrester failure energy were quite uneven. We also have observed the aspects of damaged zinc-oxide elements, and have investigated the relationship between the arrester failure energy and the failure types of zinc-oxide elements. From these results, we suggest the improvement of the energy absorption capability of distribution surge arresters, especially for the uniform energy absorption capability.

  3. AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1.

    PubMed

    Chou, Chia-Hua; Loh, Joon-Khim; Yang, Ming-Chang; Lin, Ching-Chih; Hong, Ming-Chang; Cho, Chung-Lung; Chou, An-Kuo; Wang, Chi-Huei; Lieu, Ann-Shung; Howng, Shen-Long; Hsu, Ching-Mei; Hong, Yi-Ren

    2015-01-01

    We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A-mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.

  4. The SFP1 gene product of Saccharomyces cerevisiae regulates G2/M transitions during the mitotic cell cycle and DNA-damage response.

    PubMed Central

    Xu, Z; Norris, D

    1998-01-01

    In eukaryotic cells, checkpoint pathways arrest cell-cycle progression if a particular event has failed to complete appropriately or if an important intracellular structure is defective or damaged. Saccharomyces cerevisiae strains that lack the SFP1 gene fail to arrest at the G2 DNA-damage checkpoint in response to genomic injury, but maintain their ability to arrest at the replication and spindle-assembly checkpoints. sfp1Delta mutants are characterized by a premature entrance into mitosis during a normal (undamaged) cell cycle, while strains that overexpress Sfp1p exhibit delays in G2. Sfp1p therefore acts as a repressor of the G2/M transition, both in the normal cell cycle and in the G2 checkpoint pathway. Sfp1 is a nuclear protein with two Cys2His2 zinc-finger domains commonly found in transcription factors. We propose that Sfp1p regulates the expression of gene products involved in the G2/M transition during the mitotic cell cycle and the DNA-damage response. In support of this model, overexpression of Sfp1p induces the expression of the PDS1 gene, which is known to encode a protein that regulates the G2 checkpoint. PMID:9832520

  5. Surface Electrocardiogram Predictors of Sudden Cardiac Arrest

    PubMed Central

    Abdelghani, Samy A.; Rosenthal, Todd M.; Morin, Daniel P.

    2016-01-01

    Background: Heart disease is a major cause of death in industrialized nations, with approximately 50% of these deaths attributable to sudden cardiac arrest. If patients at high risk for sudden cardiac arrest can be identified, their odds of surviving fatal arrhythmias can be significantly improved through prophylactic implantable cardioverter defibrillator placement. This review summarizes the current knowledge pertaining to surface electrocardiogram (ECG) predictors of sudden cardiac arrest. Methods: We conducted a literature review focused on methods of predicting sudden cardiac arrest through noninvasive electrocardiographic testing. Results: Several electrocardiographic-based methods of risk stratification of sudden cardiac arrest have been studied, including QT prolongation, QRS duration, fragmented QRS complexes, early repolarization, Holter monitoring, heart rate variability, heart rate turbulence, signal-averaged ECG, T wave alternans, and T-peak to T-end. These ECG findings have shown variable effectiveness as screening tools. Conclusion: At this time, no individual ECG finding has been found to be able to adequately stratify patients with regard to risk for sudden cardiac arrest. However, one or more of these candidate surface ECG parameters may become useful components of future multifactorial risk stratification calculators. PMID:27660578

  6. Mitotic Index is an Independent Predictor of Recurrence-Free Survival in Meningioma.

    PubMed

    Olar, Adriana; Wani, Khalida M; Sulman, Erik P; Mansouri, Alireza; Zadeh, Gelareh; Wilson, Charmaine D; DeMonte, Franco; Fuller, Gregory N; Aldape, Kenneth D

    2015-05-01

    While World Health Organization (WHO) grading of meningioma stratifies patients according to recurrence risk overall, there is substantial within-grade heterogeneity with respect to recurrence-free survival (RFS). Most meningiomas are graded according to mitotic counts per unit area on hematoxylin and eosin sections, a method potentially confounded by tumor cellularity, as well as potential limitations of accurate mitotic figure detection on routine histology. To refine mitotic figure assessment, we evaluated 363 meningiomas with phospho-histone H3 (Ser10) and determined the mitotic index (number of mitoses per 1000 tumor cells). The median mitotic indices among WHO grade I (n = 268), grade II (n = 84) and grade III (n = 11) tumors were 1, 4 and 12. Classification and regression tree analysis to categorize cut-offs identified three subgroups defined by mitotic indices of 0-2, 3-4 and ≥5, which on univariate analysis were associated with RFS (P < 0.01). In multivariate analysis, mitotic index subgrouped in this manner was significantly associated with RFS (P < 0.01) after adjustment for Simpson grade, WHO grade and MIB-1 index. Mitotic index was then examined within individual WHO grade, showing that for grade I and grade II meningiomas, mitotic index can add additional information to RFS risk. The results suggest that the use of a robust mitotic marker in meningioma could refine risk stratification.

  7. Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression.

    PubMed

    O'Regan, Laura; Sampson, Josephina; Richards, Mark W; Knebel, Axel; Roth, Daniel; Hood, Fiona E; Straube, Anne; Royle, Stephen J; Bayliss, Richard; Fry, Andrew M

    2015-05-11

    Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber-stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT-kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG-TACC3 complex.

  8. Sex Disparities in Arrest Outcomes for Domestic Violence

    ERIC Educational Resources Information Center

    Hamilton, Melissa; Worthen, Meredith G. F.

    2011-01-01

    Domestic violence arrests have been historically focused on protecting women and children from abusive men. Arrest patterns continue to reflect this bias with more men arrested for domestic violence compared to women. Such potential gender variations in arrest patterns pave the way to the investigation of disparities by sex of the offender in…

  9. Why a Combination of WP 631 and Epo B is an Improvement on the Drugs Singly - Involvement in the Cell Cycle and Mitotic Slippage.

    PubMed

    Bukowska, Barbara; Rogalska, Aneta; Forma, Ewa; Brys, Magdalena; Marczak, Agnieszka

    2016-01-01

    Our previous studies clearly demonstrated that a combination of WP 631 and Epo B has higher activity against ovarian cancer cells than either of these compounds used separately. In order to fully understand the exact mechanism of action in combination, we assessed effects on the cell cycle of SKOV-3 cells. We evaluated three control points essential for WP 631 and Epo B action to determine which cell cycle-regulating proteins (CDK1/cyclin B complex, EpCAM or HMGB1) mediate activity. The effects of the drug on the cell cycle were measured based on the nuclear DNA content using flow cytometry. Expression of cell cycle-regulating genes was analyzed using real-time PCR. It was discovered that WP 631, at the tested concentration, did not affect the SKOV-3 cell cycle. Epo B caused significant G2/M arrest, whereas the drug combination induced stronger apoptosis and lower mitotic arrest than Epo B alone. This is very important information from the point of view of the fight against cancer, as, while mitotic arrest in Epo B-treated cells could be overcame after DNA damage repair, apoptosis which occurs after mitotic slippage in combination-treated cells is irreversible. It clearly explains the higher activity of the drug combination in comparison to Epo B alone. Epo B acts via the CDK1/cyclin B complex and has the ability to inhibit CDK1, which may be a promising strategy for ovarian cancer treatment in the future. The drug combination diminishes EpCAM and HMGB1 expression to a greater degree than either WP 631 and Epo B alone. Owing to the fact that the high expression of these two proteins is a poor prognostic factor for ovarian cancer, a decrease in their expression, observed in our studies, may result in improved efficacy of cancer therapy. The presented findings show that the combination of WP 631 and Epo B is a better therapeutic option than either of these drugs alone.

  10. A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms.

    PubMed

    Néant, I; Charbonneau, M; Guerrier, P

    1989-04-01

    Populations of hormone-stimulated starfish oocytes and fertilized sea urchin eggs undergo synchronous meiotic and mitotic divisions. We have studied the requirement for protein phosphorylation during these events by testing the effects of 6-dimethylaminopurine (6-DMAP) upon the incorporation of [32P]orthophosphate. It was found that 6-DMAP blocked meiosis reinitiation and early cleavage and simultaneously inhibited protein phosphorylation, without changing the rate of [35S]methionine incorporation or pattern of protein synthesis. The protein, cyclin (54 kDa in starfish and 57 kDa in sea urchin), continues to be synthesized in the presence of 6-DMAP. This protein is destroyed at first and second cell cycles when 6-DMAP is added 30 min following fertilization but not when this drug is present before fertilization. Thus, cyclin breakdown does not depend on the completion of the nuclear events of M-phase, and its time of breakdown is set at an early step between fertilization and first cleavage. Using tubulin immunostaining, we found that 6-DMAP did not affect the cortical microtubules and resting female centrioles of prophase-arrested starfish oocytes, whereas it induced a precocious disappearance of spindle fibers when applied to hormone-stimulated oocytes. While an early addition of 6-DMAP precluded nuclear breakdown and spindle formation in both systems, a late treatment always allowed chromosome separation and centriole separation. Under these conditions pericentriolar tubulin persisted and could organize new spindles after the inhibitor was removed. It is suggested that (1) the assembly of cortical and centriolar-associated microtubules is not controlled by the same factors as spindle-associated tubulin; (2) specific proteins which are required for the cell to enter the following M-phase can become operative only via a process depending upon protein phosphorylation; (3) microtubule-associated kinases may play an important role in MPF function and spindle dynamics.

  11. A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

    PubMed Central

    Smith, M M; Yang, P; Santisteban, M S; Boone, P W; Goldstein, A T; Megee, P C

    1996-01-01

    The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation. PMID:8622646

  12. XCTK2: A Kinesin-related Protein That Promotes Mitotic Spindle Assembly in Xenopus laevis Egg Extracts

    PubMed Central

    Walczak, Claire E.; Verma, Suzie; Mitchison, Timothy J.

    1997-01-01

    We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central α-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor- arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles. PMID:9049251

  13. Multi-Kinase Inhibitor C1 Triggers Mitotic Catastrophe of Glioma Stem Cells Mainly through MELK Kinase Inhibition

    PubMed Central

    Joshi, Kaushal; Nakano-Okuno, Mariko; Hong, Christopher; Nguyen, Chi-Hung; Kornblum, Harley I.; Molla, Annie; Nakano, Ichiro

    2014-01-01

    Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors. PMID:24739874

  14. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

    PubMed

    Singh, Gaurav Kumar; Karade, Sharanbasappa Shrimant; Ranjan, Rajeev; Ahamad, Nafees; Ahmed, Shakil

    2017-02-01

    The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

  15. Influence of the circadian rhythm in cell division on radiation-induced mitotic delay in vivo

    SciTech Connect

    Rubin, N.H.

    1982-01-01

    Mitotic delay is described as a classical response to radiation; however, circadian rhythmicity in cell division in vivo has not been considered by many authors. The present study investigated the relation between fluctuations reported as mitotic delay and recovery in vivo and circadian oscillations in mitotic index in mouse corneal epithelium. One aspect involved single doses (approximately 600 rad) given to mice at different circadian stages. The normal circadian rhythm in cell division was never obliterated. Inhibition of mitosis was evident but unpredictable, ranging from 6 to 15 hr after irradiation. Recovery was evident only during the daily increase in mitotic index of controls. The classical interpretation of recovery from mitotic delay may be in an in vitro phenomenon not reflecting in vivo responses, which are apparently strongly circadian stage dependent. The second portion of the study demonstrated a dose-response effect on length of mitotic delay and, to a lesser extent, degree of recovery.

  16. MEN, destruction and separation: mechanistic links between mitotic exit and cytokinesis in budding yeast.

    PubMed

    Yeong, Foong May; Lim, Hong Hwa; Surana, Uttam

    2002-07-01

    Cellular events must be executed in a certain sequence during the cell division in order to maintain genome integrity and hence ensure a cell's survival. In M phase, for instance, chromosome segregation always precedes mitotic exit (characterized by mitotic kinase inactivation via cyclin destruction); this is then followed by cytokinesis. How do cells impose this strict order? Recent findings in budding yeast have suggested a mechanism whereby partitioning of chromosomes into the daughter cell is a prerequisite for the activation of mitotic exit network (MEN). So far, however, a regulatory scheme that would temporally link the initiation of cytokinesis to the execution of mitotic exit has not been determined. We propose that the requirement of MEN components for cytokinesis, their translocation to the mother-daughter neck and triggering of this translocation by inactivation of the mitotic kinase may be the three crucial elements that render initiation of cytokinesis dependent on mitotic exit.

  17. Alcohol Arrests on Campuses Jumped 10% in 1996; Drug Arrests Increased by 5%.

    ERIC Educational Resources Information Center

    Lively, Kit

    1998-01-01

    Campus police and other college officials believe the 16,237 alcohol arrests and 7,060 drug arrests on college campuses in 1996 reflect tougher enforcement, not increased usage among students. This is particularly true in states such as Michigan, where state law concerning underage drinking has changed, and in communities where enforcement is…

  18. Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells

    SciTech Connect

    Kim, Soon-hee; Park, Hee-Sook; Lee, Myoung-su; Cho, Yong-Jin; Kim, Young-Sup; Hwang, Jin-Taek; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young

    2008-07-18

    Inhibition of adipocyte differentiation is one approach among the anti-obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, inhibits adipocyte differentiation most effectively of 18 stilbenes tested. Fat accumulation and PPAR{gamma} expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly inhibited preadipocyte proliferation and consequent differentiation within the first 2 days of treatment, indicating that the anti-adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was reduced. Therefore, vitisin A seems to induce G1 arrest through p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A inhibits adipocyte differentiation through preadipocyte cell cycle arrest.

  19. Enhancement of spontaneous mitotic recombination by the meiotic mutant spo11-1 in Saccharomyces cerevisiae

    SciTech Connect

    Bruschi, C.V.; Esposito, M.S.

    1983-12-01

    Both nonreciprocal and reciprocal mitotic recombination are enhanced by the recessive mutant spo11-1, which was previously shown to affect meiosis by decreasing recombination and increasing nondisjunction. The mitotic effects are not distributed equally in all chromosomal regions. The genotypes of mitotic recombinants in spo11-1/spo11-1 diploid cells provide further evidence that widely spaced chromosomal markers undergo coincident conversion in mitosis.

  20. A pharmacologic review of cardiac arrest.

    PubMed

    Wagner, Bradley J; Yunker, Nancy S

    2014-01-01

    Cardiac arrest is manifested by arrhythmias (ventricular fibrillation or pulseless ventricular tachycardia, pulseless electrical activity, or asystole) resulting in minimal to no forward blood flow to the body's oxygen-dependent tissues. Defibrillation and cardiopulmonary resuscitation (CPR) should be initiated immediately as they have been shown to increase return of spontaneous circulation and survival to discharge rates. Cardiac arrest in the surgical patient population has devastating consequences. Data specific to the surgical patient found that 1 in 203 surgical patients experienced cardiac arrest requiring CPR within 30 days after surgery. A subgroup analysis found that 1 in 1,020 plastic surgery patients required CPR in this same time frame. Thirty-day mortality in the general surgery patient population was 72%. The American Heart Association updates the advanced cardiac life support (ACLS) guidelines every 5 years. Their latest publication in 2010 recommended that the resuscitative protocol be transitioned from its basic life support sequence of airway-breathing-chest compressions to chest compressions-airway-breathing. All health care professionals should have an understanding of the clinical presentation and medical management of cardiac arrest. Maintaining biannual basic life support and ACLS certification ensures that health care professionals remain current with American Heart Association guideline recommendations. Guideline-directed management of cardiac arrest should include timely implementation of the ACLS algorithm to maximize patient outcomes.

  1. Sudden Cardiac Arrest in Athletic Medicine

    PubMed Central

    Kyle, James M.; Ellis, James M.; Cantwell, John; Courson, Ron; Medlin, Ron

    2001-01-01

    Objective: To emphasize the importance of decreasing the response time by a trained target responder to increase the survival rate among athletes experiencing sudden cardiac arrest at an athletic event. Background: Death due to sudden cardiac arrest that is witnessed is preventable in many cases. However, most people who experience this condition die because of a prolonged response time from onset of the fatal arrhythmia to defibrillation by trained treatment providers. If athletic trainers or other members of the athletic care medical team are trained as target responders and equipped with automated electronic defibrillators, they can immediately treat an athlete who experiences a sudden, life-threatening tachyarrhythmia. This prompt response to the life-threatening emergency should result in a higher survival rate. Description: We review the causes of sudden cardiac arrest during athletic events, note some unusual clinical presentations, discuss improved methods of response and new equipment for treatment, and define the athletic trainer's role as a target responder trained to treat people experiencing sudden cardiac arrest at an athletic event. Clinical Advantages: An athletic care team willing to become part of an emergency response team can help improve the survival rate of athletes experiencing sudden cardiac arrest at an athletic event. PMID:12937464

  2. BCL-W is a regulator of microtubule inhibitor-induced mitotic cell death

    PubMed Central

    Huang, Shan; Tang, Rui; Randy, Y.C. Poon

    2016-01-01

    Microtubule inhibitors including taxanes and vinca alkaloids are among the most widely used anticancer agents. Disrupting the microtubules activates the spindle-assembly checkpoint and traps cells in mitosis. Whether cells subsequently undergo mitotic cell death is an important factor for the effectiveness of the anticancer agents. Given that apoptosis accounts for the majority of mitotic cell death induced by microtubule inhibitors, we performed a systematic study to determine which members of the anti-apoptotic BCL-2 family are involved in determining the duration of mitotic block before cell death or slippage. Depletion of several anti-apoptotic BCL-2-like proteins significantly shortened the time before apoptosis. Among these proteins, BCL-W has not been previously characterized to play a role in mitotic cell death. Although the expression of BCL-W remained constant during mitotic block, it varied significantly between different cell lines. Knockdown of BCL-W with siRNA or disruption of the BCL-W gene with CRISPR-Cas9 speeded up mitotic cell death. Conversely, overexpression of BCL-W delayed mitotic cell death, extending the mitotic block to allow mitotic slippage. Taken together, these results showed that BCL-W contributes to the threshold of anti-apoptotic activity during mitosis. PMID:27231850

  3. A Functional Mitotic Spindle Prepared from Mammalian Cells in Culture

    PubMed Central

    Cande, W. Zacheus; Snyder, Judith; Smith, Diana; Summers, Keith; McIntosh, J. R.

    1974-01-01

    Mitotic cells lysed into solutions of polymerizable microtubule protein contain a spindle which is similar to the living spindle in two respects: it will lose and gain birefringence when cooled and warmed, and it will move anaphase chromosomes to the opposite ends of the cell. Early anaphase cells lysed into buffers containing high molecular weight polyethylene glycol and nucleotide triphosphates will continue chromosome motion and spindle elongation in the absence of exogenous spindle subunits. These results suggest that while spindle growth requires microtubule polymerization, anaphase motions do not. Images PMID:4524659

  4. Mitotically unstable polyploids in the yeast Pichia guilliermondii.

    PubMed

    Klinner, U; Böttcher, F

    1992-01-01

    Attempts to obtain triploids or tetraploids of P. guilliermondii by sexual hybridization led to mitotically stable hybrids. However, their DNA content per cell was not higher than in diploids. The results of random spore analysis demonstrate that these hybrids were in fact aneuploids which obviously suffered drastic chromosome losses immediately after mating. This phenomenon could have been caused either by aneuploidy already present in the parental strains or it might have been due to a general inability of P. guilliermondii to maintain a polyploid genome.

  5. Contact line arrest in solidifying spreading drops

    NASA Astrophysics Data System (ADS)

    de Ruiter, Rielle; Colinet, Pierre; Snoeijer, Jacco; Gelderblom, Hanneke

    2016-11-01

    When does a drop, deposited on a cold substrate, stop spreading? Despite the practical relevance of this question, for example in airplane icing and 3D metal printing, the exact mechanism of arrest in solidifying spreading drops has not yet been unraveled. Here, we consider the spreading and arrest of hexadecane drops of constant volume on two smooth wettable substrates; copper with a high thermal conductivity and glass with a low thermal conductivity. We record the spreading radius and contact angle in time for a range of substrate temperatures. We show that our measurements on both copper and glass are well explained by a contact line arrest condition based on crystallization kinetics, which takes into account the effect of kinetic undercooling and the thermal conductivity of the substrate.

  6. Studies of Shuttle orbiter arrestment system

    NASA Technical Reports Server (NTRS)

    Davis, Pamela A.; Stubbs, Sandy M.

    1993-01-01

    Scale model studies of the Shuttle Orbiter Arrestment System (AS) were completed with a 1/27.5-scale model at the NASA Langley Research Center. The purpose of these studies was to determine the proper configuration for a net arrestment system to bring the orbiter to a safe stop with minimal damage in the event of a runway overrun. Tests were conducted for runway on-centerline and off-centerline engagements at simulated speeds up to approximately 100 knots (full scale). The results of these tests defined the interaction of the net and the orbiter, the dynamics of off-centerline engagements, and the maximum number of vertical net straps that may become entangled with the nose gear. In addition to these tests, a test program with a 1/8-scale model was conducted by the arrestment system contractor, and the results are presented in the appendix.

