A Abrieu

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

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Publications (22)252.87 Total impact

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    ABSTRACT: Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.Cell Death and Differentiation advance online publication, 9 August 2013; doi:10.1038/cdd.2013.105.
    Cell death and differentiation 08/2013; · 8.24 Impact Factor
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    ABSTRACT: Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.
    Current biology: CB 02/2012; 22(4):289-95. · 10.99 Impact Factor
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    ABSTRACT: A recent screen for compounds that selectively targeted pancreatic cancer cells isolated UA62784. We found that UA62784 inhibits microtubule polymerization in vitro. UA62784 interacts with tubulin dimers ten times more potently than colchicine, vinblastine, or nocodazole. Competition experiments revealed that UA62784 interacts with tubulin at or near the colchicine-binding site. Nanomolar doses of UA62784 promote the accumulation of mammalian cells in mitosis, due to aberrant mitotic spindles, as shown by immunofluorescence and live cell imaging. Treatment of cancerous cell lines with UA62784 is lethal, following activation of apoptosis signaling. By monitoring mitotic spindle perturbations and apoptosis, we found that the effects of UA62784 and of some known microtubule-depolymerizing drugs are additive. Finally, high content screening of H2B-GFP HeLa cells revealed that low doses of UA62784 and vinblastine potentiate each other to inhibit proliferation.
    Chemistry & biology 05/2011; 18(5):631-41. · 6.52 Impact Factor
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    Cell Research 12/2010; 20(12):1386-9. · 10.53 Impact Factor
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    ABSTRACT: During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.
    Molecular cell 04/2008; 29(5):637-43. · 14.61 Impact Factor
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    ABSTRACT: Whereas early cytokinesis events have been relatively well studied, little is known about its final stage, abscission. The Cdc14 phosphatase is involved in the regulation of multiple cell cycle events, and in all systems studied Cdc14 misexpression leads to cytokinesis defects. In this work, we have cloned two CDC14 cDNA from Xenopus, including a previously unreported CDC14B homologue. We use Xenopus and human cell lines and demonstrate that localization of Cdc14 proteins is independent of both cell-type and species specificity. Ectopically expressed XCdc14A is centrosomal in interphase and localizes to the midbody in cytokinesis. By using XCdc14A misregulation, we confirm its control over different cell cycle events and unravel new functions during abscission. XCdc14A regulates the G1/S and G2/M transitions. We show that Cdc25 is an in vitro substrate for XCdc14A and might be its target at the G2/M transition. Upregulated wild-type or phosphatase-dead XCdc14A arrest cells in a late stage of cytokinesis, connected by thin cytoplasmic bridges. It does not interfere with central spindle formation, nor with the relocalization of passenger protein and centralspindlin complexes to the midbody. We demonstrate that XCdc14A upregulation prevents targeting of exocyst and SNARE complexes to the midbody, both essential for abscission to occur.
    Experimental Cell Research 05/2007; 313(6):1225-39. · 3.56 Impact Factor
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    ABSTRACT: We have identified a unique human microtubule-associated protein (MAP) named ASAP for ASter-Associated Protein. ASAP localizes to microtubules in interphase, associates with the mitotic spindle during mitosis, localizes to the central body during cytokinesis and directly binds to purified microtubules by its COOH-terminal domain. Overexpression of ASAP induces profound bundling of cytoplasmic microtubules in interphase cells and aberrant monopolar spindles in mitosis. Depletion of ASAP by RNA interference results in severe mitotic defects: it provokes aberrant mitotic spindle, delays mitotic progression, and leads to defective cytokinesis or cell death. These results suggest a crucial role for ASAP in the organization of the bipolar mitotic spindle, mitosis progression, and cytokinesis and define ASAP as a key factor for proper spindle assembly.
    Proceedings of the National Academy of Sciences 09/2005; 102(32):11302-7. · 9.74 Impact Factor
  • Yinghui Mao, Ariane Abrieu, Don W Cleveland
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    ABSTRACT: The mitotic checkpoint prevents advance to anaphase prior to successful attachment of every centromere/kinetochore to mitotic spindle microtubules. Using purified components and Xenopus egg extracts, the kinetochore-associated microtubule motor CENP-E is now shown to be the activator of the essential checkpoint kinase BubR1. Since kinase activity and the checkpoint are silenced following CENP-E-dependent microtubule attachment in extracts or binding of CENP-E antibodies that do not disrupt CENP-E association with BubR1, CENP-E mediates silencing of BubR1 signaling. Checkpoint signaling requires the normal level of BubR1 containing a functional Mad3 domain implicated in Cdc20 binding, but only a small fraction need be kinase competent. This supports bifunctional roles for BubR1 in the checkpoint: an enzymatic one requiring CENP-E-dependent activation of its kinase activity at kinetochores and a stoichiometric one as a direct inhibitor of Cdc20.
    Cell 08/2003; 114(1):87-98. · 31.96 Impact Factor
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    ABSTRACT: The mitotic checkpoint acts to inhibit entry into anaphase until all chromosomes have successfully attached to spindle microtubules. Unattached kinetochores are believed to release an activated form of Mad2 that inhibits APC/C-dependent ubiquitination and subsequent proteolysis of components needed for anaphase onset. Using Xenopus egg extracts, a vertebrate homolog of yeast Mps1p is shown here to be a kinetochore-associated kinase, whose activity is necessary to establish and maintain the checkpoint. Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C. Mps1 is essential for the checkpoint because it is required for recruitment and retention of active CENP-E at kinetochores, which in turn is necessary for kinetochore association of Mad1 and Mad2.
    Cell 08/2001; 106(1):83-93. · 31.96 Impact Factor
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    A Abrieu, M Dorée, D Fisher
