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Workshop Modelos Animales en Cáncer Animal Models for Cancer Coordinadores Organizers Marcos Malumbres Alberto Martín-Pendás Francisco X. Real 22-23 Octubre/October 2004 Institut Municipal d’Investigació Mèdica (IMIM), Barcelona Workshop Animal Models for Cancer 2 Workshop Modelos Animales en Cáncer Workshop Modelos Animales en Cáncer Programa Programme 22-23 Octubre/October 2004 Institut Municipal d’Investigació Mèdica (IMIM), Barcelona 3 Workshop Animal Models for Cancer Viernes 22 octubre 2004 9:15 Bienvenida-Presentación Sala Joseph Marull de L’Imas, IMIM I. Oncogenes y Transducción de Señales Chairman: ALBERTO MARTÍN-PENDÁS 9:30 ÁNGEL NEBREDA Centro Nacional de Investigaciones Oncológicas, Madrid Signal transduction and cell cycle regulation 10:00 MIRENTXU SANTOS CIEMAT, Madrid Tumorigénesis epitelial y alteraciones en el desarrollo producidas por una forma activa de Akt 10:20 CARMEN GUERRA Centro Nacional de Investigaciones Oncológicas, Madrid Tumour induction by an endogenous K-ras oncogene 10:40 MANUEL SÁNCHEZ-MARTÍN IBMCC- CSIC/Universidad de Salamanca, Salamanca Análisis del inicio y progresión de la leucemia mediante la expresión condicional del gen BCR-ABL en ratones 11:00 CARME GALLEGO Universitat de Lleida Catalunya, Lleida Opposite effects of Kis in neuronal proliferation and differentiation 11:20 Coffee break II. Ciclo Celular Chairman: ÁNGEL NEBREDA 11:45 CAYETANO GONZÁLEZ ICREA i IRBB, Parc Científic de Barcelona, Barcelona Induction of tumoral growth by altered cell fate in Drosophila 12:15 ALBERTO MARTÍN Centro Nacional de Investigaciones Oncológicas, Madrid Cell cycle inhibition and tumor suppression by p27Kip1 and p21Cip1 are independent of Cdk2 12:35 ANXO VIDAL Universidad de Santiago de Compostela La deficiencia de p27Kip1 desenmascara la función antioncogénica de p130 y p107 12:55 ANA ZUBIAGA Universidad del País Vasco, Bilbao Dissecting the unique and shared functions of E2F transcription factors 13:15 IGNACIO PÉREZ DE CASTRO Centro Nacional de Investigaciones Oncológicas, Madrid Target validation in vivo of mitotic regulators in cancer 13:45 Lunch 4 Workshop Modelos Animales en Cáncer III. Stem Cells y Tecnología Chairman: MARCOS MALUMBRES 15:30 ISIDRO SÁNCHEZ-GARCÍA IBMCC- CSIC/Universidad de Salamanca, Salamanca Of man in mouse: modelling human cancer genotype-phenotype correlations in mice 16:00 SAGRARIO ORTEGA Centro Nacional de Investigaciones Oncológicas, Madrid Developing tools to study tumor angiogenesis in the mouse 16:30 OLGA MILLÁN Instituto de Alta Tecnología, PRBB, Barcelona Imagen molecular in vivo: abordajes multimodales para el seguimiento de tumores 16:50 MICHEL HERRANZ Centro Nacional de Investigaciones Oncológicas, Madrid MRI and PET follow-up of the anti-tumoral effect of Histone Deacetylases Inhibitors (HDACIs) in a gamma-irradiated mouse lymphoma model 17:10 17:30 Coffee break LUIS A. HERRÁEZ BARANDA ZF Biolabs, Madrid Zebrafish as an experimental model in cancer 17:50 DIRK BUSCHER Genetrix S.L., Madrid Zebrafish: more than a developmental biologist's new toy 18:10 Mesa redonda: Plataformas tecnológicas y necesidades futuras Participantes: M. ÁNGELA NIETO, ISIDRO SÁNCHEZ-GARCÍA, SAGRARIO ORTEGA, CRISTINA FILLAT, ANXO VIDAL 20:30 Cena del Meeting 5 Workshop Animal Models for Cancer Sábado 23 Octubre 2004 IV. Señalización Celular y Metástasis Chairman: RAMÓN MANGUES 9:30 CRISTINA LÓPEZ-RODRÍGUEZ Centre de Regulació Genòmica, Barcelona Loss of NFAT5 in mice causes growth deficiency and lack of expression of an osmoprotective transcription program in kidney 9:50 JOSE ARAMBURU Universitat Pompeu Fabra, Barcelona Caracterización de la interacción del factor de transcripción NFAT5 con proteínas reguladoras 10:10 MARÍA PÉREZ-CARO IBMCC- CSIC/Universidad de Salamanca, Salamanca Snail overexpression in development and cancer 10:30 MANUEL SÁNCHEZ-MARTÍN IBMCC- CSIC/Universidad de Salamanca, Salamanca Slug in cancer development 10:50 ALBERTO MARTÍN-PENDÁS Instituto Universitario de Oncología del Principado de Asturias, Oviedo Dual roles of MMPs in cancer progression 11:10 Coffee break V. Ciclo Celular e Inestabilidad Cromosómica Chairman: ISIDRO SÁNCHEZ-GARCÍA 11:30 MARÍA A. BLASCO Centro Nacional de Investigaciones