http://hdl.handle.net/10171/3489 2013-05-23T23:40:19Z http://hdl.handle.net/10171/27601 Title: Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia. Author(s) : Vilas-Zornoza, A. (Amaya); Aguirre, X. (Xavier); Abizanda, G. (Gloria); Moreno, C. (Cristina); Segura, V. (Víctor); Martino-Rodriguez, A. (Alba) de; San-Jose-Eneriz, E. (Edurne); Miranda, E. (Estibaliz); Martin-Subero, J.I. (José Ignacio); Garate, L. (Leire); Blanco-Prieto, M.J. (María José); Garcia-de-Jalon, J.A. (José A.); Rio, P. (Paula); Rifon, J. (José); Cigudosa, J.C. (Juan Cruz); Martinez-Climent, J.A. (José Angel.); Roman-Gomez, J. (José); Calasanz, M.J. (María José); Ribera, J.M. (José María); Prosper, F. (Felipe) Abstract: Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. 2011-12-31T23:00:00Z http://hdl.handle.net/10171/27601 Title: Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia. Author(s) : Vilas-Zornoza, A. (Amaya); Aguirre, X. (Xavier); Abizanda, G. (Gloria); Moreno, C. (Cristina); Segura, V. (Víctor); Martino-Rodriguez, A. (Alba) de; San-Jose-Eneriz, E. (Edurne); Miranda, E. (Estibaliz); Martin-Subero, J.I. (José Ignacio); Garate, L. (Leire); Blanco-Prieto, M.J. (María José); Garcia-de-Jalon, J.A. (José A.); Rio, P. (Paula); Rifon, J. (José); Cigudosa, J.C. (Juan Cruz); Martinez-Climent, J.A. (José Angel.); Roman-Gomez, J. (José); Calasanz, M.J. (María José); Ribera, J.M. (José María); Prosper, F. (Felipe) Abstract: Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. 2011-12-31T23:00:00Z http://hdl.handle.net/10171/21568 Title: Toward the discovery of new biomarkers of hepatocellular carcinoma by proteomics Author(s) : Santamaria, E. (Enrique); Muñoz, J. (Javier); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Corrales, F.J. (Fernando José) Abstract: Primary liver cancer is the fifth most frequent neoplasm and the third most common cause of cancer-related death, with more than 500,000 new cases diagnosed yearly. The outcome for hepatocellular carcinoma (HCC) patients still remains dismal, partly because of our limited knowledge of its molecular pathogenesis and the difficulty in detecting the disease at its early stages. Therefore, studies aimed at the definition of the mechanisms associated with HCC progression and the identification of new biomarkers leading to early diagnosis and more effective therapeutic interventions are urgently needed. Proteomics is a rapidly expanding discipline that is expected to change the way in which diseases will be diagnosed, treated, and monitored in the near future. In the last few years, HCC has been extensively investigated using different proteomic approaches on HCC cell lines, animal models, and human tumor tissues. In this review, state-of-the-art technology on proteomics is overviewed, and recent advances in liver cancer proteomics and their clinical projections are discussed. 2006-12-31T23:00:00Z http://hdl.handle.net/10171/21567 Title: Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates Author(s) : Sanchez del Pino, M.M. (Manuel M.); Perez-Mato, I. (Isabel); Sanz, J.M. (Jesús M.); Mato, J.M. (José María); Corrales, F.J. (Fernando José) Abstract: Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III. 2001-12-31T23:00:00Z http://hdl.handle.net/10171/21566 Title: Chaperone activity and structure of monomeric polypeptide binding domains of GroEL Author(s) : Zahn, R. (Ralph); Buckle, A.M. (Asley M.); Perret, S. (Sarah); Johnson, C.M. (Christopher M.); Corrales, F.J. (Fernando José); Golbik, R. (Ralph); Fersht, A.R (Alan R.) Abstract: The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions. 1995-12-31T23:00:00Z http://hdl.handle.net/10171/21565 Title: Correlation between gene expression and GO semantic similarity Author(s) : Sevilla, J.L. (José L.); Segura, V. (Víctor); Podhorski, A. (Adam); Guruceaga, E. (Elizabeth); Mato, J.M. (José María); Martinez-Cruz, L.A. (L. Alfonso); Corrales, F.J. (Fernando José); Rubio, A. (Ángel) Abstract: This research analyzes some aspects of the relationship between gene expression, gene function, and gene annotation. Many recent studies are implicitly based on the assumption that gene products that are biologically and functionally related would maintain this similarity both in their expression profiles as well as in their Gene