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Gomez R.,Autonomous University of Madrid | Jordan P.W.,The Jackson Laboratory | Viera A.,Autonomous University of Madrid | Alsheimer M.,University of Würzburg | And 6 more authors.
Journal of Cell Science | Year: 2013

Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II. © 2013. Published by The Company of Biologists Ltd.


Rivero D.,University of Florence | Herrero S.,Karlsruhe Institute of Technology | Moreno S.,Institute Biologia Molecular y Celular del Cancer
Eukaryotic Cell | Year: 2010

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2α, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2α kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2α kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2α phosphorylation. Finally, the MAPK Sty1, but not the eIF2α kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2α kinase pathways for a particular range of stress responses. © 2010, American Society for Microbiology.


Gomez R.,Autonomous University of Madrid | Viera A.,Autonomous University of Madrid | Berenguer I.,Autonomous University of Madrid | Llano E.,Institute Biologia Molecular Y Celular del Cancer | And 4 more authors.
Chromosoma | Year: 2014

Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II. © 2013 Springer-Verlag.


Cen-Pacheco F.,University of La Laguna | Villa-Pulgarin J.A.,Institute Biologia Molecular y Celular Del Cancer | Villa-Pulgarin J.A.,University of Salamanca | Mollinedo F.,Institute Biologia Molecular y Celular Del Cancer | And 3 more authors.
European Journal of Medicinal Chemistry | Year: 2011

Three new polyether squalene derivatives 15-dehydroxythyrsenol A (2), prethyrsenol A (3) and 13-hydroxyprethyrsenol A (4) have been isolated from the red alga Laurencia viridis. Their structures were determined through the interpretation of NMR spectroscopic data and chemical correlations. In addition, four semi-synthetic compounds modulating the solubility of the lead compound dehydrothyrsiferol (1) were prepared without loss of activity. The cytotoxicity of the new compounds exhibited low μM activities. In order to explain their biological properties, docking simulations of the natural and synthetic compounds onto the αvβ3 integrin binding region were carried out. © 2011 Elsevier Masson SAS. All rights reserved.


Caburet S.,University Paris Diderot | Arboleda V.A.,University of California at Los Angeles | Llano E.,University of Salamanca | Llano E.,Institute Biologia Molecular Y Celular del Cancer | And 12 more authors.
New England Journal of Medicine | Year: 2014

Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age. Copyright © 2014 Massachusetts Medical Society.


Yokotsuka M.,Tokyo Medical University | Iwaya K.,Tokyo Medical University | Iwaya K.,National Defense Medical College | Saito T.,Juntendo University | And 5 more authors.
Breast Cancer Research and Treatment | Year: 2011

The final signal for triggering the formation of lamellipodia that initiate directional migration of mammalian cells is binding of the Wiskott-Aldrich syndrome (WASP)/WASP family verproline-homologous protein 2 (WAVE2) to the actin-related protein 2 and 3 (Arp2/3) complex. This WAVE2-Arp2/3 signal is suggested to be enhanced in some breast cancers, facilitating invasion, and/or metastasis. Here, we demonstrated one cause of the enhanced signal using four breast cancer cell lines (SKBR3, AU565, MCF7, and MDA-MB-231). The WAVE2-Arp2/3 signal was estimated semi-quantitatively by counting the number of lamellipodia expressing both WAVE2 and Arp2 using high-power confocal laser microscopy. Higher expression of the WAVE2-Arp2/3 signal was detected in SKBR3 and AU565, which have HER2 gene amplification, than in the other two cell lines that lack HER2 gene amplification. Trastuzumab suppressed both the formation of lamellipodia and migration in a Boyden chamber experiment in SKBR3 and AU565. When the HER2 gene was transfected into MCF7, the number of both lamellipodia and migrated cells was increased. This enhancement of migration did not occur in the presence of extracellular matrix, and zymographic analysis showed no clear difference between HER2 gene-transfected cells and MCF7 cells. Immunohistochemical analysis of 115 cases of breast cancer revealed that coexpression of WAVE2 and Arp2 was significantly correlated with HER2-overexpression (P < 0.0001). These data indicate that an abnormal signal resulting from HER2 gene amplification activates lamellipodia formation in breast cancer cells, which initiates their metalloproteinase-independent migration. © 2011 Springer Science+Business Media, LLC.


Marcos I.S.,University of Salamanca | Moro R.F.,University of Salamanca | Costales I.,University of Salamanca | Basabe P.,University of Salamanca | And 3 more authors.
Tetrahedron | Year: 2013

Biomimetic synthesis of 12-epi-ent-pentacyclindole 7, using as key step the cyclization of 12-epi-ent-polyalthenol acetate has been carried out. This way, the structure and absolute configuration of the natural product pentacyclindole 1 has been confirmed. The synthesized indole sesquiterpenes 7, 11 and 12 show cellular proliferation inhibition of a number of human leukaemic and solid tumour cell lines. © 2013 Elsevier Ltd. All rights reserved.