Oncológicas, Madrid New mouse models of telomere dysfunction 12:00 PURA MUÑOZ-CÁNOVES Centre de Regulació Genòmica, Barcelona Sequential phosphorylation of p53 by ATM and ATR kinases in response to MNNG-induced DNA damage: activation of PAI-1 gene expression by MNNG 12:20 IGNACIO MORENO DE ALBORÁN Centro Nacional de Biotecnología, Madrid C-Myc regulates cell size and ploidy but is not essential for postnatal proliferation in liver 12:40 RAÚL MENDEZ Centre de Regulació Genòmica, Barcelona Xenopus oocytes as a model to study translational control of cell cycle 13:10 JORDI SURRALLES Universidad Autónoma de Barcelona Linking chromatin and chromosome fragility: involvement of histone H2AX in the Fanconi anemia/BRCA tumour supressor pathway 6 Workshop Modelos Animales en Cáncer 13:30 Lunch VI. Modelos Preclinicos de Terapia Antitumoral Chairman: FRANCISCO X. REAL 15:00 MANUEL GUZMÁN Universidad Complutense de Madrid Cannabinoides: ¿Posibles agentes antitumorales? 15:30 RAMON MANGUES Hospital de Sant Pau, Barcelona La expresión ectópica de E-cadherina favorece el crecimiento de metástasis retroperitoneales en un modelo ortotópico de carcinoma de colon 15:50 MARIA VIRTUDES CÉSPEDES Hospital de Sant Pau, Barcelona Desarrollo de un modelo de manipulación genética ex vivo e implantación ortotópica para el estudio de capacidad metastásica de las proteinas de la famila Rho. 16:10 JOANA VISA Institut de Recerca Oncologica, Barcelona A standarized protocol applied in the animal facility (SE-IRO) for humane endpoints in mouse models for cancer research 16:30 HÉCTOR PEINADO Instituto de Investigaciones Biomédicas, Madrid A key role for ß-catenin in mouse skin carcinogenesis 16:50 Despedida 7 Workshop Animal Models for Cancer 8 Workshop Modelos Animales en Cáncer Workshop Modelos Animales en Cáncer Resúmenes Abstracts 22-23 Octubre/October 2004 Institut Municipal d’Investigació Mèdica (IMIM), Barcelona 9 Workshop Animal Models for Cancer JOSÉ ARAMBURU [email protected] Caracterización de la interacción del factor de transcripción NFAT5 con proteínas reguladoras Cristina López-Rodríguez 2, Beatriz Morancho 1, Jordi Minguillón 1 & Jose Aramburu 1. 1 2 Departament de Ciències Experimentals i de la Salut. Universitat Pompeu Fabra, Barcelona. Cancer and Differentiation Programme. Centre de Regulació Genòmica, Barcelona. NFAT5 es un factor de transcripción de mamíferos que pertenece a la familia Rel (NF- B y NFATc). Aunque NFAT5 muestra una alta homología con los NFATc a nivel de su dominio de unión a DNA, sin embargo difiere de ellos en cuanto a su regulación. NFAT5 reconoce el mismo motivo de secuencia de DNA que los NFATc pero en contraste con éstos no interacciona con AP-1 (Jun y Fos). Otra diferencia importante entre los NFATc y NFAT5 es que éste no depende de la fosfatasa calcineurina para activarse y translocar al núcleo. NFAT5 es activado por hipertonicidad y regula la expresión de genes osmoprotectores. Además, NFAT5 puede ser activado por señales transducidas por integrinas y también es inducido en respuesta a estimulación antigénica en linfocitos. La inducción de NFAT5 via TCR en linfocitos depende en parte de la activación de la calcineurina. Aparte de la función osmoprotectora de NFAT5, se desconocen en gran medida sus mecanismos de regulación así como su papel en respuesta a otros estímulos. Dado que la expresión de NFAT5 es regulada por la calcineurina en linfocitos hemos investigado si NFAT5 es capaz de regular procesos dependientes de calcineurina. Nuestros resultados indican que NFAT5 puede interaccionar con proteínas NFATc y que es capaz de potenciar la activación de éstos dependiente de la calcineurina. Este efecto es no requiere la actividad transcripcional de NFAT5, indicando que NFAT5 podría regular la función de proteínas NFATc independientemente de su propia función transcripcional. Hemos determinado que NFAT5 también puede asociarse a la calcineurina por un mecanismo distinto al utilizado por los NFATc. Además hemos iniciado la identificación de proteínas asociadas a NFAT5 para entender cómo se regula este factor y su posible papel más allá de la respuesta a hipertonicidad. 