Ontology (GO) annotation. We analyze how accurate this assumption proves to be using real publicly available data. We also aim to validate a measure of semantic similarity for GO annotation. We use the Pearson correlation coefficient and its absolute value as a measure of similarity between expression profiles of gene products. We explore a number of semantic similarity measures (Resnik, Jiang, and Lin) and compute the similarity between gene products annotated using the GO. Finally, we compute correlation coefficients to compare gene expression similarity against GO semantic similarity. Our results suggest that the Resnik similarity measure outperforms the others and seems better suited for use in Gene Ontology. We also deduce that there seems to be correlation between semantic similarity in the GO annotation and gene expression for the three GO ontologies. We show that this correlation is negligible up to a certain semantic similarity value; then, for higher similarity values, the relationship trend becomes almost linear. These results can be used to augment the knowledge provided by clustering algorithms and in the development of bioinformatic tools for finding and characterizing gene products. 2004-12-31T23:00:00Z http://hdl.handle.net/10171/21564 Title: GARBAN II: an integrative framework for extracting biological information from proteomic and genomic data Author(s) : Segura, V. (Victoriano); Podhorski, A. (Adam); Guruceaga, E. (Elizabeth); Sevilla, J.L. (José L.); Corrales, F.J. (Fernando José); Rubio, A. (Ángel) Abstract: Genomic and proteomic analyses generate a massive amount of data that requires specific bioinformatic tools for its management and interpretation. GARBAN II, developed from the previous GARBAN platform, provides an integrated framework to simultaneously analyse and compare multiple datasets from DNA microarrays and proteomic studies. The general architecture, gene classification and comparison, and graphical representation have been redesigned to ensure a user-friendly feature and to improve the capabilities and efficiency of this system. Additionally, GARBAN II has been extended with new applications to display networks of coexpressed genes and to integrate access to BioRag and MotifScanner so as to facilitate the holistic analysis of users' data. 2005-12-31T23:00:00Z http://hdl.handle.net/10171/21563 Title: A signature of six genes highlights defects on cell growth and specific metabolic pathways in murine and human hepatocellular carcinoma Author(s) : Schröder, P.C. (Paul C.); Segura, V. (Víctor); Riezu-Boj, J.I. (José Ignacio); Santamaria, E. (Enrique); Corrales, F.J. (Fernando José); Sangro, B. (Bruno); Mato, J.M. (José María); Prieto, J. (Jesús) Abstract: Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Clinical Proteomics. 2010-12-31T23:00:00Z http://hdl.handle.net/10171/21562 Title: Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III Characterization of DDX3X as a target of S-adenosylmethionine Author(s) : Schröder, P.C. (Paul C.); Fernandez-Irigoyen, J. (Joaquín); Bigaud, Emilie; Serna, A. (Antonio); Hernandez-Alcoceba, R. (Rubén); Lu, S.C. (Shelly C.); Mato, J.M. (José María); Prieto, J. (Jesús); Corrales, F.J. (Fernando José) Abstract: Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Clinical Proteomics. 2011-12-31T23:00:00Z http://hdl.handle.net/10171/21561 Title: Proteomic analysis of liver diseases: molecular mechanisms and biomarker discovery Author(s) : Corrales, F.J. (Fernando José); Santamaria, E. (Enrique); Muñoz, J. (Javier); Fernandez-Irigoyen, J. (Joaquín); Sesma, L. (Laura); Odriozola, L.. (Leticia) Abstract: Liver diseases afflict more than 10% of the world population. Although the main risk factors are known and the population at risk is monitored, new biomarkers are urgently needed to allow early diagnosis and hence more effective therapeutic interventions. Here, we revise the contribution of proteomics to the study of liver diseases and its potential impact in the clinical practice is evaluated. 