  7. Inhibition of REV3 Expression Induces Persistent DNA Damage and Growth Arrest in Cancer Cells12

    PubMed Central

    Knobel, Philip A; Kotov, Ilya N; Felley-Bosco, Emanuela; Stahel, Rolf A; Marti, Thomas M

    2011-01-01

    REV3 is the catalytic subunit of DNA translesion synthesis polymerase ζ. Inhibition of REV3 expression increases the sensitivity of human cells to a variety of DNA-damaging agents and reduces the formation of resistant cells. Surprisingly, we found that short hairpin RNA-mediated depletion of REV3 per se suppresses colony formation of lung (A549, Calu-3), breast (MCF-7, MDA-MB-231), mesothelioma (IL45 and ZL55), and colon (HCT116 +/-p53) tumor cell lines, whereas control cell lines (AD293, LP9-hTERT) and the normal mesothelial primary culture (SDM104) are less affected. Inhibition of REV3 expression in cancer cells leads to an accumulation of persistent DNA damage as indicated by an increase in phospho-ATM, 53BP1, and phospho-H2AX foci formation, subsequently leading to the activation of the ATM-dependent DNA damage response cascade. REV3 depletion in p53-proficient cancer cell lines results in a G1 arrest and induction of senescence as indicated by the accumulation of p21 and an increase in senescence-associated β-galactosidase activity. In contrast, inhibition of REV3 expression in p53-deficient cells results in growth inhibition and a G2/M arrest. A small fraction of the p53-deficient cancer cells can overcome the G2/M arrest, which results in mitotic slippage and aneuploidy. Our findings reveal that REV3 depletion per se suppresses growth of cancer cell lines from different origin, whereas control cell lines and a mesothelial primary culture were less affected. Thus, our findings indicate that depletion of REV3 not only can amend cisplatin-based cancer therapy but also can be applied for susceptible cancers as a potential monotherapy. PMID:22028621

  8. Cognitive impairment after sudden cardiac arrest

    PubMed Central

    Jaszke-Psonka, Magdalena; Piegza, Magdalena; Pudlo, Robert; Piegza, Jacek; Badura-Brzoza, Karina; Leksowska, Aleksandra; Hese, Robert T.; Gorczyca, Piotr W.

    2016-01-01

    Aim To evaluate the incidence and severity of the impairment of selected cognitive functions in patients after sudden cardiac arrest (SCA) in comparison to patients after myocardial infarction without SCA and healthy subjects and to analyze the influence of sociodemographic and clinical parameters and the duration of cardiac arrest on the presence and severity of the described disorders. Material and methods The study group comprised 30 cardiac arrest survivors, the reference group comprised 31 survivors of myocardial infarction without cardiac arrest, and the control group comprised 30 healthy subjects. The Mini-Mental State Examination (MMSE), the Digit Span test from the Wechsler Adult Intelligence Scale, Lauretta Bender’s Visual-Motor Gestalt Test, and the Benton Visual Retention Test (BVRT) were used to assess the presence of cognitive impairment. An original questionnaire developed by the author was used for overall mental state assessment. Results The Bender test demonstrated a significant difference in the presence and severity of visual-motor skills between the study group and the control group, while BVRT and MMSE revealed increased incidence of cognitive impairment in the study group. The Bender and BVRT (D/D)/SS (version D, method D, scaled score) scales indicated cognitive impairment in 53.3% of these patients, while the BVRT (C/A)/SS test indicated cognitive impairment in 40%. For the reference group, the values were 32.3% and 12.9%, respectively. No correlation was found between the severity of cognitive impairment and the duration of cardiac arrest. Conclusions Impairment of visual-motor skills, short-term visual memory, concentration, and visual-motor coordination occurs much more frequently and is more severe in individuals after SCA than in healthy individuals. Impairment of memory trace storage and recall after delay occurs more frequently in patients after SCA than in patients after myocardial infarction without cardiac arrest and in healthy

  9. Determination of Cell Cycle Stage and Mitotic Exit Through the Quantification of the Protein Levels of Known Mitotic Regulators.

    PubMed

    Cepeda-García, Cristina

    2017-01-01

    There are multiple processes that occur at certain points during the cell cycle and that affect later steps. Impairment of such processes could cause delays or even completely abolish cell cycle progression. Therefore, it is extremely helpful in order to determine the potential consequences that interfering on a cellular process imposes on cell cycle progression to be able to precisely characterize the cell cycle stage by using molecular markers. Here, we describe the analysis of the protein levels of known mitotic regulators as molecular markers to monitor the progression of cells through the cell cycle by western blot in synchronized yeast cell cultures.

  10. Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis

    PubMed Central

    Hascoet, Pauline; Chesnel, Franck; Le Goff, Cathy; Le Goff, Xavier; Arlot-Bonnemains, Yannick

    2015-01-01

    Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel–Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell–cell adhesion and apical–basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the “non-mitotic” unconventional functions of these kinases in renal tumorigenesis. PMID:26579493

  11. Differential Mitotic Stability of Yeast Disomes Derived from Triploid Meiosis

    PubMed Central

    Campbell, Douglas; Doctor, John S.; Feuersanger, Jeane H.; Doolittle, Mark M.

    1981-01-01

    The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies employed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses (N+1→N) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability. PMID:7035289

  12. Mitotic cell death caused by follistatin-like 1 inhibition is associated with up-regulated Bim by inactivated Erk1/2 in human lung cancer cells.

    PubMed

    Bae, Kieun; Park, Kyoung Eun; Han, Jihye; Kim, Jongkwang; Kim, Kyungtae; Yoon, Kyong-Ah

    2016-04-05

    Follistatin-like 1 (FSTL1) was identified as a novel pro-inflammatory protein showing high-level expression in rheumatoid arthritis. The protective effect of FSTL1 via the inhibition of apoptosis was reported in myocardial injury. However, the functional mechanism of FSTL1 in cancer is poorly characterized, and its proliferative effects are ambiguous. Here, we examined the effects of FSTL1 on cellular proliferation and cell cycle checkpoints in lung cancer cells. FSTL1 inhibition induced the cellular portion of G2/M phase in human lung cancer cells via the accumulation of regulators of the transition through the G2/M phase, including the cyclin-dependent kinase 1 (Cdk1)-cyclin B1 complex. An increase in histone H3 phosphorylation (at Ser10), another hallmark of mitosis, indicated that the knockdown of FSTL1 in lung cancer cells stimulated a mitotic arrest. After that, apoptosis was promoted by the activation of caspase-3 and -9. Protein level of Bim, a BH3 domain-only, pro-apoptotic member and its isoforms, BimL, BimS, and BimEL were up-regulated by FSTL1 inhibition. Degradation of Bim was blocked in FSTL1-knockdown cells by decreased phosphorylation of Bim. Increased BimEL as well as decreased phosphorylated Erk1/2 is essential for cell death by FSTL1 inhibition in NCI-H460 cells. Taken together, our results suggest that the knockdown of FSTL1 induces apoptosis through a mitotic arrest and caspase-dependent cell death. FSTL1 plays the important roles in cellular proliferation and apoptosis in lung cancer cells, and thus can be a new target for lung cancer treatment.

  13. Adenovirus E4orf4 protein-induced death of p53-/- H1299 human cancer cells follows a G1 arrest of both tetraploid and diploid cells due to a failure to initiate DNA synthesis.

    PubMed

    Cabon, Lauriane; Sriskandarajah, Neera; Mui, Melissa Z; Teodoro, Jose G; Blanchette, Paola; Branton, Philip E

    2013-12-01

    The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G1 because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.

  14. Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein.

    PubMed Central

    Frattini, M G; Hurst, S D; Lim, H B; Swaminathan, S; Laimins, L A

    1997-01-01

    Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53. PMID:9029152

  15. Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression

    SciTech Connect

    Shen, Enzhi; Lei, Yan; Liu, Qian; Zheng, Yanbo; Song, Chunqing; Marc, Jan; Wang, Yongchao; Sun, Le; Liang, Qianjin

    2009-04-15

    A novel protein that associates with interphase nucleus and mitotic apparatus (INMAP) was identified by screening HeLa cDNA expression library with an autoimmune serum followed by tandem mass spectrometry. Its complete cDNA sequence of 1.818 kb encodes 343 amino acids with predicted molecular mass of 38.2 kDa and numerous phosphorylation sites. The sequence is identical with nucleotides 1-1800 bp of an unnamed gene (GenBank accession no. (7022388)) and highly homologous with the 3'-terminal sequence of POLR3B. A monoclonal antibody against INMAP reacted with similar proteins in S. cerevisiae, Mel and HeLa cells, suggesting that it is a conserved protein. Confocal microscopy using either GFP-INMAP fusion protein or labeling with the monoclonal antibody revealed that the protein localizes as distinct dots in the interphase nucleus, but during mitosis associates closely with the spindle. Double immunolabeling using specific antibodies showed that the INMAP co-localizes with {alpha}-tubulin, {gamma}-tubulin, and NuMA. INMAP also co-immunoprecipitated with these proteins in their native state. Stable overexpression of INMAP in HeLa cell lines leads to defects in the spindle, mitotic arrest, formation of polycentrosomal and multinuclear cells, inhibition of growth, and apoptosis. We propose that INMAP is a novel protein that plays essential role in spindle formation and cell-cycle progression.

  16. Validate Mitotic Checkpoint and Kinetochore Motor Proteins in Breast Cancer Cells as Targets for the Development of Novel Anti-Mitotic Drugs

    DTIC Science & Technology

    2005-07-01

    which chromosomal instability, aneuploidy, and increased tumorigenesis are prominent hallmarks. These include ataxia-telangiectasia, xeroderma ... pigmentosum , Nijmegen breakage syndromes, Bloom’s syndrome, and Werner’s syndrome, (Modesti and Kanaar, 2001; Thompson and Schild, 2002). Defects in mitotic

  17. Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

    PubMed Central

    Onishi, Masayuki; Yeong, Foong May

    2016-01-01

    Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

  18. Juvenile Arrests, 2007. Juvenile Justice Bulletin

    ERIC Educational Resources Information Center

    Puzzanchera, Charles

    2009-01-01

    This Bulletin summarizes 2007 juvenile crime and arrest data reported by local law enforcement agencies across the country and cited in the FBI report, "Crime in the United States 2007." The Bulletin describes the extent and nature of juvenile crime that comes to the attention of the justice system. It serves as a baseline for comparison for…

  19. Juvenile Arrests, 1999. Juvenile Justice Bulletin.

    ERIC Educational Resources Information Center

    Snyder, Howard N.

    This bulletin presents a summary and analysis of national and state juvenile arrest data for 1999. Data come from the FBI's annual "Crime in the United States" report, which offers the estimated number of crimes reported to law enforcement agencies. The 1999 murder rate was the lowest since 1966. Of the nearly 1,800 juveniles murdered in…

  20. 43 CFR 4770.4 - Arrest.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 2 2011-10-01 2011-10-01 false Arrest. 4770.4 Section 4770.4 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR RANGE MANAGEMENT (4000) PROTECTION, MANAGEMENT, AND CONTROL OF WILD FREE-ROAMING HORSES AND...

  1. Arrest History among Men and Sexual Orientation

    ERIC Educational Resources Information Center

    Fisher, Dennis G.; Milroy, Michael E.; Reynolds, Grace L.; Klahn, Jennifer A.; Wood, Michele M.

    2004-01-01

    This study explored associations between ever having been arrested and other variables among 490 male drug users. Participants were classified into three groups based on recent sexual history: men who had not had sex (NOSEX), men who had had sex with women (HETERO), and men who had had sex with men (MSM). We found that MSM who had been arrested…

  2. Drug and Alcohol Arrests Increased in 1999.

    ERIC Educational Resources Information Center

    Nicklin, Julie L.

    2001-01-01

    U.S. Department of Education (DOE) data showed a 1999 increase in drug and alcohol arrests on college campuses. Also, the number of reported sex offenses rose by 6 percent from 1998-99. Some experts question the validity of the year-to-year comparisons and the DOE data. Presents statistics on sex offenses, drug use, and drinking and football. (SM)

  3. The Organizational Determinants of Police Arrest Decisions

    ERIC Educational Resources Information Center

    Chappell, Allison T.; MacDonald, John M.; Manz, Patrick W.

    2006-01-01

    A limited amount of research has examined the relationship between characteristics of police organizations and policing styles. In particular, few studies have examined the link between organizational structures and police officer arrest decisions. Wilson's (1968) pioneering case study of police organizations suggested that individual police…

  4. 32 CFR 935.122 - Arrests.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS... an arrest, a peace officer may use only the degree of force needed to effect submission, and may... verbal warning. (f) A peace officer may force an entry into any building, vehicle, or aircraft...

  5. 32 CFR 935.122 - Arrests.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS... an arrest, a peace officer may use only the degree of force needed to effect submission, and may... verbal warning. (f) A peace officer may force an entry into any building, vehicle, or aircraft...

  6. 32 CFR 935.122 - Arrests.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS... an arrest, a peace officer may use only the degree of force needed to effect submission, and may... verbal warning. (f) A peace officer may force an entry into any building, vehicle, or aircraft...

  7. 32 CFR 935.122 - Arrests.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS... an arrest, a peace officer may use only the degree of force needed to effect submission, and may... verbal warning. (f) A peace officer may force an entry into any building, vehicle, or aircraft...

  8. 32 CFR 935.122 - Arrests.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS... an arrest, a peace officer may use only the degree of force needed to effect submission, and may... verbal warning. (f) A peace officer may force an entry into any building, vehicle, or aircraft...

  9. Perinatal cardiac arrest. Quality of the survivors.

    PubMed Central

    Steiner, H; Neligan, G

    1975-01-01

    Steiner, H., and Neligan, G. (1975). Archives of Disease in Childhood, 50, 696. Perinatal cardiac arrest: quality of the survivors. Twenty-two consecutive survivors of perinatal cardiac arrest have been followed to a mean age of 4 1/4 years, using methods of neurological and developmental assessment appropriate to their ages. 4 showed evidence of gross, diffuse brain-damage (2 of these died before the age of 3 years). These were the only 4 survivors of the first month of life who took more than 30 minutes to establish regular, active respiration after their heartbeat had been restored. The arrest in these cases had occurred during or within 15 minutes of delivery, and followed antepartum haemorrhage, breech delivery, or prolapsed cord. The remaining 18 were free of any evidence of brain damage. In the majority of these the arrest had occurred during shoulder dystocia or exchange transfusion, or was unexplained; the heartbeat had been restored within 5 minutes in most cases, and regular, active respiration had been established within 30 minutes thereafter in all cases. PMID:1190819

  10. Rhp51-Dependent Recombination Intermediates That Do Not Generate Checkpoint Signal Are Accumulated in Schizosaccharomyces pombe rad60 and smc5/6 Mutants after Release from Replication Arrest

    PubMed Central

    Miyabe, Izumi; Morishita, Takashi; Hishida, Takashi; Yonei, Shuji; Shinagawa, Hideo

    2006-01-01

    The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1Δ double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells. PMID:16354704

  11. Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.

    PubMed

    Heng, Boon Chin; Cao, Tong; Liu, Hua; Rufaihah, Abdul Jalil

    2005-01-01

    This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.

  12. Maternal cardiac arrest: a practical and comprehensive review.

    PubMed

    Jeejeebhoy, Farida M; Morrison, Laurie J

    2013-01-01

    Cardiac arrest during pregnancy is a dedicated chapter in the American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care; however, a robust maternal cardiac arrest knowledge translation strategy and emergency response plan is not usually the focus of institutional emergency preparedness programs. Although maternal cardiac arrest is rare, the emergency department is a high-risk area for receiving pregnant women in either prearrest or full cardiac arrest. It is imperative that institutions review and update emergency response plans for a maternal arrest. This review highlights the most recent science, guidelines, and recommended implementation strategies related to a maternal arrest. The aim of this paper is to increase the understanding of the important physiological differences of, and management strategies for, a maternal cardiac arrest, as well as provide institutions with the most up-to-date literature on which they can build emergency preparedness programs for a maternal arrest.

  13. 33 CFR 154.2106 - Detonation arresters installation.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... arrester, line size expansions must be in a straight pipe run and must be no closer than 120 times the pipe's diameter from the detonation arrester unless the manufacturer has test data to show the...

  14. Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements

    PubMed Central

    Arampatzi, Panagiota; Gialitakis, Manolis; Makatounakis, Takis; Papamatheakis, Joseph

    2013-01-01

    Transcriptional silencing during mitosis is caused by inactivation of critical transcriptional regulators and/or chromatin condensation. Inheritance of gene expression patterns through cell division involves various bookmarking mechanisms. In this report, we have examined the mitotic and post-mitotic expression of the DRA major histocompatibility class II (MHCII) gene in different cell types. During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors. In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility. Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation. PMID:23303784

  15. On generating cell exemplars for detection of mitotic cells in breast cancer histopathology images.

    PubMed

    Aloraidi, Nada A; Sirinukunwattana, Korsuk; Khan, Adnan M; Rajpoot, Nasir M

    2014-01-01

    Mitotic activity is one of the main criteria that pathologists use to decide the grade of the cancer. Computerised mitotic cell detection promises to bring efficiency and accuracy into the grading process. However, detection and classification of mitotic cells in breast cancer histopathology images is a challenging task because of the large intra-class variation in the visual appearance of mitotic cells in various stages of cell division life cycle. In this paper, we test the hypothesis that cells in histopathology images can be effectively represented using cell exemplars derived from sub-images of various kinds of cells in an image for the purposes of mitotic cell classification. We compare three methods for generating exemplar cells. The methods have been evaluated in terms of classification performance on the MITOS dataset. The experimental results demonstrate that eigencells combined with support vector machines produce reasonably high detection accuracy among all the methods.

  16. The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

    PubMed Central

    Carvalhal, Sara; Ribeiro, Susana Abreu; Arocena, Miguel; Kasciukovic, Taciana; Temme, Achim; Koehler, Katrin; Huebner, Angela; Griffis, Eric R.

    2015-01-01

    The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome. PMID:26246606

  17. Temporal and compartment-specific signals coordinate mitotic exit with spindle position

    PubMed Central

    Caydasi, Ayse Koca; Khmelinskii, Anton; Duenas-Sanchez, Rafael; Kurtulmus, Bahtiyar; Knop, Michael; Pereira, Gislene

    2017-01-01

    The spatiotemporal control of mitotic exit is crucial for faithful chromosome segregation during mitosis. In budding yeast, the mitotic exit network (MEN) drives cells out of mitosis, whereas the spindle position checkpoint (SPOC) blocks MEN activity when the anaphase spindle is mispositioned. How the SPOC operates at a molecular level remains unclear. Here, we report novel insights into how mitotic signalling pathways orchestrate chromosome segregation in time and space. We establish that the key function of the central SPOC kinase, Kin4, is to counterbalance MEN activation by the cdc fourteen early anaphase release (FEAR) network in the mother cell compartment. Remarkably, Kin4 becomes dispensable for SPOC function in the absence of FEAR. Cells lacking both FEAR and Kin4 show that FEAR contributes to mitotic exit through regulation of the SPOC component Bfa1 and the MEN kinase Cdc15. Furthermore, we uncover controls that specifically promote mitotic exit in the daughter cell compartment. PMID:28117323

  18. Microtubule organization within mitotic spindles revealed by serial block face scanning EM and image analysis.

    PubMed

    Nixon, Faye M; Honnor, Thomas R; Clarke, Nicholas I; Starling, Georgina P; Beckett, Alison J; Johansen, Adam M; Brettschneider, Julia A; Prior, Ian A; Royle, Stephen J

    2017-04-07

    Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow which are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of MTs within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.

  19. Temporal and compartment-specific signals coordinate mitotic exit with spindle position.

    PubMed

    Caydasi, Ayse Koca; Khmelinskii, Anton; Duenas-Sanchez, Rafael; Kurtulmus, Bahtiyar; Knop, Michael; Pereira, Gislene

    2017-01-24

    The spatiotemporal control of mitotic exit is crucial for faithful chromosome segregation during mitosis. In budding yeast, the mitotic exit network (MEN) drives cells out of mitosis, whereas the spindle position checkpoint (SPOC) blocks MEN activity when the anaphase spindle is mispositioned. How the SPOC operates at a molecular level remains unclear. Here, we report novel insights into how mitotic signalling pathways orchestrate chromosome segregation in time and space. We establish that the key function of the central SPOC kinase, Kin4, is to counterbalance MEN activation by the cdc fourteen early anaphase release (FEAR) network in the mother cell compartment. Remarkably, Kin4 becomes dispensable for SPOC function in the absence of FEAR. Cells lacking both FEAR and Kin4 show that FEAR contributes to mitotic exit through regulation of the SPOC component Bfa1 and the MEN kinase Cdc15. Furthermore, we uncover controls that specifically promote mitotic exit in the daughter cell compartment.

  20. Infradian biorhythms of mitotic activity esophageal epithelium in male Wistar rats.

    PubMed

    Diatroptov, M E; Makarova, O V

    2015-01-01

    Infradian rhythms of esophageal epithelium mitotic activity were studied in male Wistar rats of two age groups: 35-45 days (prepubertal) and 3-4 months (adults). Studies of the time course of esophageal epithelium mitotic indexes in adult males showed 4- and 12-day biorhythms, while prepubertal rats exhibited only 4-day infradian biorhythms of this parameter. Studies of the mitotic activity over long periods (3 years) showed 4.058- and 12.175-day periodicity of infradian biorhythms for this parameter, presumably due to external exposures synchronizing the biorhythms. Studies of the mean daily values of the Bz component of interplanetary magnetic field during the period of our research (2012-2013) showed rhythmicities analogous to changes in the mitotic activity of the epithelium. The minimum mitotic indexes were detected on the days of the most pronounced negative values of the interplanetary magnetic field Bz component.