10 Workshop Modelos Animales en Cáncer 11 Workshop Animal Models for Cancer MARÍA A. BLASCO [email protected] New mouse models of telomere dysfunction Purificación Muñoz1, Raquel Blanco1, Susana Gonzalo1, Marta Garcia-Cao1, Gunnar Schotta2, Thomas Jenuwein2, Juana M. Flores3 & María A. Blasco1 1 Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain 2 Research Institute of Molecular Pathology (IMP), Vienna Biocenter, Vienna, Austria 3 Animal Surgery and Medicine Department, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain Telomeres are chromosome end-capping structures, which protect the chromosome ends from unscheduled DNA repair and degradation. Telomeres are heterochromatic domains composed of repetitive DNA (TTAGGG repeats) bound to an array of specialized proteins. The length of telomere repeats and the integrity of telomere-binding proteins are both important for telomere protection. In addition, we have recently shown that telomere length is regulated by a number of epigenetic modifications, thus pointing to a higher-order control of telomere function. First, we will describe a novel role for the Rb family of proteins in directing full heterochromatin formation. We show that mouse embryonic fibroblasts triply deficient for Rb, p107 and p130 have a decrease in methylation of pericentric DNA. Using chromatin Immunoprecipitation (ChIP), we show that tri-methylation of H4K20 by the Suv4-20h1 and Suv4-20h2 histone methyltransferases is specifically decreased at pericentric and telomeric chromatin. These defects are independent of E2F family function, as well as of the expression and localization of the Suv4-20h enzymes, indicating a direct role of the Rb family in controlling H4-K20 tri-methylation at telomeres and centromeres by these novel HMTases. These epigenetic abnormalities are accompanied by telomere and centromere defects, by errors in chromosome segregation, and by stabilization of tetraploidy. Together, these observations indicate a role for the Rb family of proteins in maintaining overall chromatin structure and in particular that of telomeres and centromeres. These findings represent a bridge between tumor suppressor function and the epigenetic definition of heterochromatin. Secondly, we will describe the generation and characterization of mice with constitutive expression of the telomere-binding protein TRF2 under the keratin 5 promoter, K5-TRF2 mice. These mice represent the first viable mouse model with altered TRF2 expression described to date. K5TRF2 mice show a remarkable phenotype in the skin consisting of hyper-pigmentation, hair loss, dry skin, as well as spontaneous pre-neoplastic lesions, all of which are reminiscent of the skin abnormalities characteristic of Xeroderma pigmentosum (XP) syndrome. At a cellular and molecular level, the skin of these mice presents a dramatic telomere shortening, loss of the telomeric G-strand overhang, as well as numerous -H2AX foci. K5-TRF2 skin is also more sensitive to UV irradiation, as indicated by increased UV-induced DNA adducts and pre-neoplastic lesions. We propose that the XPlike skin phenotypes described here for K5-TRF2 mice are the result of a combination of defective DNA repair together with short telomeres, thus pinpointing to the roles of TRF2 in the context of the organism. In addition, this new mouse model demonstrates the impact of altered TRF2 expression both on cancer and aging. 12 Workshop Modelos Animales en Cáncer 13 Workshop Animal Models for Cancer DIRK BUSCHER [email protected] Zebrafish: more than a developmental biologist's new toy Dirk Buscher Genetrix S.L., Madrid The zebrafish (Danio rerio) has entered the club of model systems several years ago, mainly due to the extremely successful first large-scale mutagenesis screens in Tübingen (Germany) and Boston (USA). Zebrafish has primarily been used by developmental biologists, who showed a high interest in this small vertebrate. Since the 1990’s the group of researchers utilizing zebrafish for their studies has grown tremendously. Techniques and tools have been refined, new ones developed, and an increased effort can been seen to combine 21st century tools with zebrafish – the era of high throughput applications. In this presentation I want to give an overview of the model system Danio rerio, its advantages and limitations. How can the model zebrafish be applied to cellular processes and diseases such as apoptosis and cancer, respectively? Can zebrafish be more than just another vertebrate model? 