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    ABSTRACT: Throughout oocyte maturation, and subsequently during the first mitotic cell cycle, the MAP kinase cascade and cyclin-B-Cdc2 kinase are associated with the control of cell cycle progression. Many roles have been directly or indirectly attributed to MAP kinase and its influence on cyclin-B-Cdc2 kinase in different model systems; yet a principle theme does not emerge from the published literature, some of which is apparently contradictory. Interplay between these two kinases affects the major events of meiotic maturation throughout the animal kingdom, including the suppression of DNA replication, the segregation of meiotic chromosomes, and the prevention of parthenogenetic activation. Central to many of these events appears to be the control by MAP kinase of cyclin translation and degradation.
    Journal of Cell Science 02/2001; 114(Pt 2):257-67. · 5.88 Impact Factor
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    ABSTRACT: Accurate chromatid separation is monitored by a checkpoint mechanism that delays anaphase onset until all centromeres are correctly attached to the mitotic spindle. Using Xenopus egg extracts, the kinetochore-associated microtubule motor protein CENP-E is now found to be required for establishing and maintaining this checkpoint. When CENP-E function is disrupted by immunodepletion or antibody addition, extracts fail to arrest in response to spindle damage. Mitotic arrest can be restored by addition of high levels of soluble MAD2, demonstrating that the absence of CENP-E eliminates kinetochore-dependent signaling but not the downstream steps in checkpoint signal transduction. Because it directly binds both to spindle microtubules and to the kinetochore-associated checkpoint kinase BUBR1, CENP-E is a central component in the vertebrate checkpoint that modulates signaling activity in a microtubule-dependent manner.
    Cell 10/2000; 102(6):817-26. · 31.96 Impact Factor
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    ABSTRACT: Here we show that suppression of synthesis of the microtubule motor CENP-E (centromere-associated protein E), a component of the kinetochore corona fibres of mammalian centromeres, yields chromosomes that are chronically mono-orientated, with spindles that are flattened along the plane of the substrate. Despite apparently normal microtubule numbers and the continued presence at kinetochores of other microtubule motors, spindle poles fragment in the absence of CENP-E, which implicates this protein in delivery of components from kinetochores to poles. CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signalling cascade, as depletion of this motor leads to profound checkpoint activation, whereas immunoprecipitation reveals a nearly stoichiometric association of CENP-E with the checkpoint kinase BubR1 during mitosis.
    Nature Cell Biology 09/2000; 2(8):484-91. · 20.76 Impact Factor
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    ABSTRACT: Downregulation of MAP kinase is a universal consequence of fertilization in the animal kingdom. Here we show that oocytes of the starfishes Astropecten aranciacus and Marthasterias glacialis complete meiotic maturation and form a pronucleus when treated with 1-methyladenine and then complete DNA replication and arrest at G2 if not fertilized. Release of G2 by fertilization or a variety of parthenogenetic treatments is associated with inactivation of MAP kinase. Prevention of MAP kinase inactivation by microinjection of Ste11-DeltaN, a constitutively active budding yeast MAP kinase kinase kinase, arrests fertilized eggs at G2 in either the first or the second mitotic cell cycle, in a dose-dependent manner. G1 arrest is never observed. Conversely, inactivation of MAP kinase by microinjection of the MAP kinase-specific phosphatase Pyst-1 releases mature starfish oocytes from G2 arrest. The role of MAP kinase in arresting cell cycle at various stages in oocytes of different animal species is discussed.
    Developmental Biology 11/1998; 202(1):1-13. · 3.87 Impact Factor
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    ABSTRACT: Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end-directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
    The Journal of Cell Biology 10/1998; 142(6):1519-32. · 10.82 Impact Factor
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    ABSTRACT: Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
    The Journal of Cell Biology 09/1998; 142(6):1519-1532. · 10.82 Impact Factor
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    ABSTRACT: We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.
    Journal of Cell Science 07/1998; 111 ( Pt 12):1751-7. · 5.88 Impact Factor
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    ABSTRACT: Down-regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal kingdom, although its role is not known. Here we show that MAPK inactivation is essential for embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down-regulation of MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade, specifically suppresses cyclin B-cdc2 kinase activation and its consequence, entry into first mitosis. It thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. The most general effect of artificially maintaining MAPK activity after fertilization is prevention of the G2 to M-phase transition in the first mitotic cell cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell cycle is not responsible for late entry of fertilized eggs into first mitosis.
    The EMBO Journal 12/1997; · 9.82 Impact Factor
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    A Abrieu, M Dorée, A Picard
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    ABSTRACT: The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.
    Molecular Biology of the Cell 03/1997; 8(2):249-61. · 4.60 Impact Factor
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    ABSTRACT: Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.
    Journal of Cell Science 02/1996; 109 ( Pt 1):239-46. · 5.88 Impact Factor
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    ABSTRACT: It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca(2+)-dependent activation of the ubiquitin-dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We showed recently that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin-dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation.
    The EMBO Journal 10/1994; 13(18):4343-52. · 9.82 Impact Factor

Publication Stats

1k Citations
252.87 Total Impact Points

Institutions

  • 1994–2008
    • French National Centre for Scientific Research
      • Centre de Recherche de Biochimie Macromoléculaire
      Lutetia Parisorum, Île-de-France, France
  • 2000–2001
    • Ludwig Institute for Cancer Research
      La Jolla, California, United States
  • 1997
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France