  1. Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Lee, Youngyi; Bae, Eun Ju

    2013-11-01

    Adipocytes are the key player in adipose tissue inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and differentiation of preadipocytes. Fistein, a polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-diabetic effects. In this study, we aimed to investigate the effect of fisetin on adipocyte proliferation and differentiation in 3T3-L1 preadipocyte cell line and its mechanism of action. We found that fisetin inhibits adipocyte differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor γ. Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the differentiation medium. We also found that fisetin inhibition of adipocyte differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of preadipocyte proliferation at early stage of differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the inhibition of adipocyte differentiation by fisetin may be at least in part mediated by cell cycle arrest during adipogenesis.

  2. Silencing erythropoietin receptor on glioma cells reinforces efficacy of temozolomide and X-rays through senescence and mitotic catastrophe

    PubMed Central

    Pérès, Elodie A.; Gérault, Aurélie N.; Valable, Samuel; Roussel, Simon; Toutain, Jérôme; Divoux, Didier; Guillamo, Jean-Sébastien; Sanson, Marc; Bernaudin, Myriam; Petit, Edwige

    2015-01-01

    Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours. It has been recently reported that erythropoietin (EPO) and its receptor (EPOR) are involved in glioma growth. We now investigated whether EPOR signalling may modulate the efficacy of the GBM current treatment based on chemotherapy (temozolomide, TMZ) and radiotherapy (X-rays). Using RNA interference, we showed on glioma cell lines (U87 and U251) that EPOR silencing induces a G2/M cell cycle arrest, consistent with the slowdown of glioma growth induced by EPOR knock-down. In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone. In vitro, we showed that EPOR silencing not only increases the sensitivity of glioma cells to TMZ as well as X-rays but also counteracts the hypoxia-induced chemo- and radioresistance. Silencing EPOR on glioma cells exposed to conventional treatments enhances senescence and induces a robust genomic instability that leads to caspase-dependent mitotic death by increasing the number of polyploid cells and cyclin B1 expression. Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide. PMID:25544764

  3. Early Versus Late Maturation Arrest: Reproductive Outcomes of Testicular Failure

    PubMed Central

    Weedin, John W.; Bennett, Richard C.; Fenig, David M.; Lamb, Dolores J.; Lipshultz, Larry I.

    2013-01-01

    Purpose There is a paucity of data characterizing infertile men with maturation arrest. We hypothesized that men with early stage maturation arrest could be clinically distinguished from men with late maturation arrest and would have worse reproductive outcomes. Materials and Methods We retrospectively reviewed the records of all patients with nonobstructive azoospermia and cryptozoospermia who underwent testis mapping and sperm extraction from 2002 to 2009 and for whom histopathological findings were available. Patients had uniform maturation arrest if multiple biopsies revealed maturation arrest at the spermatogonia/spermatocyte (early maturation arrest) or the spermatid (late maturation arrest) stage. Clinical parameters and pregnancy outcomes of in vitro fertilization/intracytoplasmic sperm injection were examined. Statistical analysis consisted of univariate and multivariate analysis. Results Uniform maturation arrest was identified in 49 of 219 men (22.3%) undergoing testicular sperm extraction. On multivariate analysis men with maturation arrest had significantly larger testes (p = 0.01), decreased follicle-stimulating hormone (p = 0.05) and more detectable genetic abnormalities (p = 0.01) than men with other histopathological conditions. Men with late maturation arrest had decreased follicle-stimulating hormone (p = 0.02), increased testosterone (p = 0.03) and a higher sperm retrieval rate at testicular sperm extraction (p = 0.01) than men with early maturation arrest. Predictors of successful sperm retrieval were larger testes, cryptozoospermia, late maturation arrest and hypospermatogenesis (each p ≤0.05). Pregnancy outcomes for men with maturation arrest were not significantly different from those for men with other histopathological conditions. Conclusions Maturation arrest is a common, diverse histopathological subtype of severe male infertility. Compared to men with late maturation arrest those with early maturation arrest have increased follicle

  4. Okadaic acid induced cyclin B1 expression and mitotic catastrophe in rat cortex.

    PubMed

    Chen, Bo; Cheng, Min; Hong, Dao-Jun; Sun, Feng-Yan; Zhu, Cui-Qing

    2006-10-09

    Accumulating evidence indicates that the aberrant re-entry of post-mitotic neurons into the G2/M phase of cell cycle and the resulting mitotic catastrophe may contribute to the pathogenesis of Alzheimer's disease. However, the cellular event that drives the differentiated neurons to abnormally enter G2/M phase remains elusive. Similarly, whether mitotic catastrophe is indeed one of the death pathways for differentiated neurons is not clear. Previous studies revealed that okadaic acid (OA), a phosphatase inhibitor that induces AD like pathological changes, evokes mitotic changes in neuroblastoma cells. In this study, we examined the in vivo effects of OA on cyclin B1 expression, the induction of mitosis, and subsequent mitotic catastrophe. We found that cyclin B1 expression in adult neurons was significantly increased after injecting OA into rat frontal cortex, which also increased tau protein phosphorylation. Interestingly, cyclin B1 and phosphorylated tau were well co-localized around the OA injection site, but were only partially co-localized in other brain regions. Staining with toluidine blue, Giemsa dye or propidium iodide revealed typical mitotic and mitotic catastrophe-like morphological changes with irregular arrangement of condensed chromatin and chromosome fibers in a few cells. Furthermore, the strong cyclin B1 staining in these cells suggests that cyclin B1 promoted G2 to M phase transition is required for the mitotic catastrophe. The detection of neuron-specific enolase in a portion of these cells demonstrated that at least part them are neuron. All together, our results suggest that the disturbance of the protein kinase-phosphatase system caused by OA is sufficient to induce neuronal cyclin B1 expression, force neurons into the mitotic phase of cell cycle, and cause mitotic catastrophe.

  5. Mitotic rate in melanoma: prognostic value of immunostaining and computer-assisted image analysis.

    PubMed

    Hale, Christopher S; Qian, Meng; Ma, Michelle W; Scanlon, Patrick; Berman, Russell S; Shapiro, Richard L; Pavlick, Anna C; Shao, Yongzhao; Polsky, David; Osman, Iman; Darvishian, Farbod

    2013-06-01

    The prognostic value of mitotic rate in melanoma is increasingly recognized, particularly in thin melanoma in which the presence or absence of a single mitosis/mm can change staging from T1a to T1b. Still, accurate mitotic rate calculation (mitoses/mm) on hematoxylin and eosin (H&E)-stained sections can be challenging. Antimonoclonal mitotic protein-2 (MPM-2) and antiphosphohistone-H3 (PHH3) are 2 antibodies reported to be more mitosis-specific than other markers of proliferation such as Ki-67. We used light microscopy and computer-assisted image analysis software to quantify MPM-2 and PHH3 staining in melanoma. We then compared mitotic rates by each method with conventional H&E-based mitotic rate for correlation with clinical outcomes. Our study included primary tissues from 190 nonconsecutive cutaneous melanoma patients who were prospectively enrolled at New York University Langone Medical Center with information on age, gender, and primary tumor characteristics. The mitotic rate was quantified manually by light microscopy of corresponding H&E-stained, MPM-2-stained, and PHH3-stained sections. Computer-assisted image analysis was then used to quantify immunolabeled mitoses on the previously examined PHH3 and MPM-2 slides. We then analyzed the association between mitotic rate and both progression-free and melanoma-specific survival. Univariate analysis of PHH3 found significant correlation between increased PHH3 mitotic rate and decreased progression-free survival (P=0.04). Computer-assisted image analysis enhanced the correlation of PHH3 mitotic rate with progression-free survival (P=0.02). Regardless of the detection method, neither MPM-2 nor PHH3 offered significant advantage over conventional H&E determination of mitotic rate.

  6. Regulation of mitotic spindle orientation during epidermal stratification.

    PubMed

    Xie, Wei; Zhou, Jun

    2016-12-20

    The epidermis is a stratified epithelium that serves as a barrier to infection from environmental pathogens and prevents water loss. Epidermal stratification is tightly controlled during embryogenesis. Progenitor cells in the developing epidermis undergo both symmetric and asymmetric cell divisions to balance the growth of the skin surface area against the generation of differentiated cell layers. Therefore, understanding the relationship between oriented divisions of progenitor cells and the development and stratification of the epidermis is of paramount importance in the field of skin biology and pathology. We provide here an integrated view of recent studies implicating that improper orientation of the mitotic spindle contributes to disorders associated with abnormal epidermal stratification and suggesting that spindle orientation could serve as a potential therapeutic target in skin diseases.

  7. Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

    2013-03-01

    Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

  8. Aurora A's Functions During Mitotic Exit: The Guess Who Game.

    PubMed

    Reboutier, David; Benaud, Christelle; Prigent, Claude

    2015-01-01

    Until recently, the knowledge of Aurora A kinase functions during mitosis was limited to pre-metaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. However, an involvement of Aurora A in post-metaphase events was also suspected, but not clearly demonstrated due to the technical difficulty to perform the appropriate experiments. Recent developments of both an analog-specific version of Aurora A and small molecule inhibitors have led to the first demonstration that Aurora A is required for the early steps of cytokinesis. As in pre-metaphase, Aurora A plays diverse functions during anaphase, essentially participating in astral microtubules dynamics and central spindle assembly and functioning. The present review describes the experimental systems used to decipher new functions of Aurora A during late mitosis and situate these functions into the context of cytokinesis mechanisms.

  9. FTO influences adipogenesis by regulating mitotic clonal expansion.

    PubMed

    Merkestein, Myrte; Laber, Samantha; McMurray, Fiona; Andrew, Daniel; Sachse, Gregor; Sanderson, Jeremy; Li, Mengdi; Usher, Samuel; Sellayah, Dyan; Ashcroft, Frances M; Cox, Roger D

    2015-04-17

    The fat mass and obesity-associated (FTO) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression (FTO-4) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout (FTO-KO) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity.

  10. Forces positioning the mitotic spindle: Theories, and now experiments.

    PubMed

    Wu, Hai-Yin; Nazockdast, Ehssan; Shelley, Michael J; Needleman, Daniel J

    2017-02-01

    The position of the spindle determines the position of the cleavage plane, and is thus crucial for cell division. Although spindle positioning has been extensively studied, the underlying forces ultimately responsible for moving the spindle remain poorly understood. A recent pioneering study by Garzon-Coral et al. uses magnetic tweezers to perform the first direct measurements of the forces involved in positioning the mitotic spindle. Combining this with molecular perturbations and geometrical effects, they use their data to argue that the forces that keep the spindle in its proper position for cell division arise from astral microtubules growing and pushing against the cell's cortex. Here, we review these ground-breaking experiments, the various biomechanical models for spindle positioning that they seek to differentiate, and discuss new questions raised by these measurements.

  11. Arrests for Major Crimes: Trends and Patterns for Elderly Offenders.

    ERIC Educational Resources Information Center

    Sapp, Allen D.

    1989-01-01

    Examined data from the Uniform Crime Reports of the Federal Bureau of Investigation for 1972 through 1981. Findings indicated that the percentage of all arrests that were arrests of the elderly was declining while the elderly population itself was rapidly increasing; and the percentage of elderly arrests for index (major) crimes was increasing…

  12. 29 CFR 1915.159 - Personal fall arrest systems (PFAS).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Personal fall arrest systems (PFAS). 1915.159 Section 1915... Protective Equipment (PPE) § 1915.159 Personal fall arrest systems (PFAS). The criteria of this section apply... acceptable as part of a personal fall arrest system. (a) Criteria for connectors and anchorages....

  13. Sulforaphane, a Dietary Isothiocyanate, Induces G2/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association

    PubMed Central

    Cheng, Ya-Min; Tsai, Ching-Chou; Hsu, Yi-Chiang

    2016-01-01

    Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN) is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G2/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa). We found that cytotoxicity is associated with an accumulation of cells in the G2/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G2/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins. PMID:27626412

  14. Taxol induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells

    NASA Astrophysics Data System (ADS)

    Guo, Wen-jing; Chen, Tong-sheng

    2010-02-01

    Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

  15. FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G₂arrest in RCS chondrocytes.

    PubMed

    Tran, Tri; Kolupaeva, Victoria; Basilico, Claudio

    2010-11-01

    Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G₂ arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest. Additionally our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

  16. Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle.

    PubMed

    Lorca, Thierry; Bernis, Cyril; Vigneron, Suzanne; Burgess, Andrew; Brioudes, Estelle; Labbé, Jean-Claude; Castro, Anna

    2010-07-01

    Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.

  17. Prevention of mammalian DNA reduplication, following the release from the mitotic spindle checkpoint, requires p53 protein, but not p53-mediated transcriptional activity.

    PubMed

    Notterman, D; Young, S; Wainger, B; Levine, A J

    1998-11-26

    The tumor suppressor p53 has been identified as a component of a mitotic spindle checkpoint. When exposed to a spindle-disrupting drug such as nocodazole, fibroblasts derived from mice having wild-type p53 are blocked with a 4N content of DNA. Conversely, fibroblasts from p53-deficient mice become polyploid. To learn if transcriptional activation of downstream genes by p53 plays a role in this putative checkpoint, three cell lines were exposed to nocodazole. In one line, p53 protein is not expressed, while the other two cell lines over-express p53. In one of these two lines, the N-terminal transactivation domain is wild-type and in the second, this region contains a mutation that eliminates the ability of the protein to act as a transcription factor. Incubation with nocodazole of cells containing wild-type p53 results in accumulation of both 2N and 4N populations of cells. Under the same conditions, cells containing a transactivation-deficient mutant of p53 accumulate a 4N population of cells, but not a 2N population of cells. Cells entirely deficient in p53 protein become hyperdiploid, and display 8N to 16N DNA content. In all three cell lines, nocodazole elicited an initial increase in mitotic cells, but within 24 h the mitotic index returned to baseline. Expression patterns of cyclins B and D indicated that following entry into mitosis, the cells returned to a G1 state but with 4N DNA content. Subsequent re-duplication of DNA beyond 4N is prevented in cells containing either wild-type or transcriptionally inactive p53 protein. In cells entirely lacking p53 protein, DNA is re-duplicated (without an intervening mitosis) and the cells become hyperdiploid. These experiments indicate that p53 does not participate in the transient mitotic arrest that follows spindle disruption, but is essential to prevent subsequent reduplication of DNA and the resulting hyperdiploid state. This function is intact in a mutant that is transcriptionally inactive.

  18. Inducing Therapeutic Hypothermia in Cardiac Arrest Caused by Lightning Strike.

    PubMed

    Scantling, Dane; Frank, Brian; Pontell, Mathew E; Medinilla, Sandra

    2016-09-01

    Only limited clinical scenarios are grounds for induction of therapeutic hypothermia. Its use in traumatic cardiac arrests, including those from lightning strikes, is not well studied. Nonshockable cardiac arrest rhythms have only recently been included in resuscitation guidelines. We report a case of full neurological recovery with therapeutic hypothermia after a lightning-induced pulseless electrical activity cardiac arrest in an 18-year-old woman. We also review the important pathophysiology of lightning-induced cardiac arrest and neurologic sequelae, elaborate upon the mechanism of therapeutic hypothermia, and add case-based evidence in favor of the use of targeted temperature management in lightning-induced cardiac arrest.

  19. Crack propagation and arrest in pressurized containers

    NASA Technical Reports Server (NTRS)

    Erdogan, F.; Delale, F.; Owczarek, J. A.

    1976-01-01

    The problem of crack propagation and arrest in a finite volume cylindrical container filled with pressurized gas is considered. It is assumed that the cylinder contains a symmetrically located longitudinal part-through crack with a relatively small net ligament. The net ligament suddenly ruptures initiating the process of fracture propagation and depressurization in the cylinder. Thus the problem is a coupled gas dynamics and solid mechanics problem the exact formulation of which does not seem to be possible. The problem is reduced to a proper initial value problem by introducing a dynamic fracture criterion which relates the crack acceleration to the difference between a load factor and the corresponding strength parameter. The results indicate that generally in gas filled cylinders fracture arrest is not possible unless the material behaves in a ductile manner and the container is relatively long.

  20. Psychopathology in women arrested for domestic violence.

    PubMed

    Stuart, Gregory L; Moore, Todd M; Gordon, Kristina Coop; Ramsey, Susan E; Kahler, Christopher W

    2006-03-01

    This study examined the prevalence of psychopathology among women arrested for violence and whether the experience of intimate partner violence (IPV) was associated with Axis I psychopathology. Women who were arrested for domestic violence perpetration and court referred to violence intervention programs (N= 103) completed measures of IPV victimization, perpetration, and psychopathology. Results revealed high rates of posttraumatic stress disorder (PTSD), depression, generalized anxiety disorder (GAD), panic disorder, substance use disorders, borderline personality disorder, and antisocial personality disorder. Violence victimization was significantly associated with symptoms of psychopathology. Logistic regression analyses showed that sexual and psychological abuse by partners were associated with the presence of PTSD, depression, and GAD diagnoses. Results highlight the potential importance of the role of violence victimization in psychopathology. Results suggest that Axis I and Axis II psychopathology should routinely be assessed as part of violence intervention programs for women and that intervention programs could be improved by offering adjunct or integrated mental health treatment.

  1. Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC)

    PubMed Central

    Tipton, Aaron R; Tipton, Michael; Yen, Tim

    2011-01-01

    The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint—the mitotic checkpoint complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity toward APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC. PMID:22037211

  2. Dental Calculus Arrest of Dental Caries

    PubMed Central

    Keyes, Paul H.; Rams, Thomas E.

    2016-01-01

    Background An inverse relationship between dental calculus mineralization and dental caries demineralization on teeth has been noted in some studies. Dental calculus may even form superficial layers over existing dental caries and arrest their progression, but this phenomenon has been only rarely documented and infrequently considered in the field of Cariology. To further assess the occurrence of dental calculus arrest of dental caries, this study evaluated a large number of extracted human teeth for the presence and location of dental caries, dental calculus, and dental plaque biofilms. Materials and methods A total of 1,200 teeth were preserved in 10% buffered formal saline, and viewed while moist by a single experienced examiner using a research stereomicroscope at 15-25× magnification. Representative teeth were sectioned and photographed, and their dental plaque biofilms subjected to gram-stain examination with light microscopy at 100× magnification. Results Dental calculus was observed on 1,140 (95%) of the extracted human teeth, and no dental carious lesions were found underlying dental calculus-covered surfaces on 1,139 of these teeth. However, dental calculus arrest of dental caries was found on one (0.54%) of 187 evaluated teeth that presented with unrestored proximal enamel caries. On the distal surface of a maxillary premolar tooth, dental calculus mineralization filled the outer surface cavitation of an incipient dental caries lesion. The dental calculus-covered carious lesion extended only slightly into enamel, and exhibited a brown pigmentation characteristic of inactive or arrested dental caries. In contrast, the tooth's mesial surface, without a superficial layer of dental calculus, had a large carious lesion going through enamel and deep into dentin. Conclusions These observations further document the potential protective effects of dental calculus mineralization against dental caries. PMID:27446993

  3. Nuclear reactor melt arrest and coolability device

    DOEpatents

    Theofanous, Theo G.; Dinh, Nam Truc; Wachowiak, Richard M.

    2016-06-14

    Example embodiments provide a Basemat-Internal Melt Arrest and Coolability device (BiMAC) that offers improved spatial and mechanical characteristics for use in damage prevention and risk mitigation in accident scenarios. Example embodiments may include a BiMAC having an inclination of less than 10-degrees from the basemat floor and/or coolant channels of less than 4 inches in diameter, while maintaining minimum safety margins required by the Nuclear Regulatory Commission.

  4. Sulfur dioxide converter and pollution arrester system

    SciTech Connect

    Montalvo, V.H.

    1983-12-06

    A sulphur dioxide converter and pollution arrester system are disclosed which involves the treatment of smoke and/or contaminated air emanating from a combustion area by passage through a zone achieving turbulence into a water spray contained first treating chamber. The turbulence zone, into which an atomized catalyst is introduced, serves to create a longer path for cooling as well as increased centrifugal motion to the solid particles in the contaminated air and also the formation of sulphur trioxide. In other words, the arrangement is such that pollution arresting action is provided in the form of ''slinging'' resulting from tangential directional movement and, when combining with the water spray in the first treating chamber, the ultimate formation of sulphuric acid. Subsequently, the contaminated air, containing amounts of sulphurous and sulphuric acids, passes through a second treating chamber, where airflow throughout the system is occasioned by action at the outlet end, such as the vacuum created by a flue and not by independent mechanical means. The arrangement serves to a twofold purpose, i.e. to minimize or arrest pollution and to convert sulphur dioxide, a component of high sulphur coal, into commercially valuable sulphuric acid.

  5. A chemical tool box defines mitotic and interphase roles for Mps1 kinase

    PubMed Central

    Lan, Weijie

    2010-01-01

    In this issue, three groups (Hewitt et al. 2010. J. Cell Biol. doi:10.1083/jcb.201002133; Maciejowski et al. 2010. J. Cell Biol. doi:10.1083/jcb.201001050; Santaguida et al. 2010. J. Cell Biol. doi:10.1083/jcb.201001036) use chemical inhibitors to analyze the function of the mitotic checkpoint kinase Mps1. These studies demonstrate that Mps1 kinase activity ensures accurate chromosome segregation through its recruitment to kinetochores of mitotic checkpoint proteins, formation of interphase and mitotic inhibitors of Cdc20, and correction of faulty microtubule attachments. PMID:20624898

  6. Mitotic spindle assembly on chromatin patterns made with deep UV photochemistry.

    PubMed

    Tarnawska, Katarzyna; Pugieux, Céline; Nédélec, François

    2014-01-01

    We provide a detailed method to generate arrays of mitotic spindles in vitro. Spindles are formed in extract prepared from unfertilized Xenopus laevis eggs, which contain all the molecular ingredients of mitotic spindles. The method is based on using deep UV photochemistry to attach chromatin-coated beads on a glass surface according to a pattern of interest. The immobilized beads act as artificial chromosomes, and induce the formation of mitotic spindles in their immediate vicinity. To perform the experiment, a chamber is assembled over the chromatin pattern, Xenopus egg extract is flowed in and after incubation the spindles are imaged with a confocal microscope.