2007-12-31T23:00:00Z http://hdl.handle.net/10171/21560 Title: Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases Author(s) : Santamaria, E. (Enrique); Muñoz, J. (Javier); Fernandez-Irigoyen, J. (Joaquín); Sesma, L. (Laura); Mora, M.I. (María I.); Berasain, C. (Carmen); Lu, S.C. (Shelly C.); Mato, J.M. (José María); Prieto, J. (Jesús); Avila, M.A. (Matías Antonio); Corrales, F.J. (Fernando José) Abstract: S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders. 2005-12-31T23:00:00Z http://hdl.handle.net/10171/21559 Title: Identification of replication-competent HSV-1 Cgal+ strain signaling targets in human hepatoma cells by functional organelle proteomics Author(s) : Santamaria, E. (Enrique); Mora, M.I. (María I.); Potel, C. (Corinne); Fernandez-Irigoyen, J. (Joaquín); Carro-Roldan, E. (Elvira); Hernandez-Alcoceba, R. (Rubén); Prieto, J. (Jesús); Epstein, A.L. (Alberto L.); Corrales, F.J. (Fernando José) Abstract: In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest. 2008-12-31T23:00:00Z http://hdl.handle.net/10171/21558 Title: Regulation of stathmin phosphorylation in mouse liver progenitor-29 cells during proteasome inhibition Author(s) : Santamaria, E. (Enrique); Mora, M.I. (María I.); Muñoz, J. (Javier); Sanchez-Quiles, V. (Virginia); Fernandez-Irigoyen, J. (Joaquín); Prieto, J. (Jesús); Corrales, F.J. (Fernando José) Abstract: Proteasome inhibitors are potential therapeutic agents in the treatment of hepatocarcinoma and other liver diseases. The analysis of alternative protein phosphorylation states might contribute to elucidate the underlying mechanisms of proteasome inhibitor-induced apoptosis. We have investigated the response of mouse liver progenitor-29 (MLP-29) cells to MG132 using a combination of phosphoprotein affinity chromatography, DIGE, and nano LC-MS/MS. Thirteen unique deregulated phosphoproteins involved in chaperone activity, stress response, mRNA processing and cell cycle control were unambiguously identified. Alterations in NDRG1 and stathmin suggest new mechanisms associated to proteasome inhibitor-induced apoptosis in MLP-29 cells. Particularly, a transient modification of the phosphorylation state of Ser(16), Ser(25) and Ser(38), which are involved in the regulation of stathmin activity, was detected in three distinct isoforms upon proteasome inhibition. The parallel deregulation of calcium/calmodulin-activated protein kinase II, extracellular regulated kinase-1/2 and cyclin-dependent kinase-2, might explain the modified phosphorylation pattern of stathmin. Interestingly, stathmin phosphorylation profile was also modified in response to epoxomicin treatment, a more specific proteasome inhibitor. In summary, we report here data supporting that regulation of NDRG1 and stathmin by phosphorylation at specific Ser/Thr residues may participate in the cellular response induced by proteasome inhibitors. 2008-12-31T23:00:00Z http://hdl.handle.net/10171/21557 Title: Identification of replication-competent HSV-1 Cgal+ strain targets in a mouse model of human hepatocarcinoma xenograft Author(s) : Santamaria, E. (Enrique); Mora, M.I. (María I.); Carro-Roldan, E. (Elvira); Molina, M. (Manuela); Fernandez-Irigoyen, J. (Joaquín); Marconi, P. (Peggy); Manservigi, R. (Roberto); Greco, A. (Anna); Epstein, A.L. (Alberto L.); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Corrales, F.J. (Fernando José) Abstract: Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal(+) strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal(+) strain during 24h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal(+) targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal(+) infection, suggesting regulatory mechanisms based on post-translational modification events. 2008-12-31T23:00:00Z http://hdl.handle.net/10171/21554 Title: Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine Author(s) : Santamaria, E. (Enrique); Avila, M.A. (Matías Antonio); Latasa, M.U. (María Ujué); Rubio, A. (Ángel); Martin-Duce, A. (Antonio); Lu, S.C. (Shelly C.); Mato, J.M. (José María); Corrales, F.J. (Fernando José) Abstract: Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis (MAT1A(-/-)) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when MAT1A(-/-) expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at approximately 8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase beta-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in obob mice and obese patients who are at risk for nonalcoholic steatohepatitis. 2002-12-31T23:00:00Z