14 Workshop Modelos Animales en Cáncer 15 Workshop Animal Models for Cancer MARÍA VIRTUDES CÉSPEDES [email protected] Desarrollo de un modelo de manipulación genética ex vivo e implantación ortotópica para el estudio de capacidad metastásica de las proteinas de la famila Rho M.V. Céspedes1, T. Gómez2, C. Espina2, A. Boluda2, F.J. Sancho1, M. Trias1, R. Mangues1 & J.C. Lacal2 1 2 Lab. d’Investigació Gastrointestinal, Institut de Recerca, Hospital Sant Pau, Barcelona Instituto de Investigaciones Biomédicas, Madrid Uno de los mayores problemas que tiene la oncología experimental es la falta de modelos de experimentación in vivo que simulen lo más posible la respuesta de los tumores al tratamiento en pacientes. Una solución es el establecimiento de sistemas de tumores humanos implantados ortotópicamente en ratones, cuyo crecimiento simula el de su ubicación original en pacientes, y que permiten analizar la actividad antitumoral de nuevos fármacos. En este estudio se pretende poner a punto un modelo experimental que permita analizar la actividad in vivo de fármacos frente a tumores en los que las GTPasas Rho juegan un papel relevante. Para ello, se ha desarrollado un modelo que combina la manipulación genética ex vivo de células de carcinoma de colon humano, seguido de xenotrasplante ortotópico en ratón atímico. Este, nos permite estudiar las alteraciones del fenotipo tumoral (crecimiento, invasión y metástasis) y los efectos tras un tratamiento. Se han utilizado las líneas celulares derivadas de tumores colorrectales humanos, HCT-116, SW620 y DLD1, con distinta morfología y agresividad caracterizando su patrón de tumorigenicidad y metástasis y la supervivencia de los animales. Estas líneas han sido manipuladas genéticamente mediante transfección, alterando la expresión de genes silvestres que codifican para GTPasas de la familia Rho. Se ha establecido, por tanto, un modelo que combina la manipulación ex vivo con la inyección ortotópica in vivo. Este sistema facilitará el estudio de la actividad antitumoral de nuevos fármacos que actúan en las rutas de señalización de las GTPasas Rho. 16 Workshop Modelos Animales en Cáncer 17 Workshop Animal Models for Cancer GARDENIA FRESNEDA [email protected] Proteolysis as a target for cancer therapy: Cdc20 and the Anaphase Promoting Complex Gardenia Fresneda1, Irene García-Higuera2, Sergio Moreno2 & Marcos Malumbres1 1 2 Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas, Madrid Centro de Investigación del Cáncer-CSIC, Salamanca The anaphase promoting complex (APC) is a multimeric complex that ubiquitinate cyclins and other cell cycle regulators. APC activity rises abruptly at metaphase, resulting in the destruction of proteins that inhibit sister-chromatid separation. APC-dependent destruction of additional regulators initiates spindle disassembly, cytokinesis and the resetting of replication origins for the next cell-division cycle. Cdc20 and Fzr1 (also named as Cdh1) are activators and substrate-specific adaptor proteins for APC. Cdc20 activity is replaced by that of Fzr1 during mitotic exit, and the role of Fzr1 in suppressing mitotic cyclins is essential to establish the G1 phase of the cell cycle. However, this switch from Cdc20 to Fzr1 is thought to allow degradation of many additional substrates because APC/CFzr1 has been shown to have broader substrate specificity than APC/CCdc20. Amongst the regulators degraded during mitotic exit in mammalian cells are B-type cyclins, securin, the polo-like kinase 1 (Plk1), Aurora kinases, and the CENP-E motor. Since many of these substrates are putative oncogenes, both Cdc20 and Fzr1 can act as tumor suppressor proteins in cancer development. 