  7. [Mitotic activity of the lymphocytes of the thymus cortex in hypokinesia during the period of readaptation].

    PubMed

    Kharlova, G V; Li, S E

    1979-10-01

    The changes in the weight and mitotic index were studied in the cortex of the thymus of Wistar rats during 10-day hypokinesia and 10-day readaptation (restoration). 24 hours after immobilization of the animals the mitotic index was 2 times as lower. No complete readaptation was attained during 10-day hypokinesia. No readaptation was attained during 10-day hypokinesia. In readaptation the stage of secondary stress was found (the mitotic index was 3.5 times as reduced), the stage of genuine restoration being revealed after 10 days.

  8. A grading system combining architectural features and mitotic count predicts recurrence in stage I lung adenocarcinoma.

    PubMed

    Kadota, Kyuichi; Suzuki, Kei; Kachala, Stefan S; Zabor, Emily C; Sima, Camelia S; Moreira, Andre L; Yoshizawa, Akihiko; Riely, Gregory J; Rusch, Valerie W; Adusumilli, Prasad S; Travis, William D

    2012-08-01

    The International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) has recently proposed a new lung adenocarcinoma classification. We investigated whether nuclear features can stratify prognostic subsets. Slides of 485 stage I lung adenocarcinoma patients were reviewed. We evaluated nuclear diameter, nuclear atypia, nuclear/cytoplasmic ratio, chromatin pattern, prominence of nucleoli, intranuclear inclusions, mitotic count/10 high-power fields (HPFs) or 2.4 mm(2), and atypical mitoses. Tumors were classified into histologic subtypes according to the IASLC/ATS/ERS classification and grouped by architectural grade into low (adenocarcinoma in situ, minimally invasive adenocarcinoma, or lepidic predominant), intermediate (papillary or acinar), and high (micropapillary or solid). Log-rank tests and Cox regression models evaluated the ability of clinicopathologic factors to predict recurrence-free probability. In univariate analyses, nuclear diameter (P=0.007), nuclear atypia (P=0.006), mitotic count (P<0.001), and atypical mitoses (P<0.001) were significant predictors of recurrence. The recurrence-free probability of patients with high mitotic count (≥5/10 HPF: n=175) was the lowest (5-year recurrence-free probability=73%), followed by intermediate (2-4/10 HPF: n=106, 80%), and low (0-1/10 HPF: n=204, 91%, P<0.001). Combined architectural/mitotic grading system stratified patient outcomes (P<0.001): low grade (low architectural grade with any mitotic count and intermediate architectural grade with low mitotic count: n=201, 5-year recurrence-free probability=92%), intermediate grade (intermediate architectural grade with intermediate-high mitotic counts: n=206, 78%), and high grade (high architectural grade with any mitotic count: n=78, 68%). The advantage of adding mitotic count to architectural grade is in stratifying patients with intermediate architectural grade into two prognostically

  9. Arrest History and Intimate Partner Violence Perpetration in a Sample of Men and Women Arrested for Domestic Violence

    PubMed Central

    Shorey, Ryan C.; Ninnemann, Andrew; Elmquist, Joanna; Labrecque, Lindsay; Zucosky, Heather; Febres, Jeniimarie; Brasfield, Hope; Temple, Jeff R.; Stuart, Gregory L.

    2014-01-01

    Intimate partner violence (IPV) is a serious and prevalent problem throughout the United States. Currently, individuals arrested for domestic violence are often court mandated to batterer intervention programs (BIPs). However, little is known about the arrest histories of these individuals, especially women. The current study examined the arrest histories of men (n = 303) and women (n = 82) arrested for domestic violence and court-referred to BIPs. Results demonstrated that over 30% of the entire sample had been previously arrested for a non-violent offense, and over 25% of the participants had been previously arrested for a violent offense other than domestic violence. Moreover, men were arrested significantly more frequently for violence-related and non-violent offenses than their female counterparts. In addition, men were more likely than women to have consumed binge-levels of alcohol prior to the offense that led to their most recent arrest and court-referral to a BIP. Lastly, arrest history was positively associated with physical and psychological aggression perpetration against an intimate partner for men only, such that more previous arrests were associated with more frequent aggression. These results provide evidence that many men and women arrested for domestic violence have engaged in a number of diverse criminal acts during their lifetimes, suggesting that BIPs may need to address general criminal behavior. PMID:25379068

  10. Arrest History and Intimate Partner Violence Perpetration in a Sample of Men and Women Arrested for Domestic Violence.

    PubMed

    Shorey, Ryan C; Ninnemann, Andrew; Elmquist, Joanna; Labrecque, Lindsay; Zucosky, Heather; Febres, Jeniimarie; Brasfield, Hope; Temple, Jeff R; Stuart, Gregory L

    2012-01-01

    Intimate partner violence (IPV) is a serious and prevalent problem throughout the United States. Currently, individuals arrested for domestic violence are often court mandated to batterer intervention programs (BIPs). However, little is known about the arrest histories of these individuals, especially women. The current study examined the arrest histories of men (n = 303) and women (n = 82) arrested for domestic violence and court-referred to BIPs. Results demonstrated that over 30% of the entire sample had been previously arrested for a non-violent offense, and over 25% of the participants had been previously arrested for a violent offense other than domestic violence. Moreover, men were arrested significantly more frequently for violence-related and non-violent offenses than their female counterparts. In addition, men were more likely than women to have consumed binge-levels of alcohol prior to the offense that led to their most recent arrest and court-referral to a BIP. Lastly, arrest history was positively associated with physical and psychological aggression perpetration against an intimate partner for men only, such that more previous arrests were associated with more frequent aggression. These results provide evidence that many men and women arrested for domestic violence have engaged in a number of diverse criminal acts during their lifetimes, suggesting that BIPs may need to address general criminal behavior.

  11. Arresting developments: trends in female arrests for domestic violence and proposed explanations.

    PubMed

    Deleon-Granados, William; Wells, William; Binsbacher, Ruddyard

    2006-04-01

    This article represents an effort to generate more systematic and specified discussion on the topic of unintended consequences in the movement to decrease violence against women. In this case, the consequence is increases in female arrests for domestic violence. This article builds on recent discussions by first using a sample of data to examine felony domestic violence arrest rates for men and women. The data support the conclusion that domestic violence arrests of women have increased. Second, the article presents six explanations that are derived from existing literature. Although the authors do not offer empirical tests of these explanations, this presentation can play an important part in better understanding the outcomes of criminal justice policies that are aimed at increasing victim safety.

  12. Modeling cardiac arrest and resuscitation in the domestic pig

    PubMed Central

    Cherry, Brandon H; Nguyen, Anh Q; Hollrah, Roger A; Olivencia-Yurvati, Albert H; Mallet, Robert T

    2015-01-01

    Cardiac arrest remains a leading cause of death and permanent disability worldwide. Although many victims are initially resuscitated, they often succumb to the extensive ischemia-reperfusion injury inflicted on the internal organs, especially the brain. Cardiac arrest initiates a complex cellular injury cascade encompassing reactive oxygen and nitrogen species, Ca2+ overload, ATP depletion, pro- and anti-apoptotic proteins, mitochondrial dysfunction, and neuronal glutamate excitotoxity, which injures and kills cells, compromises function of internal organs and ignites a destructive systemic inflammatory response. The sheer complexity and scope of this cascade challenges the development of experimental models of and effective treatments for cardiac arrest. Many experimental animal preparations have been developed to decipher the mechanisms of damage to vital internal organs following cardiac arrest and cardiopulmonary resuscitation (CPR), and to develop treatments to interrupt the lethal injury cascades. Porcine models of cardiac arrest and resuscitation offer several important advantages over other species, and outcomes in this large animal are readily translated to the clinical setting. This review summarizes porcine cardiac arrest-CPR models reported in the literature, describes clinically relevant phenomena observed during cardiac arrest and resuscitation in pigs, and discusses numerous methodological considerations in modeling cardiac arrest/CPR. Collectively, published reports show the domestic pig to be a suitable large animal model of cardiac arrest which is responsive to CPR, defibrillatory countershocks and medications, and yields extensive information to foster advances in clinical treatment of cardiac arrest. PMID:25685718

  13. Arrests of women for driving under the influence.

    PubMed

    Shore, E R; McCoy, M L; Toonen, L A; Kuntz, E J

    1988-01-01

    Police records of arrests of women in Wichita, Kansas for driving under the influence (DUI) of alcohol for a 5-year period (1980-1984) were studied. The proportion of arrests of women increased from 10.6 to 14.5% of total arrested. Women in their 20s comprised the largest age group; single women were greatly overrepresented. More than one-half of the arrested women were employed outside the home; a substantial proportion (30.8%) were unemployed at the time of arrest. The average blood alcohol level of those tested was 183 mg/dl. Characteristics of arrestees are discussed in terms of changes in the social roles and expectations of women. Although time of arrest was similar to that of men (i.e., night), arrests of women were more evenly spread across the days of the week. Within the 5-year period, the rate of recidivism for DUI was 7.43%. The implications of arrest and recidivism patterns are discussed. A change in legal and arrest procedures was found to have the same effect on arrests of women as it had on those of men, suggesting that the changes did not produce differential treatment by police.

  14. Fast Track intervention effects on youth arrests and delinquency

    PubMed Central

    2010-01-01

    This paper examines the effects of the Fast Track preventive intervention on youth arrests and self-reported delinquent behavior through age 19. High-risk youth randomly assigned to receive a long-term, comprehensive preventive intervention from 1st grade through 10th grade at four sites were compared to high-risk control youth. Findings indicated that random assignment to Fast Track reduced court-recorded juvenile arrest activity based on a severity weighted sum of juvenile arrests. Supplementary analyses revealed an intervention effect on the reduction in the number of court-recorded moderate-severity juvenile arrests, relative to control children. In addition, among youth with higher initial behavioral risk, the intervention reduced the number of high-severity adult arrests relative to the control youth. Survival analyses examining the onset of arrests and delinquent behavior revealed a similar pattern of findings. Intervention decreased the probability of any juvenile arrest among intervention youth not previously arrested. In addition, intervention decreased the probability of a self-reported high-severity offense among youth with no previous self-reported high-severity offense. Intervention effects were also evident on the onset of high-severity court-recorded adult arrests among participants, but these effects varied by site. The current findings suggest that comprehensive preventive intervention can prevent juvenile arrest rates, although the presence and nature of intervention effects differs by outcome. PMID:20577576

  15. Mitotic and meiotic chromosome studies in silky anteater Cyclopes didactylus (Myrmecophagidae: Xenarthra).

    PubMed

    Jorge, W

    2000-01-01

    The karyotype of a male pigmy anteater, Cyclopes didactylus, an endangered species from the Amazon region, is described. The size and morphology of the X and Y chromosomes in mitotic and meiotic analyses is recorded and discussed.

  16. Selective extraction of isolated mitotic apparatus. Evidence that typical microtubule protein is extracted by organic mercurial.

    PubMed

    Bibring, T; Baxandall, J

    1971-02-01

    Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

  17. Suspension of mitotic activity in dentate gyrus of the hibernating ground squirrel.

    PubMed

    Popov, Victor I; Kraev, Igor V; Ignat'ev, Dmitri A; Stewart, Michael G

    2011-01-01

    Neurogenesis occurs in the adult mammalian hippocampus, a region of the brain important for learning and memory. Hibernation in Siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h (from 35-36°C to +4-6°C) permits accumulation of mitotic cells at different stages of the cell cycle. Histological methods used to study adult neurogenesis are limited largely to fixed tissue, and the mitotic state elucidated depends on the specific phase of mitosis at the time of day. However, using an immunohistochemical study of doublecortin (DCX) and BrdU-labelled neurons, we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity. Our data suggest that doublecortin-labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus.

  18. Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

    PubMed Central

    Nishino, Yoshinori; Eltsov, Mikhail; Joti, Yasumasa; Ito, Kazuki; Takata, Hideaki; Takahashi, Yukio; Hihara, Saera; Frangakis, Achilleas S; Imamoto, Naoko; Ishikawa, Tetsuya; Maeshima, Kazuhiro

    2012-01-01

    How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures. PMID:22343941

  19. Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus

    PubMed Central

    Grenfell, Andrew W.

    2016-01-01

    Transcription at the centromere of chromosomes plays an important role in kinetochore assembly in many eukaryotes, and noncoding RNAs contribute to activation of the mitotic kinase Aurora B. However, little is known about how mitotic RNA processing contributes to spindle assembly. We found that inhibition of transcription initiation or RNA splicing, but not translation, leads to spindle defects in Xenopus egg extracts. Spliceosome inhibition resulted in the accumulation of high molecular weight centromeric transcripts, concomitant with decreased recruitment of the centromere and kinetochore proteins CENP-A, CENP-C, and NDC80 to mitotic chromosomes. In addition, blocking transcript synthesis or processing during mitosis caused accumulation of MCAK, a microtubule depolymerase, on the spindle, indicating misregulation of Aurora B. These findings suggest that co-transcriptional recruitment of the RNA processing machinery to nascent mitotic transcripts is an important step in kinetochore and spindle assembly and challenge the idea that RNA processing is globally repressed during mitosis. PMID:27402954

  20. A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Lee, Phoebe S.; Greenwell, Patricia W.; Dominska, Margaret; Gawel, Malgorzata; Hamilton, Monica; Petes, Thomas D.

    2009-01-01

    Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle. PMID:19282969

  1. Dimerization of TRAF-interacting protein (TRAIP) regulates the mitotic progression.

    PubMed

    Park, I Seul; Jo, Ku-Sung; Won, Hyung-Sik; Kim, Hongtae

    2015-08-07

    The homo- or hetero-dimerization of proteins plays critical roles in the mitotic progression. The TRAF-interacting protein (TRAIP) is crucial in early mitotic progression and chromosome alignment defects in the metaphase. The TRAIP is a 469 amino acid protein, including the Really Interesting New Gene (RING), coiled-coil (CC), and leucine zipper (LZ) domain. In general, the CC or LZ domain containing proteins forms homo- or hetero-dimerization to achieve its activity. In this study, a number of TRAIP mutants were used to define the TRAIP molecular domains responsible for its homo-dimerization. A co-immunoprecipitation assay indicated that the TRAIP forms homo-dimerization through the CC domain. The cells, expressing the CC domain-deleted mutant that could not form a homo-dimer, increased the mitotic index and promoted mitotic progression.

  2. Anti-mitotic agents: Are they emerging molecules for cancer treatment?

    PubMed

    Penna, Larissa Siqueira; Henriques, João Antonio Pêgas; Bonatto, Diego

    2017-02-04

    Mutations in cancer cells frequently result in cell cycle alterations that lead to unrestricted growth compared to normal cells. Considering this phenomenon, many drugs have been developed to inhibit different cell-cycle phases. Mitotic phase targeting disturbs mitosis in tumor cells, triggers the spindle assembly checkpoint and frequently results in cell death. The first anti-mitotics to enter clinical trials aimed to target tubulin. Although these drugs improved the treatment of certain cancers, and many anti-microtubule compounds are already approved for clinical use, severe adverse events such as neuropathies were observed. Since then, efforts have been focused on the development of drugs that also target kinases, motor proteins and multi-protein complexes involved in mitosis. In this review, we summarize the major proteins involved in the mitotic phase that can also be targeted for cancer treatment. Finally, we address the activity of anti-mitotic drugs tested in clinical trials in recent years.

  3. Poleward microtubule flux mitotic spindles assembled in vitro

    PubMed Central

    1991-01-01

    In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase. PMID:1999464

  4. Physical Description of Mitotic Spindle Orientation During Cell Division

    NASA Astrophysics Data System (ADS)

    Jiménez-Dalmaroni, Andrea; Théry, Manuel; Racine, Victor; Bornens, Michel; Jülicher, Frank

    2009-03-01

    During cell division, the duplicated chromosomes are physically separated by the action of the mitotic spindle. The spindle is a dynamic structure of the cytoskeleton, which consists of two microtubule asters. Its orientation defines the axis along which the cell divides. Recent experiments show that the spindle orientation depends on the spatial distribution of cell adhesion sites. Here we show that the experimentally observed spindle orientation can be understood as the result of the action of cortical force generators acting on the spindle. We assume that the local activity of force generators is controlled by the spatial distribution of cell adhesion sites determined by the particular geometry of the adhesive substrate. We develop a simple physical description of the spindle mechanics, which allows us to calculate the torque acting on the spindle, as well as the energy profile and the angular distribution of spindle orientation. Our model accounts for the preferred spindle orientation, as well as the full shape of the angular distributions of spindle orientation observed in a large variety of pattern geometries. M. Th'ery, A. Jim'enez-Dalmaroni, et al., Nature 447, 493 (2007).

  5. Mitotic abnormalities leading to cancer predisposition and progression.

    PubMed

    Cavenee, W K

    1989-01-01

    The development of human cancer is generally thought to entail a series of events that cause a progressively more malignant phenotype. Such a hypothesis predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each of the events less the last one, and so on. That is to say a dissection of the pathway from a normal cell to a fully malignant tumor may be viewed as the unraveling of a nested set of aberrations. In experiments designed to elucidate these events, we have compared genotypic combinations at genomic loci defined by restriction endonuclease recognition site variation in normal and tumor tissues from patients with various forms and stages of cancer. The first step, inherited predisposition, is best described for retinoblastoma in which a recessive mutation of a locus residing in the 13q14 region of the genome is unmasked by aberrant, but specific, mitotic chromosomal segregation. A similar mechanism involving the distal short arm of chromosome 17 is apparent in astrocytic tumors and the event is shared by cells in each malignancy stage. This is distinct from a loss of heterozygosity for loci on chromosome 10 which is restricted to the ultimate stage, glioblastoma multiforme. These results suggest a genetic approach to defining degrees of tumor progression and means for determining the genomic locations of genes involved in the pathway as a prelude to their molecular isolation and characterization.

  6. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

    PubMed Central

    1991-01-01

    A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore- enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover. PMID:1991791

  7. Control of the mitotic exit network during meiosis.

    PubMed

    Attner, Michelle A; Amon, Angelika

    2012-08-01

    The mitotic exit network (MEN) is an essential GTPase signaling pathway that triggers exit from mitosis in budding yeast. We show here that during meiosis, the MEN is dispensable for exit from meiosis I but contributes to the timely exit from meiosis II. Consistent with a role for the MEN during meiosis II, we find that the signaling pathway is active only during meiosis II. Our analysis further shows that MEN signaling is modulated during meiosis in several key ways. Whereas binding of MEN components to spindle pole bodies (SPBs) is necessary for MEN signaling during mitosis, during meiosis MEN signaling occurs off SPBs and does not require the SPB recruitment factor Nud1. Furthermore, unlike during mitosis, MEN signaling is controlled through the regulated interaction between the MEN kinase Dbf20 and its activating subunit Mob1. Our data lead to the conclusion that a pathway essential for vegetative growth is largely dispensable for the specialized meiotic divisions and provide insights into how cell cycle regulatory pathways are modulated to accommodate different modes of cell division.

  8. The Prp19 complex directly functions in mitotic spindle assembly.

    PubMed

    Hofmann, Jennifer C; Tegha-Dunghu, Justus; Dräger, Stefanie; Will, Cindy L; Lührmann, Reinhard; Gruss, Oliver J

    2013-01-01

    The conserved Prp19 (pre-RNA processing 19) complex is required for pre-mRNA splicing in eukaryotic nuclei. Recent RNAi screens indicated that knockdown of Prp19 complex subunits strongly delays cell proliferation. Here we show that knockdown of the smallest subunit, BCAS2/Spf27, destabilizes the entire complex and leads to specific mitotic defects in human cells. These could result from splicing failures in interphase or reflect a direct function of the complex in open mitosis. Using Xenopus extracts, in which cell cycle progression and spindle formation can be reconstituted in vitro, we tested Prp19 complex functions during a complete cell cycle and directly in open mitosis. Strikingly, immunodepletion of the complex either before or after interphase significantly reduces the number of intact spindles, and increases the percentage of spindles with lower microtubule density and impaired metaphase alignment of chromosomes. Our data identify the Prp19 complex as the first spliceosome subcomplex that directly contributes to mitosis in vertebrates independently of its function in interphase.