Teodosio C.,Institute Biologia Molecular y Celular del Cancer | Garcia-Montero A.C.,Institute Biologia Molecular y Celular del Cancer | Jara-Acevedo M.,Institute Biologia Molecular y Celular del Cancer | Sanchez-Munoz L.,Hospital Virgen del Valle | And 6 more authors.
Journal of Allergy and Clinical Immunology | Year: 2010

Background: Systemic mastocytosis (SM) is a heterogeneous group of disorders with distinct clinical and biological behavior. Despite this, little is known about the immunophenotypic features of the distinct diagnostic categories of SM. Objective: To analyze the immunophenotypic characteristics of bone marrow (BM) mast cells (MCs) of different subtypes of SM. Methods: Bone marrow samples from 123 patients with different subtypes of SM and 92 controls were analyzed for a broad panel of immunophenotypic markers by flow cytometry. Results: Three clearly different maturation-associated immunophenotypic profiles were found for BMMCs in SM. These different profiles were associated with both genetic markers of the disease and its clinical behavior. BMMCs from poor-prognosis categories of SM (aggressive SM and MC leukemia) typically showed an immature phenotype with clonal involvement of all myeloid lineages by the D816V stem cell growth factor receptor gene (KIT) mutation. In turn, a mature activated versus resting BMMC immunophenotype was commonly found among patients with good-prognosis subtypes of SM depending on whether they carried (indolent SM and clonal MC activation disorders) or not (well differentiated SM) the D816V KIT mutation. Conclusion: Bone marrow MCs from SM show 3 different maturation-related immunophenotypic profiles that are associated with both the genetic markers of the disease and its clinical behavior. © 2010 American Academy of Allergy, Asthma & Immunology.


Leis O.,Regulation of Cell Growth Laboratory | Eguiara A.,Regulation of Cell Growth Laboratory | Lopez-Arribillaga E.,Regulation of Cell Growth Laboratory | Alberdi M.J.,Onkologikoa | And 5 more authors.
Oncogene | Year: 2012

The cancer stem cell (CSC) model does not imply that tumours are generated from transformed tissue stem cells. The target of transformation could be a tissue stem cell, a progenitor cell, or a differentiated cell that acquires self-renewal ability. The observation that induced pluripotency reprogramming and cancer are related has lead to the speculation that CSCs may arise through a reprogramming-like mechanism. Expression of pluripotency genes (Oct4, Nanog and Sox2) was tested in breast tumours by immunohistochemistry and it was found that Sox2 is expressed in early stage breast tumours. However, expression of Oct4 or Nanog was not found. Mammosphere formation in culture was used to reveal stem cell properties, where expression of Sox2, but not Oct4 or Nanog, was induced. Over-expression of Sox2 increased mammosphere formation, effect dependent on continuous Sox2 expression; furthermore, Sox2 knockdown prevented mammosphere formation and delayed tumour formation in xenograft tumour initiation models. Induction of Sox2 expression was achieved through activation of the distal enhancer of Sox2 promoter upon sphere formation, the same element that controls Sox2 transcription in pluripotent stem cells. These findings suggest that reactivation of Sox2 represents an early step in breast tumour initiation, explaining tumour heterogeneity by placing the tumour-initiating event in any cell along the axis of mammary differentiation. © 2012 Macmillan Publishers Limited All rights reserved.


Melo-Lima S.,Institute Biologia Molecular y Celular Del Cancer | Melo-Lima S.,University of Coimbra | Lopes M.C.,University of Coimbra | Mollinedo F.,Institute Biologia Molecular y Celular Del Cancer | Mollinedo F.,Hospital Universitario Of Salamanca
Pharmacological Research | Year: 2015

Glioblastoma is characterized by constitutive apoptosis resistance and survival signaling expression, but paradoxically is a necrosis-prone neoplasm. Incubation of human U118 glioblastoma cells with the antitumor alkylphospholipid analog edelfosine induced a potent necrotic cell death, whereas apoptosis was scarce. Preincubation of U118 cells with the selective MEK1/2 inhibitor U0126, which inhibits MEK1/2-mediated activation of ERK1/2, led to a switch from necrosis to caspase-dependent apoptosis following edelfosine treatment. Combined treatment of U0126 and edelfosine totally inhibited ERK1/2 phosphorylation, and led to RIPK1 and RelA/NF-κB degradation, together with a strong activation of caspase-3 and -8. This apoptotic response was accompanied by the activation of the intrinsic apoptotic pathway with mitochondrial transmembrane potential loss, Bcl-xL degradation and caspase-9 activation. Inhibition of ERK phosphorylation also led to a dramatic increase in edelfosine-induced apoptosis when the alkylphospholipid analog was used at a low micromolar range, suggesting that ERK phosphorylation acts as a potent regulator of apoptotic cell death in edelfosine-treated U118 cells. These data show that inhibition of MEK1/2-ERK1/2 signaling pathway highly potentiates edelfosine-induced apoptosis in glioblastoma U118 cells and switches the type of edelfosine-induced cell death from necrosis to apoptosis. © 2015 Elsevier Ltd. All rights reserved.

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