  9. Maintaining Genome Stability in Defiance of Mitotic DNA Damage

    PubMed Central

    Ferrari, Stefano; Gentili, Christian

    2016-01-01

    The implementation of decisions affecting cell viability and proliferation is based on prompt detection of the issue to be addressed, formulation and transmission of a correct set of instructions and fidelity in the execution of orders. While the first and the last are purely mechanical processes relying on the faithful functioning of single proteins or macromolecular complexes (sensors and effectors), information is the real cue, with signal amplitude, duration, and frequency ultimately determining the type of response. The cellular response to DNA damage is no exception to the rule. In this review article we focus on DNA damage responses in G2 and Mitosis. First, we set the stage describing mitosis and the machineries in charge of assembling the apparatus responsible for chromosome alignment and segregation as well as the inputs that control its function (checkpoints). Next, we examine the type of issues that a cell approaching mitosis might face, presenting the impact of post-translational modifications (PTMs) on the correct and timely functioning of pathways correcting errors or damage before chromosome segregation. We conclude this essay with a perspective on the current status of mitotic signaling pathway inhibitors and their potential use in cancer therapy. PMID:27493659

  10. Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

    PubMed

    Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

    2008-03-01

    The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

  11. Prognostic value of mitotic index and Bcl2 expression in male breast cancer.

    PubMed

    Lacle, Miangela M; van der Pol, Carmen; Witkamp, Arjen; van der Wall, Elsken; van Diest, Paul J

    2013-01-01

    The incidence of male breast cancer (MBC) is rising. Current treatment regimens for MBC are extrapolated from female breast cancer (FBC), based on the assumption that FBC prognostic features and therapeutic targets can be extrapolated to MBC. However, there is yet little evidence that prognostic features that have been developed and established in FBC are applicable to MBC as well. In a recent study on FBC, a combination of mitotic index and Bcl2 expression proved to be of strong prognostic value. Previous papers on Bcl2 expression in MBC were equivocal, and the prognostic value of Bcl2 combined with mitotic index has not been studied in MBC. The aim of the present study was therefore to investigate the prognostic value of Bcl2 in combination with mitotic index in MBC. Immunohistochemical staining for Bcl2 was performed on tissue microarrays of a total of 151 male breast cancer cases. Mitotic index was scored. The prognostic value of Bcl2 expression and Bcl2/mitotic index combinations was evaluated studying their correlations with clinicopathologic features and their prediction of survival. The vast majority of MBC (94%) showed Bcl2 expression, more frequently than previously described for FBC. Bcl2 expression had no significant associations with clinicopathologic features such as tumor size, mitotic count and grade. In univariate survival analysis, Bcl2 had no prognostic value, and showed no additional prognostic value to tumor size and histological grade in Cox regression. In addition, the Bcl2/mitotic index combination as opposed to FBC did not predict survival in MBC. In conclusion, Bcl2 expression is common in MBC, but is not associated with major clinicopathologic features and, in contrast to FBC, does not seem to have prognostic value, also when combined with mitotic index.

  12. Mitotane sensitizes adrenocortical cancer cells to ionizing radiations by involvement of the cyclin B1/CDK complex in G2 arrest and mismatch repair enzymes modulation.

    PubMed

    Cerquetti, Lidia; Sampaoli, Camilla; Amendola, Donatella; Bucci, Barbara; Misiti, Silvia; Raza, Giorgio; De Paula, Ugo; Marchese, Rodolfo; Brunetti, Ercole; Toscano, Vincenzo; Stigliano, Antonio

    2010-08-01

    Mitotane inhibits steroid synthesis by an action on steroidogenic enzymes, as 11beta-hydroxylase and cholesterol side chain cleavage. It also has a cytotoxic effect on the adrenocortical cells and represents a primary drug used in the adrenocortical carcinoma (ACC). H295R and SW13 cell lines were treated with mitotane 10(-5) M and ionizing radiations (IR) in combination therapy, inducing an irreversible inhibition of cell growth in both adrenocortical cancer cells. As shown in a previous report, mitotane/IR combination treatment induced a cell accumulation in the G2 phase. Here, we report the radiosensitizing properties of mitotane in two different ACC cell lines. The drug reveals the effectiveness to enhance the cytotoxic effects of IR by attenuating DNA repair and interfering on the activation of mitosis promoting factor (MPF), mainly regulated by the degradation of cyclin B1 in the mitotic process. These events may explain the inappropriate activation of cdc2, implicated in G2/M phase arrest and probably induced by the mitotane and IR in the combined treatment. Indeed, treatment with purvalanol, a cdc2-inhibitor prevents cell cycle arrest, triggering the G2/M transition. The observation that mitotane and IR in combination treatment amplifies the activation level of cyclin B/cdc2 complexes contributing to cell cycle arrest, suggests that the MPF could function as a master signal for controlling the temporal order of different mitotic events. Moreover, we report that mitotane interferes in modulation of mismatch repair (MMR) enzymes, revealing radiosensitizing drug ability.

  13. Hydroxylated PBDEs induce developmental arrest in zebrafish

    SciTech Connect

    Usenko, Crystal Y. Hopkins, David C.; Trumble, Stephen J. Bruce, Erica D.

    2012-07-01

    The ubiquitous spread of polybrominated diphenyl ethers (PBDEs) has led to concerns regarding the metabolites of these congeners, in particular hydroxylated PBDEs. There are limited studies regarding the biological interactions of these chemicals, yet there is some concern they may be more toxic than their parent compounds. In this study three hydroxylated PBDEs were assessed for toxicity in embryonic zebrafish: 3-OH-BDE 47, 5-OH-BDE 47, and 6-OH-BDE 47. All three congeners induced developmental arrest in a concentration-dependent manner; however, 6-OH-BDE 47 induced adverse effects at lower concentrations than the other congeners. Furthermore, all three induced cell death; however apoptosis was not observed. In short-term exposures (24–28 hours post fertilization), all hydroxylated PBDEs generated oxidative stress in the region corresponding to the cell death at 5 and 10 ppm. To further investigate the short-term effects that may be responsible for the developmental arrest observed in this study, gene regulation was assessed for embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 hpf. Genes involved in stress response, thyroid hormone regulation, and neurodevelopment were significantly upregulated compared to controls; however, genes related to oxidative stress were either unaffected or downregulated. This study suggests that hydroxylated PBDEs disrupt development, and may induce oxidative stress and potentially disrupt the cholinergic system and thyroid hormone homeostasis. -- Highlights: ► OH-PBDEs induce developmental arrest in a concentration-dependent manner. ► Hydroxyl group location influences biological interaction. ► OH-PBDEs induce oxidative stress. ► Thyroid hormone gene regulation was disrupted following exposure. ► To our knowledge, this is the first whole organism study of OH-PBDE toxicity.

  14. Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding.

    PubMed

    Fykerud, Tone A; Knudsen, Lars M; Totland, Max Z; Sørensen, Vigdis; Dahal-Koirala, Shiva; Lothe, Ragnhild A; Brech, Andreas; Leithe, Edward

    2016-11-01

    In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed "mitotic nanotubes," were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.

  15. Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding

    PubMed Central

    Fykerud, Tone A.; Knudsen, Lars M.; Totland, Max Z.; Dahal-Koirala, Shiva; Lothe, Ragnhild A.; Brech, Andreas; Leithe, Edward

    2016-01-01

    ABSTRACT In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding. PMID:27625181

  16. The Mitotic Checkpoint Gene, SIL is Regulated by E2F1

    PubMed Central

    Erez, Ayelet; Chaussepied, Marie; Tina, Colaizzo-Anas; Aplan, Peter; Ginsberg, Doron; Izraeli, Shai

    2009-01-01

    The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in-vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F. PMID:18649360

  17. Coupling growth arrest and adipocyte differentiation.

    PubMed Central

    Ailhaud, G; Dani, C; Amri, E Z; Djian, P; Vannier, C; Doglio, A; Forest, C; Gaillard, D; Négrel, R; Grimaldi, P

    1989-01-01

    The complete differentiation program of preadipose cells can be divided into early and late events. The expression of early markers takes place at growth arrest (G1/S boundary), whereas that of late markers, leading to terminal differentiation, takes place after a limited number of mitoses of early marker-containing cells. Only terminal differentiation requires the presence of growth hormone and triiodothyronine and results in the formation of triacylglycerol-filled, nondividing cells. The events of adipose cell differentiation which take place in vitro allow a better understanding of the development of adipose tissue in vivo. Images FIGURE 1. FIGURE 3. FIGURE 5. PMID:2647477

  18. [Identifying children at risk for cardiorespiratory arrest].

    PubMed

    Carrillo Alvarez, A; Martínez Gutiérrez, A; Salvat Germán, F

    2004-08-01

    Cardiorespiratory arrest in children with severe disease does not usually present suddenly or unexpectedly but is often the result of a progressive deterioration of respiratory and/or circulatory function. Before failure of these functions occurs, there is a series of clinical signs that serve as a warning. Health professionals should not only evaluate clinical signs of respiratory and/or circulatory insufficiency but should also be able to identify these warning signs as early as possible, preferably in the compensation phase, given that the possibility that this process can be reversed by therapeutic measures decreases as the process progresses.

  19. Optimal Protective Hypothermia in Arrested Mammalian Hearts

    PubMed Central

    Villet, Outi M.; Ge, Ming; Sekhar, Laigam N.; Corson, Marshall A.; Tylee, Tracy S.; Fan, Lu-Ping; Yao, Lin; Zhu, Chun; Olson, Aaron K.; Buroker, Norman E.; Xu, Cheng-Su; Anderson, David L.; Soh, Yong-Kian; Wang, Elise; Chen, Shi-Han; Portman, Michael A.

    2015-01-01

    Many therapeutic hypothermia recommendations have been reported, but the information supporting them is sparse, and reveals a need for the data of target therapeutic hypothermia (TTH) from well-controlled experiments. The core temperature ≤35°C is considered as hypothermia, and 29°C is a cooling injury threshold in pig heart in vivo. Thus, an optimal protective hypothermia (OPH) should be in the range 29–35°C. This study was conducted with a pig cardiopulmonary bypass preparation to decrease the core temperature to 29–35°C range at 20 minutes before and 60 minutes during heart arrest. The left ventricular (LV) developed pressure, maximum of the first derivative of LV (dP/dtmax), cardiac power, heart rate, cardiac output, and myocardial velocity (Vmax) were recorded continuously via an LV pressure catheter and an aortic flow probe. At 20 minutes of off-pump during reperfusion after 60 minutes arrest, 17 hypothermic hearts showed that the recovery of Vmax and dP/dtmax established sigmoid curves that consisted of two plateaus: a good recovery plateau at 29–30.5°C, the function recovered to baseline level (BL) (Vmax=118.4%±3.9% of BL, LV dP/dtmax=120.7%±3.1% of BL, n=6); another poor recovery plateau at 34–35°C (Vmax=60.2%±2.8% of BL, LV dP/dtmax=28.0%±5.9% of BL, p<0.05, n=6; ), which are similar to the four normothermia arrest (37°C) hearts (Vmax=55.9%±4.8% of BL, LV dP/dtmax=24.5%±2.1% of BL, n=4). The 32–32.5°C arrest hearts showed moderate recovery (n=5). A point of inflection (around 30.5–31°C) existed at the edge of a good recovery plateau followed by a steep slope. The point presented an OPH that should be the TTH. The results are concordant with data in the mammalian hearts, suggesting that the TTH should be initiated to cool core temperature at 31°C. PMID:25514569

  20. POG1, a novel yeast gene, promotes recovery from pheromone arrest via the G1 cyclin CLN2.

    PubMed Central

    Leza, M A; Elion, E A

    1999-01-01

    In the absence of a successful mating, pheromone-arrested Saccharomyces cerevisiae cells reenter the mitotic cycle through a recovery process that involves downregulation of the mating mitogen-activated protein kinase (MAPK) cascade. We have isolated a novel gene, POG1, whose promotion of recovery parallels that of the MAPK phosphatase Msg5. POG1 confers alpha-factor resistance when overexpressed and enhances alpha-factor sensitivity when deleted in the background of an msg5 mutant. Overexpression of POG1 inhibits alpha-factor-induced G1 arrest and transcriptional repression of the CLN1 and CLN2 genes. The block in transcriptional repression occurs at SCB/MCB promoter elements by a mechanism that requires Bck1 but not Cln3. Genetic tests strongly argue that POG1 promotes recovery through upregulation of the CLN2 gene and that the resulting Cln2 protein promotes recovery primarily through an effect on Ste20, an activator of the mating MAPK cascade. A pog1 cln3 double mutant displays synthetic mutant phenotypes shared by cell-wall integrity and actin cytoskeleton mutants, with no synthetic defect in the expression of CLN1 or CLN2. These and other results suggest that POG1 may regulate additional genes during vegetative growth and recovery. PMID:9927449

  1. Post-cardiac arrest brain injury: pathophysiology and treatment.

    PubMed

    Chalkias, Athanasios; Xanthos, Theodoros

    2012-04-15

    Cardiac arrest is a leading cause of death that affects more than a million individuals worldwide every year. Despite the recent advancement in the field of cardiac arrest and resuscitation, the management and prognosis of post-cardiac arrest brain injury remain suboptimal. The pathophysiology of post-cardiac arrest brain injury involves a complex cascade of molecular events, most of which remain unknown. Considering that a potentially broad therapeutic window for neuroprotective drug therapy is offered in most successfully resuscitated patient after cardiac arrest, the need for further research is imperative. The aim of this article is to present the major pathophysiological disturbances leading to post-cardiac arrest brain injury, as well as to review the available pharmacological therapies.

  2. The psychological and legal aftermath of false arrest and imprisonment.

    PubMed

    Simon, R I

    1993-01-01

    False arrest and imprisonment can be an extraordinarily stressful psychological trauma. This is clearly demonstrated in the evaluation of forensic cases alleging false arrest and imprisonment, a review of the recent forensic psychiatric literature and reported legal cases. A clinical vignette is presented that illustrates the psychological trauma and sequelae associated with false arrest and imprisonment. Psychiatric treatment of these individuals is discussed. A number of these cases are litigated.

  3. Modes of induced cardiac arrest: hyperkalemia and hypocalcemia - Literature review

    PubMed Central

    de Oliveira, Marcos Aurélio Barboza; Brandi, Antônio Carlos; dos Santos, Carlos Alberto; Botelho, Paulo Henrique Husseini; Cortez, José Luis Lasso; Braile, Domingo Marcolino

    2014-01-01

    The entry of sodium and calcium play a key effect on myocyte subjected to cardiac arrest by hyperkalemia. They cause cell swelling, acidosis, consumption of adenosine triphosphate and trigger programmed cell death. Cardiac arrest caused by hypocalcemia maintains intracellular adenosine triphosphate levels, improves diastolic performance and reduces oxygen consumption, which can be translated into better protection to myocyte injury induced by cardiac arrest. PMID:25372919

  4. The analysis on nonlinear control of the aircraft arresting system

    NASA Astrophysics Data System (ADS)

    Song, Jinchun; Du, Tianrong

    2005-12-01

    The aircraft arresting system is a complicated nonlinear system. This paper analyzes the mechanical-hydraulic structure of aircraft arresting system composed of electro hydraulic valve and establishes the dynamic equation of the aircraft arresting system. Based on the state-feedback linearization of nonlinear system, a PD-based controller is synthesized. Simulation studies indicate, while arresting the different type aircraft, the proposed controller has fast response, good tracking performance and strong robustness. By tuning the parameters of the PD controller, a satisfactory control performance can be guaranteed.

  5. Hydroxylated PBDEs induce developmental arrest in zebrafish.

    PubMed

    Usenko, Crystal Y; Hopkins, David C; Trumble, Stephen J; Bruce, Erica D

    2012-07-01

    The ubiquitous spread of polybrominated diphenyl ethers (PBDEs) has led to concerns regarding the metabolites of these congeners, in particular hydroxylated PBDEs. There are limited studies regarding the biological interactions of these chemicals, yet there is some concern they may be more toxic than their parent compounds. In this study three hydroxylated PBDEs were assessed for toxicity in embryonic zebrafish: 3-OH-BDE 47, 5-OH-BDE 47, and 6-OH-BDE 47. All three congeners induced developmental arrest in a concentration-dependent manner; however, 6-OH-BDE 47 induced adverse effects at lower concentrations than the other congeners. Furthermore, all three induced cell death; however apoptosis was not observed. In short-term exposures (24-28 hours post fertilization), all hydroxylated PBDEs generated oxidative stress in the region corresponding to the cell death at 5 and 10 ppm. To further investigate the short-term effects that may be responsible for the developmental arrest observed in this study, gene regulation was assessed for embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 hpf. Genes involved in stress response, thyroid hormone regulation, and neurodevelopment were significantly upregulated compared to controls; however, genes related to oxidative stress were either unaffected or downregulated. This study suggests that hydroxylated PBDEs disrupt development, and may induce oxidative stress and potentially disrupt the cholinergic system and thyroid hormone homeostasis.

  6. Nitrogen deficiency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates.

    PubMed

    Kavanová, Monika; Lattanzi, Fernando Alfredo; Schnyder, Hans

    2008-06-01

    Nitrogen deficiency severely inhibits leaf growth. This response was analysed at the cellular level by growing Lolium perenne L. under 7.5 mM (high) or 1 mM (low) nitrate supply, and performing a kinematic analysis to assess the effect of nitrogen status on cell proliferation and cell growth in the leaf blade epidermis. Low nitrogen supply reduced leaf elongation rate (LER) by 43% through a similar decrease in the cell production rate and final cell length. The former was entirely because of a decreased average cell division rate (0.023 versus 0.032 h(-1)) and thus longer cell cycle duration (30 versus 22 h). Nitrogen status did not affect the number of division cycles of the initial cell's progeny (5.7), and accordingly the meristematic cell number (53). Meristematic cell length was unaffected by nitrogen deficiency, implying that the division and mitotic growth rates were equally impaired. The shorter mature cell length arose from a considerably reduced post-mitotic growth rate (0.033 versus 0.049 h(-1)). But, nitrogen stress did not affect the position where elongation stopped, and increased cell elongation duration. In conclusion, nitrogen deficiency limited leaf growth by increasing the cell cycle duration and decreasing mitotic and post-mitotic elongation rates, delaying cell maturation.

  7. Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.

    PubMed

    Morelli, Maria Beatrice; Amantini, Consuelo; Santoni, Matteo; Soriani, Alessandra; Nabissi, Massimo; Cardinali, Claudio; Santoni, Angela; Santoni, Giorgio

    2015-11-03

    Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC.Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy.On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner.Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity.

  8. Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells

    PubMed Central

    Morelli, Maria Beatrice; Amantini, Consuelo; Santoni, Matteo; Soriani, Alessandra; Nabissi, Massimo; Cardinali, Claudio; Santoni, Angela; Santoni, Giorgio

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC. Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy. On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner. Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity. PMID:26474283

  9. Salinomycin sensitizes antimitotic drugs-treated cancer cells by increasing apoptosis via the prevention of G2 arrest

    SciTech Connect

    Kim, Ju-Hwa; Yoo, Hye-In; Kang, Han Sung; Ro, Jungsil; Yoon, Sungpil

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Sal sensitizes antimitotic drugs-treated cancer cells. Black-Right-Pointing-Pointer Sal sensitizes them by prevention of G2 arrest and reduced cyclin D1 levels. Black-Right-Pointing-Pointer Sal also sensitizes them by increasing DNA damage and reducing p21 level. Black-Right-Pointing-Pointer A low concentration of Sal effectively sensitized the cancer cells to antimitotic drugs. -- Abstract: Here, we investigated whether Sal could sensitize cancer cells to antimitotic drugs. We demonstrated that Sal sensitized paclitaxcel (PAC)-, docetaxcel (DOC)-, vinblastin (VIN)-, or colchicine (COL)-treated cancer cell lines, suggesting that Sal has the ability to sensitize the cells to any form of microtubule-targeting drugs. Sensitization to the antimitotic drugs could be achieved with very low concentrations of Sal, suggesting that there is a possibility to minimize Sal toxicity associated with human cancer patient treatments. Sensitization by Sal increased apoptosis, which was observed by C-PARP production. Sal sensitized the cancer cells to antimitotic drugs by preventing G2 arrest, suggesting that Sal contributes to the induction of mitotic catastrophe. Sal generally reduced cyclin D1 levels in PAC-, DOC-, and VIN-treated cells. In addition, Sal treatment increased pH2AX levels and reduced p21 levels in antimitotic drugs-treated cells. These observations suggest that the mechanisms underlying Sal sensitization to DNA-damaging compounds, radiation, and microtubule-targeting drugs are similar. Our data demonstrated that Sal sensitizes cancer cells to antimitotic drugs by increasing apoptosis through the prevention of G2 arrest via conserved Sal-sensitization mechanisms. These results may contribute to the development of Sal-based chemotherapy for cancer patients treated with antimitotic drugs.

  10. Newly synthesized podophyllotoxin derivative, LJ12, induces apoptosis and mitotic catastrophe in non-small cell lung cancer cells in vitro.

    PubMed

    Hui, Ling; Sang, Chunyan; Wang, Donghong; Wang, Xiaohui; Wang, Meiliang; Jia, Qinghua; Ma, Mingren; Chen, Shiwu

    2016-01-01

    Deoxypodophyllotoxin (DPT), an active compound isolated from a number of herbs and used in traditional medicine, has been reported to exhibit promising anti‑tumor activity. A newly synthesized derivative, N-(1-oxyl‑4'-demethyl-4-deoxyp odophyllic)-L‑methine-4'-piperazine carbamate (LJ12) may have improved antitumor activity and fewer side effects. The present study assessed the effect of LJ12 on cell viability, apoptosis, cell cycle distribution and mitotic catastrophe in A549 human lung cancer cells in vitro. The molecular mechanisms underlying the antitumor activity of LJ12 were also examined. The results demonstrated that LJ12 reduced A549 cell viability in a time‑ and dose‑dependent manner, with a lower half maximal inhibitory concentration of ~0.1 µM, compared with another known DPT derivative, etoposide (10 µM). Flow cytometric analysis showed that LJ12 induced tumor cell arrest at the G2/M phase of the cell cycle. The present study also observed an expected concomitant decrease in the numbers of cells cells in the G0/G1 and S phases. LJ12 was found to upregulate the protein expression levels of Cdc2 and Cyclin B1. Furthermore, LJ12 induced tumor cell apoptosis and the protein expression of B cell lymphoma‑2‑associated X protein, caspase‑3 and p53. The present study also observed the formation of giant, multinucleated cells, indicating that LJ12 induced mitotic catastrophe in the tumor cells. These results indicated that LJ12 has anti‑non‑small cell lung cancer activity in vitro. Further investigations aim to develop LJ12 as a therapeutic agent for the treatment of lung cancer.

  11. JNK inhibitor SP600125 promotes the formation of polymerized tubulin, leading to G2/M phase arrest, endoreduplication, and delayed apoptosis.

    PubMed

    Moon, Dong Oh; Kim, Mun Ock; Kang, Chang Hee; Lee, Jae Dong; Choi, Yung Hyun; Kim, Gi Young

    2009-09-30

    The JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking cell-cycle progression and inducing apoptosis. Despite extensive study, the mechanism by which SP600125 inhibits mitosis-related effects in human leukemia cells remains unclear. We investigated the effects of SP600125 on the inhibition of cell proliferation and the cell cycle, and on microtubule dynamics in vivo and in vitro. Treatment of synchronized leukemia cells with varying concentrations of SP600125 results in significant G2/M cell cycle arrest with elevated p21 levels, phosphorylation of histone H3 within 24 h, and endoreduplication with elevated Cdk2 protein levels after 48 h. SP600125 also induces significant abnormal microtubule dynamics in vivo. High concentrations of SP600125 (200 microM) were required to disorganize microtubule polymerization in vitro. Additionally, SP600125- induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activity in the late phase (at 72 h). Endoreduplication showed a greater increase in ectopic Bcl-2-expressing U937 cells at 72 h than in wild-type U937 cells without delayed apoptosis. These results indicate that Bcl-2 suppresses apoptosis and SP600125-induced G2/M arrest and endoreduplication. Therefore, we suggest that SP600125 induces mitotic arrest by inducing abnormal spindle microtubule dynamics.

  12. The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

    PubMed Central

    Bhide, Shreerang A.; Eccles, Suzanne A.; Workman, Paul; Nutting, Christopher M.; Huddart, Robert A.; Harrington, Kevin J.

    2012-01-01

    Background Heat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies. Principal Findings NVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001). NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent. Conclusions These results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G2/M arrest, but that the contribution of cell cycle perturbation to

  13. The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

    PubMed Central

    Baker, Bruce S.; Carpenter, Adelaide T. C.; Ripoll, P.

    1978-01-01

    To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts

  14. Sex Differences in Urban Arrest Patterns, 1934-79.

    ERIC Educational Resources Information Center

    Steffensmeier, Darrell J.; Cobb, Michael J.

    1981-01-01

    Federal Bureau of Investigation statistics show that women have made large gains in arrests for petty property crimes and smaller gains for other offenses. However, alternate sources of data as well as changes in reporting and statistical coverage suggest that female arrest gains are more apparent than real. (Author/GC)

  15. A remote tester for surge arresters: Final report

    SciTech Connect

    Shaw, J.H.

    1986-12-01

    Laboratory studies show that the most probable indication that a surge arrester is failing is electromagnetic energy emission. In field trials by eight utilities, a tester designed to detect radiofrequency emissions located defective arresters, but stray emissions in the environment limited its performance.

  16. Colleges Report Increases in Arrests for Drug and Alcohol Violations.

    ERIC Educational Resources Information Center

    Nicklin, Julie L.

    1999-01-01

    Arrests for violations of drug/alcohol laws at colleges and universities rose 7.2 and 3.6%, respectively, from 1996 to 1997. Campus police attribute the increases not to increased drug and alcohol use but to more aggressive enforcement. However, some health researchers feel usage has risen. Campus weapons violations and forcible rape arrests have…

  17. Nitrite therapy is neuroprotective and safe in cardiac arrest survivors.

    PubMed

    Dezfulian, Cameron; Alekseyenko, Aleksey; Dave, Kunjan R; Raval, Ami P; Do, Rose; Kim, Francis; Perez-Pinzon, Miguel A

    2012-05-15

    Cardiac arrest results in significant mortality after initial resuscitation due in most cases to ischemia-reperfusion induced brain injury and to a lesser degree myocardial dysfunction. Nitrite has previously been shown to protect against reperfusion injury in animal models of focal cerebral and heart ischemia. Nitrite therapy after murine cardiac arrest improved 22 h survival through improvements in myocardial contractility. These improvements accompanied transient mitochondrial inhibition which reduced oxidative injury to the heart. Based on preliminary evidence that nitrite may also protect against ischemic brain injury, we sought to test this hypothesis in a rat model of asphyxia cardiac arrest with prolonged survival (7d). Cardiac arrest resulted in hippocampal CA1 delayed neuronal death well characterized in this and other cardiac arrest models. Nitrite therapy did not alter post-arrest hemodynamics but did result in significant (75%) increases in CA1 neuron survival. This was associated with increases in hippocampal nitrite and S-nitrosothiol levels but not cGMP shortly after therapy. Mitochondrial function 1h after resuscitation trended towards improvement with nitrite therapy. Based on promising preclinical data, the first ever phase I trial of nitrite infusions in human cardiac arrest survivors has been undertaken. We present preliminary data showing low dose nitrite infusion did not result in hypotension or cause methemoglobinemia. Nitrite thus appears safe and effective for clinical translation as a promising therapy against cardiac arrest mediated heart and brain injury.

  18. Evolution of the dragonfly head-arresting system

    PubMed Central

    Gorb, S. N.

    1999-01-01

    The arrester or fixation system of the head in adult Odonata is unique among arthropods. This system involves the organs of two body segments: the head and the neck. It consists of a skeleton–muscle apparatus that sets the arrester parts in motion. The parts comprise formations covered with complicated microstructures: fields of microtrichia on the rear surface of the head and post-cervical sclerites of the neck. The arrester immobilizes the head during feeding or when the dragonfly is in tandem flight. Thus, it may serve as an adaptation to save the head from violent mechanical disturbance and to stabilize gaze in a variety of behavioural situations. This study shows the evolutionary trend of the arrester in the order Odonata by using scanning electron microscopy and measurements of arrester structures in 227 species from 26 odonate families. The arrester design occurring in the Epiophlebiidae, Gomphidae, Neopetaliidae, Petaluridae and Chlorogomphinae is suggested to be the basic one. Two convergent pathways of head-arrester evolution among Zygoptera and Anisoptera are proposed. The possible functional significance of the arrester system is discussed.

  19. 32 CFR 935.125 - Citation in place of arrest.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ....125 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS WAKE ISLAND CODE Peace Officers § 935.125 Citation in place of arrest. In any case in which a peace officer may make an arrest without a warrant, he may issue and serve a citation if...

  20. 32 CFR 935.125 - Citation in place of arrest.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ....125 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS WAKE ISLAND CODE Peace Officers § 935.125 Citation in place of arrest. In any case in which a peace officer may make an arrest without a warrant, he may issue and serve a citation if...

  1. 32 CFR 935.125 - Citation in place of arrest.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ....125 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS WAKE ISLAND CODE Peace Officers § 935.125 Citation in place of arrest. In any case in which a peace officer may make an arrest without a warrant, he may issue and serve a citation if...

  2. 32 CFR 935.125 - Citation in place of arrest.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ....125 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS WAKE ISLAND CODE Peace Officers § 935.125 Citation in place of arrest. In any case in which a peace officer may make an arrest without a warrant, he may issue and serve a citation if...

  3. 32 CFR 935.125 - Citation in place of arrest.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ....125 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE TERRITORIAL AND INSULAR REGULATIONS WAKE ISLAND CODE Peace Officers § 935.125 Citation in place of arrest. In any case in which a peace officer may make an arrest without a warrant, he may issue and serve a citation if...

  4. 41. #1 ARRESTING GEAR ENGINE AFT LOOKING FORWARD PORT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    41. #1 ARRESTING GEAR ENGINE - AFT LOOKING FORWARD PORT TO STARBOARD SHOWING ARRESTING GEAR ENGINE ACCUMULATOR, AIR FLASK, CONTROL VALVE, WITH CONTROL RAM, SHEAVES AND WIRES UNDERNEATH ENGINE STAND. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  5. The arrest of Agulhas retroflection during glaciations

    NASA Astrophysics Data System (ADS)

    Zharkov, V.; Nof, D.; Ortiz, J. D.; Paldor, N.; Chassignet, E.

    2011-12-01

    Paleoceanographic proxy data indicate that the Agulhas leakage into the South Atlantic was dramatically reduced during glacial times, thus probably resulting in the collapse of the Atlantic Meridional Overturning Circulation. In our former papers, we hypothesized that this was due to a northward shift of the zero wind stress curl that, in turn, forced the retroflection to occur farther north, where the slant of the coastline relative to the north is steep. Here we propose that strong westerlies (0.4 Pa implying a wind speed of ~ 12 m/s at zero degrees centigrade), which were supposedly common during glaciations, also could have arrested the leakage. This arrest occurs because the wind stress opposes the momentum flux associated with the retroflection and, therefore, the retroflection does not shift in latitude. We use a simple, nonlinear, "reduced gravity" model to show analytically and numerically that, under the above conditions, the eastward wind stress compensates for the zonal westward flow-force associated with the retroflection, thus avoiding the development and shedding of rings. For a nearly zonal wall, westerly winds, and small upper layer thickness along the wall, the arresting wind stress is found, theoretically, to be, τx~0.042α3/2ρf[(2fQ)3/g']1/4 where α is twice the retroflection eddy vorticity, ρ the water density, and Q the Agulhas Current volume flux; the remaining notation is conventional. According to this formula, wind typical for the Agulhas region during glacial times (0.4Pa) significantly affects the moderately strong Agulhas rings of large PV (α=0.1) but, with increasing α, the influence of wind quickly decreases, and becomes negligible for α>0.2. This theoretical result is in agreement with the results of the numerical simulations that we conducted. The numerics show that the wind tends to destroy the detached rings by squeezing them onto the wall, a result that is valid in both the straight and the kinked coast cases. In the

  6. UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores.

    PubMed

    Zhang, Xiaojuan; Ling, Youguo; Wang, Wenjun; Zhang, Yanhong; Ma, Qingjun; Tan, Pingping; Song, Ting; Wei, Congwen; Li, Ping; Liu, Xuedong; Ma, Runlin Z; Zhong, Hui; Cao, Cheng; Xu, Quanbin

    2013-04-15

    The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.

  7. PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells

    PubMed Central

    Rogers, Samuel; Fey, Dirk; McCloy, Rachael A.; Parker, Benjamin L.; Mitchell, Nicholas J.; Payne, Richard J.; Daly, Roger J.; James, David E.; Caldon, C. Elizabeth; Watkins, D. Neil; Croucher, David R.; Burgess, Andrew

    2016-01-01

    ABSTRACT Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit. PMID:26872783

  8. PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells.

    PubMed

    Rogers, Samuel; Fey, Dirk; McCloy, Rachael A; Parker, Benjamin L; Mitchell, Nicholas J; Payne, Richard J; Daly, Roger J; James, David E; Caldon, C Elizabeth; Watkins, D Neil; Croucher, David R; Burgess, Andrew

    2016-04-01

    Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit.

  9. Greatwall dephosphorylation and inactivation upon mitotic exit is triggered by PP1.

    PubMed

    Ma, Sheng; Vigneron, Suzanne; Robert, Perle; Strub, Jean Marc; Cianferani, Sara; Castro, Anna; Lorca, Thierry

    2016-04-01

    Entry into mitosis is induced by the activation of cyclin-B-Cdk1 and Greatwall (Gwl; also known as MASTL in mammals) kinases. Cyclin-B-Cdk1 phosphorylates mitotic substrates, whereas Gwl activation promotes the phosphorylation of the small proteins Arpp19 and ENSA. Phosphorylated Arpp19 and/or ENSA bind to and inhibit PP2A comprising the B55 subunit (PP2A-B55; B55 is also known as PPP2R2A), the phosphatase responsible for cyclin-B-Cdk1 substrate dephosphorylation, allowing the stable phosphorylation of mitotic proteins. Upon mitotic exit, cyclin-B-Cdk1 and Gwl kinases are inactivated, and mitotic substrates are dephosphorylated. Here, we have identified protein phosphatase-1 (PP1) as the phosphatase involved in the dephosphorylation of the activating site (Ser875) of Gwl. Depletion of PP1 from meioticXenopusegg extracts maintains phosphorylation of Ser875, as well as the full activity of this kinase, resulting in a block of meiotic and mitotic exit. By contrast, preventing the reactivation of PP2A-B55 through the addition of a hyperactive Gwl mutant (GwlK72M) mainly affected Gwl dephosphorylation on Thr194, resulting in partial inactivation of Gwl and in the incomplete exit from mitosis or meiosis. We also show that when PP2A-B55 is fully reactivated by depleting Arpp19, this protein phosphatase is able to dephosphorylate both activating sites, even in the absence of PP1.

  10. Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

    PubMed Central

    Lindqvist, Arne; van Zon, Wouter; Karlsson Rosenthal, Christina; Wolthuis, Rob M. F

    2007-01-01

    Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression. PMID:17472438

  11. Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements.

    PubMed

    Kawamura, Ryo; Pope, Lisa H; Christensen, Morten O; Sun, Mingxuan; Terekhova, Ksenia; Boege, Fritz; Mielke, Christian; Andersen, Anni H; Marko, John F

    2010-03-08

    We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II-specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.

  12. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.

    PubMed

    Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A; Gerlich, Daniel W

    2016-07-14

    Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

  13. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes

    PubMed Central

    Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z.; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A.; Gerlich, Daniel W.

    2016-01-01

    Summary Eukaryotic genomes are partitioned into chromosomes, which during mitosis form compact and spatially well-separated mechanical bodies1–3.This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes4 and the discovery of proteins at the chromosome surface3,5,6, the molecular and biophysical basis of mitotic chromosome individuality have remained unclear. We report that Ki-67, a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of Ki-67 is not confined within a specific protein domain but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrical barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-color labeling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic for polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery and suggests that natural proteins can function as surfactants in intracellular compartmentalization. PMID:27362226

  14. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

    PubMed Central

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-01-01

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.09384.001 PMID:26406118

  15. Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells *

    PubMed Central

    Min, Mingwei; Mayor, Ugo; Dittmar, Gunnar; Lindon, Catherine

    2014-01-01

    Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells. PMID:24857844

  16. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

    SciTech Connect

    Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko; Ueda, Keiji

    2016-09-15

    In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.

  17. Studies on the control of mitotic activity in excised roots. I. The experimental system.

    PubMed

    WILSON, G B; MORRISON, J H; KNOBLOCH, N

    1959-05-25

    The mitotic characteristics of excised roots of the garden pea, Pisum sativum, have been studied under conditions of controlled nutrition. The excised root system was tested with regard to its ability to respond, mitotically, to various carbon sources. Sucrose, glucose, fructose, and DL-glyceraldehyde were found to support mitotic activity in excised roots, galactose and 2-deoxy-D-glucose were toxic, and mannose ineffective. Initiation of mitotic activity in the presence of glucose was inhibited by the respiratory poisons, KCN and malonic acid, the uncoupling agent, 2,4-dinitrophenol, but was not notably affected by the protein synthesis inhibitor, chloramphenicol. The glucose-induced response in mitotic activity was not affected by the carcinogen, urethan, and indeed, there is some evidence that the response was actually enhanced. The fact that KCN, malonic acid, and probably 2,4-dinitrophenol, in suitable concentrations inhibit the onset of cell division suggests that some level of operation of the Krebs' cycle is essential for commission of cells into mitosis. Likewise, failure to inhibit cells in the process of active mitosis by KCN and malonic acid is not inconsistent with the idea that there is a shift from reliance on aerobic to anaerobic respiration between antephase and active mitosis.

  18. Kin4 kinase delays mitotic exit in response to spindle alignment defects.

    PubMed

    Pereira, Gislene; Schiebel, Elmar

    2005-07-22

    For many polarized cells, it is critical that the mitotic spindle becomes positioned relative to the polarity axis. This is especially important in yeast, where the site of cytokinesis is predetermined. The spindle position checkpoint (SPOC) therefore delays mitotic exit of cells with a mispositioned spindle. One component of the SPOC is the Bub2-Bfa1 complex, an inhibitor of the mitotic exit network (MEN). Here, we show that the Kin4 kinase is a component of the SPOC and as such is essential to delay cell cycle progression of cells with a misaligned spindle. When spindles are correctly oriented, Kin4 and Bub2-Bfa1 are asymmetrically localized to opposite spindle pole bodies (SPBs). Bub2-Bfa1 then becomes inhibited by Cdc5 polo kinase with anaphase onset, a prerequisite for mitotic exit. In response to spindle misalignment, Kin4 and Bub2-Bfa1 are brought together at both SPBs. Kin4 now maintains Bub2-Bfa1 activity by counteracting Cdc5, thereby inhibiting mitotic exit.

  19. Electro-acoustic behavior of the mitotic spindle: a semi-classical coarse-grained model.

    PubMed

    Havelka, Daniel; Kučera, Ondřej; Deriu, Marco A; Cifra, Michal

    2014-01-01

    The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells--a strategy used in novel methods for cancer treatment.

  20. Electro-Acoustic Behavior of the Mitotic Spindle: A Semi-Classical Coarse-Grained Model

    PubMed Central

    Havelka, Daniel; Kučera, Ondřej; Deriu, Marco A.; Cifra, Michal

    2014-01-01

    The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells—a strategy used in novel methods for cancer treatment. PMID:24497952

  1. Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

    PubMed Central

    Lim, Nicholas R.; Yeap, Yvonne Y. C.; Ang, Ching-Seng; Williamson, Nicholas A.; Bogoyevitch, Marie A.; Quinn, Leonie M.; Ng, Dominic C. H.

    2016-01-01

    ABSTRACT Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization. PMID:26713495

  2. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

    PubMed

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-09-25

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division.

  3. The effect of magnesium on mitotic spindle formation in Schizosaccharomyces pombe.

    PubMed

    Uz, Gulsen; Sarikaya, Aysegul Topal

    2016-01-01

    Magnesium (Mg2+), an essential ion for cells and biological systems, is involved in a variety of cellular processes, including the formation and breakdown of microtubules. The results of a previous investigation suggested that as cells grow the intracellular Mg2+ concentration falls, thereby stimulating formation of the mitotic spindle. In the present work, we used a Mg2+-deficient Schizosaccharomyces pombe strain GA2, in which two essential membrane Mg2+ transporter genes (homologs of ALR1 and ALR2 in Saccharomyces cerevisae) were deleted, and its parental strain Sp292, to examine the extent to which low Mg2+ concentrations can affect mitotic spindle formation. The two S. pombe strains were transformed with a plasmid carrying a GFP-α2-tubulin construct to fluorescently label microtubules. Using the free Mg2+-specific fluorescent probe mag-fura-2, we confirmed that intracellular free Mg2+ levels were lower in GA2 than in the parental strain. Defects in interphase microtubule organization, a lower percentage of mitotic spindle formation and a reduced mitotic index were also observed in the GA2 strain. Although there was interphase microtubule polymerization, the lower level of mitotic spindle formation in the Mg2+-deficient strain suggested a greater requirement for Mg2+ in this phenomenon than previously thought.

  4. Wnt activation followed by Notch inhibition promotes mitotic hair cell regeneration in the postnatal mouse cochlea

    PubMed Central

    Li, Wenyan; Chen, Yan; Zhang, Shasha; Tang, Mingliang; Sun, Shan; Chai, Renjie; Li, Huawei

    2016-01-01

    Hair cell (HC) loss is the main cause of permanent hearing loss in mammals. Previous studies have reported that in neonatal mice cochleae, Wnt activation promotes supporting cell (SC) proliferation and Notch inhibition promotes the trans-differentiation of SCs into HCs. However, Wnt activation alone fails to regenerate significant amounts of new HCs, Notch inhibition alone regenerates the HCs at the cost of exhausting the SC population, which leads to the death of the newly regenerated HCs. Mitotic HC regeneration might preserve the SC number while regenerating the HCs, which could be a better approach for long-term HC regeneration. We present a two-step gene manipulation, Wnt activation followed by Notch inhibition, to accomplish mitotic regeneration of HCs while partially preserving the SC number. We show that Wnt activation followed by Notch inhibition strongly promotes the mitotic regeneration of new HCs in both normal and neomycin-damaged cochleae while partially preserving the SC number. Lineage tracing shows that the majority of the mitotically regenerated HCs are derived specifically from the Lgr5+ progenitors with or without HC damage. Our findings suggest that the co-regulation of Wnt and Notch signaling might provide a better approach to mitotically regenerate HCs from Lgr5+ progenitor cells. PMID:27564256

  5. Growth arrest lines and recurrent patellar dislocation: a new sign.

    PubMed

    Abraham, A; Macnicol, M F

    2001-06-01

    The phenomenon of growth arrest lines has been widely described in the medical literature. They are usually found at the metaphysis of growing long bones and are the result of short periods of partial growth arrest. Recurrent dislocation of the patella is a well-recognised problem, particularly in adolescents. Several radiological features have been reported in association with patellar dislocation or instability. We have reported a hitherto undescribed radiological sign of patellar growth arrest lines on the skyline radiographs of two patients with this condition. The shape of the patella when symptoms were at their worst corresponded remarkably closely to the outline of the subsequent growth arrest line. We postulated that repeated dislocations adversely affect the process of normal maturation of the patella. With the resolution of symptoms, patella ossification resumes, leaving the telltale sign of previous injury in the form of a growth arrest line and an improvement in bone density once the patella has been stabilised and tracks normally.

  6. Substance Use Disorders, Comorbidity, and Arrest among Indigenous Adolescents*

    PubMed Central

    Hartshorn, Kelley J. Sittner; Whitbeck, Les B.; Prentice, Patricia

    2011-01-01

    Indigenous adolescents are overrepresented at multiple stages of the justice system, but we know very little about the role that mental health, particularly substance use disorder, plays in Indigenous pathways to arrest. This study examined the association between substance use disorder, its comorbidity with other disorders, and arrest using a longitudinal sample of Indigenous youth from the Northern Midwest and Canada. Of the 16% of youth who reported at least one arrest at Wave 5, half met criteria for substance abuse/dependence, and slightly more for conduct disorder. Substance abuse/dependence and conduct disorder were each associated with an increased risk of arrest, although co-occurring disorders were not. The reciprocal effects of arrest and mental disorder are discussed. PMID:26759503

  7. Flashback flame arrester devices for fuel cargo tank vapor vents

    NASA Astrophysics Data System (ADS)

    Bjorklund, R. A.; Kushida, R. O.

    1981-03-01

    The flame quenching capability of four types of flame arresting devices suitable for installation on fuel cargo tank vents of marine transport vessels is evaluated. A single 30 mesh screen, a dual 20 mesh screen, a spiral wound crimped metal ribbon, and a packed bed of ballast rings were tested. Flame speed and flame penetration of the test arresters were determined. Eight fuels representative of bulk cargoes were tested. The test arresters quenched a minimum of three flashback flames from all eight fuels, with one exception: high speed ethylene flames penetrated the dual 20 mesh screen on three tests. The arresters withstood the sustained flame from a propane/air mixture for 30 minutes. None of the arresters withstood the sustained flame from an ethylene/air mixture for more than 7 minutes.

  8. Flashback flame arrester devices for fuel cargo tank vapor vents

    NASA Technical Reports Server (NTRS)

    Bjorklund, R. A.; Kushida, R. O.

    1981-01-01

    The flame quenching capability of four types of flame arresting devices suitable for installation on fuel cargo tank vents of marine transport vessels is evaluated. A single 30 mesh screen, a dual 20 mesh screen, a spiral wound crimped metal ribbon, and a packed bed of ballast rings were tested. Flame speed and flame penetration of the test arresters were determined. Eight fuels representative of bulk cargoes were tested. The test arresters quenched a minimum of three flashback flames from all eight fuels, with one exception: high speed ethylene flames penetrated the dual 20 mesh screen on three tests. The arresters withstood the sustained flame from a propane/air mixture for 30 minutes. None of the arresters withstood the sustained flame from an ethylene/air mixture for more than 7 minutes.

  9. Cremophor EL stimulates mitotic recombination in uvsH//uvsH diploid strain of Aspergillus nidulans.

    PubMed

    Busso, Cleverson; Castro-Prado, Marialba A A

    2004-03-01

    Cremophor EL is a solubilizer and emulsifier agent used in the pharmaceutical and foodstuff industries. The solvent is the principal constituent of paclitaxel's clinical formulation vehicle. Since mitotic recombination plays a crucial role in multistep carcinogenesis, the study of the recombinagenic potential of chemical compounds is of the utmost importance. In our research genotoxicity of cremophor EL has been studied by using an uvsH//uvsH diploid strain of Aspergillus nidulans. Since it spends a great part of its cell cycle in the G2period, this fungus is a special screening system for the study of mitotic recombination induced by chemical substances. Homozygotization Indexes (HI) for paba and bi markers from heterozygous B211//A837 diploid strain were determined for the evaluation of the recombinagenic effect of cremophor EL. It has been shown that cremophor EL induces increase in mitotic crossing-over events at nontoxic concentrations (0.05 and 0.075% v/v).

  10. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

    PubMed Central

    Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

    2015-01-01

    Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. PMID:26602832

  11. Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

    NASA Astrophysics Data System (ADS)

    Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

    2015-11-01

    Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

  12. Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies.

    PubMed

    Scarfone, Ilaria; Piatti, Simonetta

    2017-01-01

    Most proteins of the Mitotic Exit Network (MEN) and their upstream regulators localize at spindle pole bodies (SPBs) at least in some stages of the cell cycle. Studying the SPB localization of MEN factors has been extremely useful to elucidate their biological roles, organize them in a hierarchical pathway, and define their dynamics under different conditions.Recruitment to SPBs of the small GTPase Tem1 and the downstream kinases Cdc15 and Mob1/Dbf2 is thought to be essential for Cdc14 activation and mitotic exit, while that of the upstream Tem1 regulators (the Kin4 kinase and the GTPase activating protein Bub2-Bfa1) is important for MEN inhibition upon spindle mispositioning. Here, we describe the detailed fluorescence microscopy procedures that we use in our lab to analyze the localization at SPBs of Mitotic Exit Network (MEN) components tagged with GFP or HA epitopes.

  13. Mitotic spindle orients perpendicular to the forces imposed by dynamic shear.

    PubMed

    Fernandez, Pablo; Maier, Matthias; Lindauer, Martina; Kuffer, Christian; Storchova, Zuzana; Bausch, Andreas R

    2011-01-01

    Orientation of the division axis can determine cell fate in the presence of morphogenetic gradients. Understanding how mitotic cells integrate directional cues is therefore an important question in embryogenesis. Here, we investigate the effect of dynamic shear forces on confined mitotic cells. We found that human epithelial cells (hTERT-RPE1) as well as MC3T3 osteoblasts align their mitotic spindle perpendicular to the external force. Spindle orientation appears to be a consequence of cell elongation along the zero-force direction in response to the dynamic shear. This process is a nonlinear response to the strain amplitude, requires actomyosin activity and correlates with redistribution of myosin II. Mechanosteered cells divide normally, suggesting that this mechanism is compatible with biological functions.

  14. The post-mitotic state in neurons correlates with a stable nuclear higher-order structure

    PubMed Central

    Aranda-Anzaldo, Armando

    2012-01-01

    Neurons become terminally differentiated (TD) post-mitotic cells very early during development yet they may remain alive and functional for decades. TD neurons preserve the molecular machinery necessary for DNA synthesis that may be reactivated by different stimuli but they never complete a successful mitosis. The non-reversible nature of the post-mitotic state in neurons suggests a non-genetic basis for it since no set of mutations has been able to revert it. Comparative studies of the nuclear higher-order structure in neurons and cells with proliferating potential suggest that the non-reversible nature of the post-mitotic state in neurons has a structural basis in the stability of the nuclear higher-order structure. PMID:22808316

  15. Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression.

    PubMed

    Kachaner, David; Filipe, Josina; Laplantine, Emmanuel; Bauch, Angela; Bennett, Keiryn L; Superti-Furga, Giulio; Israël, Alain; Weil, Robert

    2012-02-24

    Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.

  16. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    PubMed

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

  17. Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs

    PubMed Central

    McCloy, Rachael A.; Parker, Benjamin L.; Rogers, Samuel; Chaudhuri, Rima; Gayevskiy, Velimir; Hoffman, Nolan J.; Ali, Naveid; Watkins, D. Neil; Daly, Roger J.; James, David E.; Lorca, Thierry; Castro, Anna; Burgess, Andrew

    2015-01-01

    Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (∼10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical

  18. Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs.

    PubMed

    McCloy, Rachael A; Parker, Benjamin L; Rogers, Samuel; Chaudhuri, Rima; Gayevskiy, Velimir; Hoffman, Nolan J; Ali, Naveid; Watkins, D Neil; Daly, Roger J; James, David E; Lorca, Thierry; Castro, Anna; Burgess, Andrew

    2015-08-01

    Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (∼ 10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical

  19. Reversible hypercondensation and decondensation of mitotic chromosomes studied using combined chemical-micromechanical techniques.

    PubMed

    Poirier, Michael G; Monhait, Tamar; Marko, John F

    2002-01-01

    We show that the chromatin in mitotic chromosomes can be drastically overcompacted or unfolded by temporary shifts in ion concentrations. By locally 'microspraying' reactants from micron-size pipettes, while simultaneously monitoring the size of and tension in single chromosomes, we are able to quantitatively study the dynamics of these reactions. The tension in a chromosome is monitored through observation and calibration of bending of the glass pipettes used to manipulate the chromosomes. For concentrations > 500 mM of NaCl and > 200 mM of MgCl2, we find that the initially applied tensions of approximately 500 pN relax to zero and that mitotic chromatin temporarily disperses in agreement with previous work (Maniotis et al. [1997] J. Cell. Biochem. 65:114-130). This unfolding occurs in about 1 s, and is reversible once the charge density is returned to physiological levels, if the exposure is not longer than approximately 1 min. Low concentrations of NaCl (< 30 mM) also induces a decrease in tension and increase in size. We observe this swelling to be isotropic in experiments on chromosomes under zero tension, a behavior inconsistent with the existence of a well-defined central chromosome 'scaffold'. By contrast 10 mM of divalent cations (MgCl2 and CaCl2) induces an extremely rapid and reversible increase in tension and a reduction in the size of mitotic chromosomes. Hexaminecobalt trichloride (trivalent cation) has the same effect as MgCl2 and CaCl2, except the magnitude of force increase and size change are much larger. Hexaminecobalt trichloride reduces mitotic chromosomes to 65% of their original volume, indicating that at least 1/3 of their apparent volume is aqueous solution. These results indicate that chromatin inside mitotic chromatids has a large amount of conformational freedom allowing dynamic unfolding and refolding and that charge interactions play a central role in maintaining mitotic chromosome structure.

  20. Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800

    PubMed Central

    1990-01-01

    Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo. PMID:2211822

  1. The Caenorhabditis elegans THO Complex Is Required for the Mitotic Cell Cycle and Development

    PubMed Central

    Castellano-Pozo, Maikel; García-Muse, Tatiana; Aguilera, Andrés

    2012-01-01

    THO is a conserved eukaryotic complex involved in mRNP biogenesis and RNA export that plays an important role in preventing transcription- and RNA-mediated genome instability in mitosis and meiosis. In mammals THO is essential for embryogenesis, which limits our capacity to analyze the physiological relevance of THO during development and in adult organisms. Using Caenorhabditis elegans as a model system we show that the THO complex is essential for mitotic genome integrity and the developmentally regulated mitotic cell cycles occurring during late postembryonic stages. PMID:23285047

  2. Mitotic behavior in root tips of Brachiaria genotypes with meiotic chromosome elimination during microsporogenesis.

    PubMed

    Felismino, M F; Silva, N; Pagliarini, M S; Valle, C B

    2008-04-15

    Three accessions of Brachiaria brizantha, three of B. humidicola, and two interspecific hybrids between B. ruziziensis and B. brizantha were analyzed with regard to their mitotic behavior in root tips. All these genotypes revealed chromosome elimination or lack of chromosome affinity in previous analyses of microsporogenesis. Analyses of root tips showed a normal mitotic division in all accessions and hybrids, reinforcing the notion that the genetic control of meiosis is totally independent of that of mitosis. The implications of these findings for the Brachiaria breeding program are discussed.

  3. Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression.

    PubMed

    Funk, Laura C; Zasadil, Lauren M; Weaver, Beth A

    2016-12-19

    Errors in chromosome segregation during mitosis have been recognized as a hallmark of tumor cells since the late 1800s, resulting in the long-standing hypothesis that mitotic abnormalities drive tumorigenesis. Recent work has shown that mitotic defects can promote tumors, suppress them, or do neither, depending on the rate of chromosome missegregation. Here we discuss the causes of chromosome missegregation, their effects on tumor initiation and progression, and the evidence that increasing the rate of chromosome missegregation may be an effective chemotherapeutic strategy.

  4. The spoilage yeast Zygosaccharomyces bailii forms mitotic spores: a screening method for haploidization.

    PubMed

    Rodrigues, Fernando; Ludovico, Paula; Sousa, Maria João; Steensma, H Yde; Côrte-Real, Manuela; Leão, Cecília

    2003-01-01

    Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z. bailii. This new set of Z. bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.

  5. A closer look at arrested spinodal decomposition in protein solutions.

    PubMed

    Gibaud, Thomas; Schurtenberger, Peter

    2009-08-12

    Concentrated aqueous solutions of the protein lysozyme undergo a liquid-solid transition upon a temperature quench into the unstable spinodal region below a characteristic arrest temperature of T(f) = 15 °C. We use video microscopy and ultra-small angle light scattering in order to investigate the arrested structures as a function of initial concentration, quench temperature and rate of the temperature quench. We find that the solid-like samples show all the features of a bicontinuous network that is formed through an arrested spinodal decomposition process. We determine the correlation length ξ and demonstrate that ξ exhibits a temperature dependence that closely follows the critical scaling expected for density fluctuations during the early stages of spinodal decomposition. These findings are in agreement with an arrest scenario based on a state diagram where the arrest or gel line extends far into the unstable region below the spinodal line. Arrest then occurs when during the early stage of spinodal decomposition the volume fraction φ(2) of the dense phase intersects the dynamical arrest threshold φ(2,Glass), upon which phase separation gets pinned into a space-spanning gel network with a characteristic length ξ.

  6. [Prevalence of supraventricular tachycardia and tachyarrhythmias in resuscitated cardiac arrest].

    PubMed

    Brembilla-Perrot, B; Marcon, O; Blangy, H; Terrier de la Chaise, A; Louis, P; Sadoul, N; Claudon, O; Nippert, M; Popovic, B; Belhakem, H

    2006-01-01

    Supraventricular arrhythmias are considered to be benign when the ventricular rate is slowed and treated by anticoagulants. The aim of this study was to determine the possible influence of these arrhythmias in resuscitated cardiac arrest. Between 1980 and 2002, 151 patients were admitted after a cardiac arrest. Supraventricular arrhythrmias were identified as a possible cause of the cardiac arrest in 21 patients. They underwent echocardiography, exercise stress test, Holter ECG monitoring , coronary angiography and electrophysiological investigation. After these investigations, three patients had a malignant form of the Wolff-Parkinson-White syndrome, two were asymptomatic and, in the third patient, ventricular fibrillation was induced by treatment with diltiazem. In 8 patients, a rapid supraventricular arrhythmia was considered to be the cause of cardiac arrest by cardiogenic shock; 2 patients had hypertrophic cardiomyopathy, 5 had severe dilated cardiomyopathy which regressed in one patient. In ten patients, cardiac arrest due to ventricular tachycardia or fibrillation was provoked by a rapid (> 220 beats/min) supraventricular arrhythmia; two patients had no apparent underlying cardiac pathology. In the others, myocardial ischaemia or acute cardiac failure were considered to be the cause of the cardiac arrest. The authors conclude that rapid supraventricular arrhythmias may cause cardiac arrest either by cardiogenic shock or degenerescence to ventricular tachycardia or fibrillation. Usually, this event occurs in patients with severe cardiac disease but it may occur in subjects without cardiac disease or by an arrhythmia-induced cardiomyopathy.

  7. Induced rates of mitotic crossing over and possible mitotic gene conversion per wing anlage cell in Drosophila melanogaster by X rays and fission neutrons

    SciTech Connect

    Ayaki, T.; Fujikawa, K.; Ryo, H.; Itoh, T.; Kondo, S. )

    1990-09-01

    As a model for chromosome aberrations, radiation-induced mitotic recombination of mwh and flr genes in Drosophila melanogaster strain (mwh +/+ flr) was quantitatively studied. Fission neutrons were five to six times more effective than X rays per unit dose in producing either crossover-mwh/flr twins and mwh singles-or flr singles, indicating that common processes are involved in the production of crossover and flr singles. The X-ray-induced rate/wing anlage cell/Gy for flr singles was 1 X 10(-5), whereas that of crossover was 2 x 10(-4); the former and the latter rate are of the same order of magnitude as those of gene conversion and crossover in yeast, respectively. Thus, we conclude that proximal-marker flr singles induced in the transheterozygote are gene convertants. Using the model based on yeast that recombination events result from repair of double-strand breaks or gaps, we propose that mitotic recombination in the fly is a secondary result of recombinational DNA repair. Evidence for recombinational misrepair in the fly is given. The relative ratio of radiation-induced mitotic crossover to spontaneous meiotic crossover is one order of magnitude higher in the fly than in yeast and humans.

  8. Epidemiology and management of cardiac arrest: what registries are revealing.

    PubMed

    Gräsner, Jan-Thorsten; Bossaert, Leo

    2013-09-01

    Major European institutions report cardiovascular disease (CVD) as the first cause of death in adults, with cardiac arrest and sudden death due to coronary ischaemia as the primary single cause. Global incidence of CVD is decreasing in most European countries, due to prevention, lifestyle and treatment. Mortality of acute coronary events inside the hospital decreases more rapidly than outside the hospital. To improve the mortality of cardiac arrest outside the hospital, reliable epidemiological and process figures are essential: "we can only manage what we can measure". Europe is a patchwork of 47 countries (total population of 830 million), with a 10-fold difference in incidence of coronary heart disease between North and South, East and West, and a 5-fold difference in number of EMS-treated cardiac arrest (range 17-53/1000,000/year). Epidemiology of cardiac arrest should not be calculated as a European average, but it is appropriate to describe the incidence of cardiac arrest, the resuscitation process, and the outcome in each of the European regions, for benchmarking and quality management. Epidemiological reports of cardiac arrest should specify definitions, nominator (number of cases) and denominator (study population). Recently some regional registries in North America, Japan and Europe fulfilled these conditions. The European Registry of Cardiac Arrest (EuReCa) has the potential to achieve these objectives on a pan-European scale. For operational applications, the Utstein definition of "Cardiac arrest" is used which includes the potential of survival. For application in community health, the WHO definition of "sudden death" is frequently used, describing the mode of death. There is considerable overlap between both definitions. But this explains that no single method can provide all information. Integrating data from multiple sources (local, national, multinational registries and surveys, death certificates, post-mortem reports, community statistics, medical

  9. p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line

    PubMed Central

    Badie, C; Bourhis, J; Sobczak-Thépot, J; Haddada, H; Chiron, M; Janicot, M; Janot, F; Tursz, T; Vassal, G

    2000-01-01

    In vivo transfer of wild-type (wt) p53 gene via a recombinant adenovirus has been proposed to induce apoptosis and increase radiosensitivity in several human carcinoma models. In the context of combining p53 gene transfer and irradiation, we investigated the consequences of adenoviral-mediated wtp53 gene transfer on the cell cycle and radiosensitivity of a human head and neck squamous cell carcinoma line (SCC97) with a p53 mutated phenotype. We showed that ectopic expression of wtp53 in SCC97 cells resulted in a prolonged G1 arrest, associated with an increased expression of the cyclin-dependent kinase inhibitor WAF1/p21 target gene. A transient arrest in G2 but not in G1 was observed after irradiation. This G2 arrest was permanent when exponentially growing cells were transduced by Ad5CMV- p53 (RPR/INGN201) immediately after irradiation with 5 or 10 Gy. Moreover, levels of cyclins A2 and B1, which are known to regulate the G2/M transition, dramatically decreased as cells arrived in G2, whereas maximal levels of expression were observed in the absence of wtp53. In conclusion, adenoviral mediated transfer of wtp53 in irradiated SCC97 cells, which are mutated for p53, appeared to increase WAF1/p21 expression and decrease levels of the mitotic cyclins A2 and B1. These observations suggest that the G2 arrest resulted from a p53-dependent premature inactivation of the mitosis promoting factor. © 2000 Cancer Research Campaign PMID:10682678

  10. "Aren't I a victim?": Notes on identity challenges relating to police action in a mandatory arrest jurisdiction.

    PubMed

    Rajah, Valli; Frye, Victoria; Haviland, Mary

    2006-10-01

    The mandated arrest of domestic violence perpetrators is a policy response to the problem of partner violence. Mandatory arrest can result, however, in unintended and sometimes undesirable arrest outcomes, including dual arrests (when both parties are arrested), retaliatory arrests (when the perpetrator has his or her partner wrongfully arrested), and failures to make an arrest (when one is warranted by law). Using an interactionist perspective, this research focuses on one negative effect of mandatory arrest: the identity challenge faced by female victims of domestic violence who experience undesirable arrest outcomes. The authors discuss policy implications, focusing on the potential empowerment effects of mandatory arrest.

  11. Spindle checkpoint protein Bub1 corrects mitotic aberrancy induced by human T-cell leukemia virus type I Tax.

    PubMed

    Sasaki, M; Sugimoto, K; Tamayose, K; Ando, M; Tanaka, Y; Oshimi, K

    2006-06-22

    Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named 'mitotic lobulation' in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.

  12. Pollution performance of 110 kV metal oxide arresters

    SciTech Connect

    Chrzan, K.; Pohl, Z.; Grzybowski, S.; Koehler, W.

    1997-04-01

    Pollution test results of single unit 110 kV metal oxide surge arresters with porcelain housing according to the solid layer and salt fog methods are presented. During 6 hours of testing, the internal and external charge and maximum temperature along the varistor column were measured. The formation of single stable dry bands on the housing was often observed, especially during salt fog tests. In such cases, the varistor temperature can reach about 70 C. The simple electrical model of the arrester enabling calculations of voltages and currents as a function of arrester and pollution parameters is shown.

  13. Behavior of zinc oxide surge arresters under pollution

    SciTech Connect

    Feser, F.; Kohler, W.; Qiu, D. ); Chrzan, K. )

    1991-04-01

    This paper presents results of pollution tests with AC voltages which were carried out with a multi-unit zinc oxide arrester. The interaction between the polluted porcelain housing and the inner varistor column due to capacitive coupling has been found to be responsible for the temperature rise of varistor elements. The different voltage distribution between inside and outside of the arrester also causes a high radial electric field which can lead to internal discharges if the radial insulation system is not properly designed. These internal discharges may damage varistor elements which are not adequately coated and may cause a total destruction of the arrester.

  14. Neuroprotective strategies and neuroprognostication after cardiac arrest.

    PubMed

    Taccone, Fabio Silvio; Crippa, Ilaria Alice; Dell'Anna, Antonio Maria; Scolletta, Sabino

    2015-12-01

    Neurocognitive disturbances are common among survivors of cardiac arrest (CA). Although initial management of CA, including bystander cardiopulmonary resuscitation, optimal chest compression, and early defibrillation, has been implemented continuously over the last years, few therapeutic interventions are available to minimize or attenuate the extent of brain injury occurring after the return of spontaneous circulation. In this review, we discuss several promising drugs that could provide some potential benefits for neurological recovery after CA. Most of these drugs have been investigated exclusively in experimental CA models and only limited clinical data are available. Further research, which also considers combined neuroprotective strategies that target multiple pathways involved in the pathophysiology of postanoxic brain injury, is certainly needed to demonstrate the effectiveness of these interventions in this setting. Moreover, the evaluation of neurological prognosis of comatose patients after CA remains an important challenge that requires the accurate use of several tools. As most patients with CA are currently treated with targeted temperature management (TTM), combined with sedative drug therapy, especially during the hypothermic phase, the reliability of neurological examination in evaluating these patients is delayed to 72-96 h after admission. Thus, additional tests, including electrophysiological examinations, brain imaging and biomarkers, have been largely implemented to evaluate earlier the extent of brain damage in these patients.

  15. Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome.

    PubMed

    Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

    2015-08-14

    PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome.

  16. Loading of PAX3 to Mitotic Chromosomes Is Mediated by Arginine Methylation and Associated with Waardenburg Syndrome*

    PubMed Central

    Wu, Tsu-Fang; Yao, Ya-Li; Lai, I-Lu; Lai, Chien-Chen; Lin, Pei-Lun; Yang, Wen-Ming

    2015-01-01

    PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome. PMID:26149688

  17. Cells transformed by PLC-gamma 1 overexpression are highly sensitive to clostridium difficile toxin A-induced apoptosis and mitotic inhibition.

    PubMed

    Nam, Hyo Jung; Kang, Jin Ku; Chang, Jong Soo; Lee, Min Soo; Nam, Seung Taek; Jung, Hyun Woo; Kim, Sung-Kuk; Ha, Eun-Mi; Seok, Heon; Son, Seung Woo; Park, Young Joo; Kim, Ho

    2012-01-01

    Phospholipase C-γl (PLC-γl) expression is associated with cellular transformation. Notably, PLC-gamma is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-γl overexpression. We found that PLC-γl-transformed cells, but not vectortransformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-γl-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-γl-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-γl is highly up-regulated.

  18. Phytonutrients Differentially Stimulate NAD(P)H:Quinone Oxidoreductase, Inhibit Proliferation, and Trigger Mitotic Catastrophe in Hepa1c1c7 Cells

    PubMed Central

    Singletary, Keith W.; Murphy, Laura L.; Venema, Richard C.; Young, Andrew J.

    2016-01-01

    Abstract Phytonutrients have rapidly emerged as natural food chemicals possessing multifaceted biological actions that may support beneficial health outcomes. Among the vast array of phytonutrients currently being studied, sulforaphane, curcumin, quercetin, and resveratrol have been frequently reported to stimulate the expression of endogenous detoxification enzymes and may thereby facilitate the neutralization of otherwise harmful environmental agents. Some of these same phytonutrients, however, have also been implicated in disrupting normal cell proliferation and hence may possess toxic properties in and of themselves. In this study, we characterize the respective minimum threshold concentrations of the aforementioned phytonutrients in Hepa1c1c7 cells that stimulate NAD(P)H:quinone oxidoreductase (NQO1), a key enzyme in the hepatic neutralization of menadione, other biological oxidants, and some environmental carcinogens. Moreover, our findings demonstrate that relatively low concentrations of either sulforaphane or curcumin significantly (P < .05) increase NQO1 protein expression and activity without triggering G2/M cell cycle arrest or mitotic catastrophe. The minimal quercetin concentration inducing NQO1, however, was 100-fold higher than that which disrupted mitosis. Also, while resveratrol modestly stimulated NQO1, the minimally effective resveratrol concentration concomitantly induced evidence of cellular apoptosis. Taken together, these findings indicate that only particular phytonutrients are likely efficacious in upregulating NQO1 activity without also leading to hepatic cytotoxicity. PMID:26623679

  19. A mitotic nuclear envelope tether for Gle1 also affects nuclear and nucleolar architecture

    PubMed Central

    Chemudupati, Mahesh; Osmani, Aysha H.; Osmani, Stephen A.

    2016-01-01

    During Aspergillus nidulans mitosis, peripheral nuclear pore complex (NPC) proteins (Nups) disperse from the core NPC structure. Unexpectedly, one predicted peripheral Nup, Gle1, remains at the mitotic nuclear envelope (NE) via an unknown mechanism. Gle1 affinity purification identified mitotic tether for Gle1 (MtgA), which tethers Gle1 to the NE during mitosis but not during interphase when Gle1 is at NPCs. MtgA is the orthologue of the Schizosaccharomyces pombe telomere-anchoring inner nuclear membrane protein Bqt4. Like Bqt4, MtgA has meiotic roles, but it is functionally distinct from Bqt4 because MtgA is not required for tethering telomeres to the NE. Domain analyses showed that MtgA targeting to the NE requires its C-terminal transmembrane domain and a nuclear localization signal. Of importance, MtgA functions beyond Gle1 mitotic targeting and meiosis and affects nuclear and nucleolar architecture when deleted or overexpressed. Deleting MtgA generates small, round nuclei, whereas overexpressing MtgA generates larger nuclei with altered nuclear compartmentalization resulting from NE expansion around the nucleolus. The accumulation of MtgA around the nucleolus promotes a similar accumulation of the endoplasmic reticulum (ER) protein Erg24, reducing its levels in the ER. This study extends the functions of Bqt4-like proteins to include mitotic Gle1 targeting and modulation of nuclear and nucleolar architecture. PMID:27630260

  20. Effects of ovariectomy on estrogen uptake capacity, mitotic index and morphology of immunocytochemically-identified gonadotropes

    SciTech Connect

    Smith, P.F.

    1985-01-01

    The primary objective of these studies was to examine the effects of ovariectomy on the pituitary gonadotrope population in the rat. Several parameters were examined including morphology, mitotic index and ability of individual cells to concentrate estrogen. Adult, female rats which had been ovariectomized 3, 14, or 50 previously, were injected with /sup 3/H-estradiol (i.v.) and killed 1 hour later. Pituitaries were excised and immediately hemisected (mid-sagittal cut). Trunk blood was collected for subsequent radioimmunoassay of serum LH levels to assess the activity of the pituitary gonadotropes. Frozen pituitaries were sectioned and processed for dry-mount autoradiography. Estrogen uptake capacity of gonadotropes increased with time after ovariectomy. This increase was not seen in male rats after castration. Hemi-pituitaries were sectioned (1 ..mu..m) and analyzed for the number of mitotic figures per mm/sup 2/ and dividing cells were characterized as to their hormonal content. Ovariectomy induced an increase in the mitotic index of the pituitary gland. Furthermore, a majority of the mitotic futures seen in the ovariectomized rat were found in cells containing LH-immunoreactivity. Electron microscopic examination of dividing gonadotropes revealed that these cells contained large amounts of vesiculated endoplasmic reticumum typical of post-castration gonadotropes.

  1. Direct preparation protocol to obtain mitotic chromosomes from canine mammary tumors.

    PubMed

    Morais, C S D; Affonso, P R A M; Bitencourt, J A; Wenceslau, A A

    2015-12-29

    Currently, mammary neoplasms in female canines are a serious problem in veterinary clinics. In addition, the canine species is an excellent disease model for human oncology because of the biological and genetic similarities between the species. Cytogenetics has allowed further study of the characterization of neoplasms in canines. We hypothesized that the use of a direct preparation protocol for mitotic chromosome analysis would provide a simple and low cost protocol for use in all laboratories. The objective of this method is to display in a few hours of dividing cells just like the time of collection since cell division in tissue can be obtained. Ten female canines with the spontaneous occurrence of mammary neoplasia were used to test a pioneering direct preparation protocol to obtain mitotic chromosomes. The excised breast tumor tissue fragments were subjected to the protocol consisting of treatment with colchicine, treatment with hypotonic solution, and fixation. Mitotic chromosomes were absent in cell suspensions of only two samples among the 10 materials analyzed, based on the analysis of five blades for each preparation obtained. So, the cell suspension obtained allowed for the observation of eight tissue samples viable for cytogenetic analysis, five of which had excellent numbers of mitotic chromosomes. However, the technique was unsuccessful in producing high-quality cell suspensions because of inadequate condensation and scattering of chromosomes. While adjustments to methodological procedures are needed, this protocol represents a low cost and simplified method to study the cytogenetics of canine tumors.

  2. Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum.

    PubMed

    Paul, E C; Roobol, A; Foster, K E; Gull, K

    1987-01-01

    Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle. We have shown that all four tubulin isotypes identified in the plasmodium (alpha 1, alpha 2, beta 1 and beta 2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: beta 2 greater than alpha 1 greater than alpha 2 greater than beta 1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.

  3. The deubiquitinating enzyme complex BRISC is required for proper mitotic spindle assembly in mammalian cells

    PubMed Central

    Yan, Kaowen; Li, Li; Wang, Xiaojian; Hong, Ruisha; Zhang, Ying; Yang, Hua; Lin, Ming; Zhang, Sha; He, Qihua; Zheng, Duo; Tang, Jun; Yin, Yuxin

    2015-01-01

    Deubiquitinating enzymes (DUBs) negatively regulate protein ubiquitination and play an important role in diverse physiological processes, including mitotic division. The BRCC36 isopeptidase complex (BRISC) is a DUB that is specific for lysine 63–linked ubiquitin hydrolysis; however, its biological function remains largely undefined. Here, we identify a critical role for BRISC in the control of mitotic spindle assembly in cultured mammalian cells. BRISC is a microtubule (MT)-associated protein complex that predominantly localizes to the minus ends of K-fibers and spindle poles and directly binds to MTs; importantly, BRISC promotes the assembly of functional bipolar spindle by deubiquitinating the essential spindle assembly factor nuclear mitotic apparatus (NuMA). The deubiquitination of NuMA regulates its interaction with dynein and importin-β, which are required for its function in spindle assembly. Collectively, these results uncover BRISC as an important regulator of the mitotic spindle assembly and cell division, and have important implications for the development of anticancer drugs targeting BRISC. PMID:26195665

  4. Subamolide a induces mitotic catastrophe accompanied by apoptosis in human lung cancer cells.

    PubMed

    Hung, Jen-Yu; Wen, Ching-Wen; Hsu, Ya-Ling; Lin, En-Shyh; Huang, Ming-Shyan; Chen, Chung-Yi; Kuo, Po-Lin

    2013-01-01

    This study investigated the anticancer effects of subamolide A (Sub-A), isolated from Cinnamomum subavenium, on human nonsmall cell lung cancer cell lines A549 and NCI-H460. Treatment of cancer cells with Sub-A resulted in decreased cell viability of both lung cancer cell lines. Sub-A induced lung cancer cell death by triggering mitotic catastrophe with apoptosis. It triggered oxidant stress, indicated by increased cellular reactive oxygen species (ROS) production and decreased glutathione level. The elevated ROS triggered the activation of ataxia-telangiectasia mutation (ATM), which further enhanced the ATF3 upregulation and subsequently enhanced p53 function by phosphorylation at Serine 15 and Serine 392. The antioxidant, EUK8, significantly decreased mitotic catastrophe by inhibiting ATM activation, ATF3 expression, and p53 phosphorylation. The reduction of ATM and ATF3 expression by shRNA decreased Sub-A-mediated p53 phosphorylation and mitotic catastrophe. Sub-A also caused a dramatic 70% reduction in tumor size in an animal model. Taken together, cell death of lung cancer cells in response to Sub-A is dependent on ROS generation, which triggers mitotic catastrophe followed by apoptosis. Therefore, Sub-A may be a novel anticancer agent for the treatment of nonsmall cell lung cancer.

  5. A role for vasa in regulating mitotic chromosome condensation in Drosophila.

    PubMed

    Pek, Jun Wei; Kai, Toshie

    2011-01-11

    Vasa (Vas) is a conserved DEAD-box RNA helicase expressed in germline cells that localizes to a characteristic perinuclear structure called nuage. Previous studies have shown that Vas has diverse functions, with roles in regulating mRNA translation, germline differentiation, pole plasm assembly, and piwi-interacting RNA (piRNA)-mediated transposon silencing. Although vas has also been implicated in the regulation of germline proliferation in Drosophila and mice, little is known about whether Vas plays a role during the mitotic cell cycle. Here, we report a translation-independent function of vas in regulating mitotic chromosome condensation in the Drosophila germline. During mitosis, Vas facilitates robust chromosomal localization of the condensin I components Barren (Barr) and CAP-D2. Vas specifically associates with Barr and CAP-D2, but not with CAP-D3 (a condensin II component). The mitotic function of Vas is mediated by the formation of perichromosomal Vas bodies during mitosis, which requires the piRNA pathway components aubergine and spindle-E. Our results suggest that Vas functions during mitosis and may link the piRNA pathway to mitotic chromosome condensation in Drosophila.

  6. Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes

    PubMed Central

    Shahbazi, Marta N.; Peña-Jimenez, Daniel; Antonucci, Francesca; Drosten, Matthias

    2017-01-01

    ABSTRACT Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mechanisms involved in this process are not yet fully understood. Using cultured primary keratinocytes, here we report that the expression of the mammalian microtubule and kinetochore-associated protein Clasp2 is intimately associated with the basal proliferative makeup of keratinocytes, and its deficiency leads to premature differentiation. Clasp2-deficient keratinocytes exhibit increased centrosomal numbers and numerous mitotic alterations, including multipolar spindles and chromosomal misalignments that overall result in mitotic stress and a high DNA content. Such mitotic block prompts premature keratinocyte differentiation in a p53-dependent manner in the absence of cell death. Our findings reveal a new role for Clasp2 in governing keratinocyte undifferentiated features and highlight the presence of surveillance mechanisms that prevent cell cycle entry in cells that have alterations in the DNA content. PMID:28069833

  7. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    SciTech Connect

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa; Yoon, Hyun-Joo; Yoo, Hae Yong; Choi, Cheol Yong

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  8. Emerging molecular mechanisms that power and regulate the anastral mitotic spindle of flowering plants.

    PubMed

    Bannigan, Alex; Lizotte-Waniewski, Michelle; Riley, Margaret; Baskin, Tobias I

    2008-01-01

    Flowering plants, lacking centrosomes as well as dynein, assemble their mitotic spindle via a pathway that is distinct visually and molecularly from that of animals and yeast. The molecular components underlying mitotic spindle assembly and function in plants are beginning to be discovered. Here, we review recent evidence suggesting the preprophase band in plants functions analogously to the centrosome in animals in establishing spindle bipolarity, and we review recent progress characterizing the roles of specific motor proteins in plant mitosis. Loss of function of certain minus-end-directed KIN-14 motor proteins causes a broadening of the spindle pole; whereas, loss of function of a KIN-5 causes the formation of monopolar spindles, resembling those formed when the homologous motor protein (e.g., Eg5) is knocked out in animal cells. We present a phylogeny of the kinesin-5 motor domain, which shows deep divergence among plant sequences, highlighting possibilities for specialization. Finally, we review information concerning the roles of selected structural proteins at mitosis as well as recent findings concerning regulation of M-phase in plants. Insight into the mitotic spindle will be obtained through continued comparison of mitotic mechanisms in a diversity of cells.

  9. Mitotic Phosphorylation of TREX1 C Terminus Disrupts TREX1 Regulation of the Oligosaccharyltransferase Complex.

    PubMed

    Kucej, Martin; Fermaintt, Charles S; Yang, Kun; Irizarry-Caro, Ricardo A; Yan, Nan

    2017-03-14

    TREX1 mutations are associated with several autoimmune and inflammatory diseases. The N-terminal DNase domain of TREX1 is important for preventing self-DNA from activating the interferon response. The C terminus of TREX1 is required for ER localization and regulation of oligosacchariyltransferase (OST) activity. Here, we show that during mitosis TREX1 is predominately phosphorylated at the C-terminal Serine-261 by Cyclin B/CDK1. TREX1 is dephosphorylated quickly at mitotic exit, likely by PP1/PP2-type serine/threonine phosphatase. Mitotic phosphorylation does not affect TREX1 DNase activity. Phosphomimetic mutations of mitotic phosphorylation sites in TREX1 disrupted the interaction with the OST subunit RPN1. RNA-seq analysis of Trex1(-/-) mouse embryonic fibroblasts expressing TREX1 wild-type or phosphor-mutants revealed a glycol-gene signature that is elevated when TREX1 mitotic phosphorylation sites are disrupted. Thus, the cell-cycle-dependent post-translation modification of TREX1 regulates its interaction with OST, which may have important implications for immune disease associated with the DNase-independent function of TREX1.

  10. Miniaturization of mitotic index cell-based assay using "wall-less" plate technology.

    PubMed

    Le Guezennec, Xavier; Phong, Mark; Nor, Liyana; Kim, Namyong

    2014-03-01

    The use of microscopic imaging for the accurate assessment of cells in mitosis is hampered by the round morphology of mitotic cells, which renders them poorly adherent and highly susceptible to loss during the washing stage of cell-based assays. Here, to circumvent these limitations, we make use of DropArray, a recent technology that allows high retention of weakly adherent cells and suspension cells. DropArray offers the competitive advantage of maintaining the classic high throughput format of microtiter plates while reducing classic microwell volume by up to 90% by using a drop format. Here, we present a mitotic index cell-based assay using the mitosis marker phospho histone H3 at serine 10 on a DropArray 384-well plate format. Dose-response curve analysis of the mitotic index assay with an antimitotic drug (docetaxel) on DropArray is presented that shows an effective dosage compared to previous established results similar to those obtained with conventional microtiter plates. The mitotic index assay with DropArray showed a Z-factor >0.6. Our results validate DropArray as a suitable platform for high throughput screening for compounds affecting mitosis or the cell cycle.

  11. 157. Detail of lightning arresters; looking west. Photo by Jet ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    157. Detail of lightning arresters; looking west. Photo by Jet Lowe, HAER, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA

  12. 156. Detail of lightning arrester on hillside above powerhouse; looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    156. Detail of lightning arrester on hillside above powerhouse; looking west. Photo by Jet Lowe, HAER, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA

  13. 154. Detail of lightning arrester on hillside above powerhouse; looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    154. Detail of lightning arrester on hillside above powerhouse; looking north. Photo by Jet Lowe, HAER, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA

  14. 53. NEW BCB AND LIGHTNING ARRESTER ARRANGEMENT, SANTA ANA RIVER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    53. NEW BCB AND LIGHTNING ARRESTER ARRANGEMENT, SANTA ANA RIVER NO. 2, JAN. 24, 1977. SCE drawing no. 455670-0. - Santa Ana River Hydroelectric System, SAR-2 Powerhouse, Redlands, San Bernardino County, CA

  15. 155. Detail of lightning arrester on hillside above powerhouse; looking ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    155. Detail of lightning arrester on hillside above powerhouse; looking north. Photo by Jet Lowe, HAER, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA

  16. [Effect of phenibut on the respiratory arrest caused by serotonin].

    PubMed

    Tarakanov, I A; Tarasova, N N; Belova, E A; Safonov, V A

    2006-01-01

    The role of the GABAergic system in mechanisms of the respiratory arrest caused by serotonin administration was studied in anaesthetized rats. Under normal conditions, the systemic administration of serotonin (20-60 mg/kg, i.v.) resulted in drastic changes of the respiratory pattern, whereby the initial phase of increased respiratory rate was followed by the respiratory arrest. The preliminary injection of phenibut (400 mg/kg, i.p.) abolished or sharply reduced the duration of the respiratory arrest phase induced by serotonin. Bilateral vagotomy following the phenibut injection potentiated the anti-apnoesic effect of phenibut, which was evidence of the additive action of vagotomy and phenibut administration. The mechanism of apnea caused by serotonin administration is suggested to include a central GABAergic element, which is activated by phenibut so as to counteract the respiratory arrest.

  17. 32 CFR 553.9 - Power of arrest.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... are authorized to arrest any person who willfully destroys, cuts, breaks, injures, or removes any tree, shrub, or plant within the limits of the cemetery and to bring that person before any United...

  18. Warning Signs of Heart Attack, Stroke and Cardiac Arrest

    MedlinePlus

    ... a Heart Attack WARNING SIGNS OF HEART ATTACK, STROKE & CARDIAC ARREST HEART ATTACK WARNING SIGNS CHEST DISCOMFORT ... nausea or lightheadedness. Learn more about heart attack STROKE WARNING SIGNS Spot a stroke F.A.S.T.: - ...

  19. How Can Death Due to Sudden Cardiac Arrest Be Prevented?

    MedlinePlus

    ... Trials Links Related Topics Arrhythmia Automated External Defibrillator Coronary Heart Disease Heart Failure Long QT Syndrome Send a link ... First Sudden Cardiac Arrest If you have severe coronary heart disease (CHD), you're at increased risk for SCA. ...

  20. Heart Attack or Sudden Cardiac Arrest: How Are They Different?

    MedlinePlus

    ... Peripheral Artery Disease Venous Thromboembolism Aortic Aneurysm More Heart Attack or Sudden Cardiac Arrest: How Are They Different? ... to heart disease and stroke. Start exploring today ! Heart Attack • Home • About Heart Attacks Acute Coronary Syndrome (ACS) ...