18 Workshop Modelos Animales en Cáncer 19 Workshop Animal Models for Cancer JAVIER GALÁN [email protected] Lack of protein versus lack of function: Generation of gene-targeted conditional knock-out and kinase-dead knock-in models in the mouse Javier Galán, David Santamaría & Mariano Barbacid Experimental Oncology Group, Centro Nacional de Investigaciones Oncológicas, Madrid Cancer disease is characterized by uncontrolled cell proliferation. Cell cycle is a process very tightly regulated in eukaryotic organisms by several kinases such as the Cdk proteins (Cyclin-dependent kinase). Cdks consists of a family of Ser/Thr kinases responsible for the phosphorylation of certain key substrates which, eventually, leads to progression through the different phases of cell cycle. Completion of G1 phase and entry into S phase require the activity of at least two different types of Cdks: Cyclin D-type (Cdk4 and Cdk6) and Cyclin E-type Cdks (Cdk2). All of them phosphorylate Rb protein in order to inactivate it, allowing the release of different transcription factors. Until very recently Cdk4 and Cdk6 were considered essential for cell cycle progression. We have recently shown that single Cdk41 and Cdk62 knock out mice are viable and that primary cultures are able to survive and progress through cell cycle without either of these Cdks. However double KO animals are embryonic lethal. This embryonic lethal phenotype makes impossible testing whether these Cdks are necessary in normal cells or tumor cells in adult mice. To this end we are generating Cdk4 conditional knock out mice using Cre/loxP system in order to obtain double Cdk4/6 knock out adult animals. Using this model will also allow us to study Cdk4 as a potential therapeutical target for cancer. However, several data suggest that these knock out models do not mimic exactly therapeutic treatments, where the targeted protein is inhibited but not eliminated. To ascertain whether there are any differences in both scenarios we are also generating a conditional Cdk4 K35M mutant mouse that results in a kinase dead form of Cdk4. 1) Rane et al. Nat. Genet. 22, 44-52, 1999. 2) Malumbres et al. Cell 118, 493-504, 2004. 20 Workshop Modelos Animales en Cáncer 21 Workshop Animal Models for Cancer CARME GALLEGO [email protected] Opposite effects of Kis in neuronal proliferation and differentiation Serafí Cambray, Martí Aldea & Carme Gallego Cell Cycle Group, Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida Catalunya, Lleida As a cellular differentiation process, neurogenesis requires the precise coordination of cell cycle exit, maintenance of conditions that prevent apoptosis, and expression of a set of genes that commit the precursor cell to specific neuronal cell types. That the control of these processes is extremely important is underlined by pathologies such as neuroblastoma, a precocious cancer, or neurodegenerative illnesses, as neuronal survival also seems to depend on the ability of cells to remain fully differentiated. Our group is using cortical precursor cells from E12.5 mouse embryos two study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Published data as well as our own results suggest that FGF and BDNF signaling molecules induce completely opposite effects in cortical precursor cells. While FGF activates proliferation, BDNF behaves as a very active differentiating agent. Our hypothesis proposes that FGF and BDNF induce such disparate processes through specific activation or inhibition of nucleo-cytoplasmic localization of p27Kip1 and the activity of Kis (kinase interacting with stathmin). Our data suggest that 1) bFGF requires Kis to prevent accumulation of p27Kip1 in the nucleus and 2) Kis is essential for survival of differentiated neurons. Elucidation of the molecular mechanism whereby Kis participates in the regulation of this two process and how Kis is regulated will allow fundamental insights into the coupling of terminal mitosis and induction of the neuronal phenotype. 22 Workshop Modelos Animales en Cáncer 23 Workshop Animal Models for Cancer CAYETANO GONZÁLEZ [email protected] Induction of tumoral growth by altered cell fate in Drosophila Cayetano González Cell Division Group. ICREA & IRBB, Parc Científic de Barcelona, Barcelona Modelling cancer in Drosophila: We are starting to exploit Drosophila to learn some basic principles regarding cell proliferation and malignant growth. As a first step we have designed a model that reproduces the most significant landmarks of cancer, including genomic instability. 24 Workshop Modelos Animales en Cáncer 25 Workshop Animal Models for Cancer CARMEN GUERRA [email protected] Tumour induction by an endogenous K-ras oncogene Carmen Guerra, Nieves Mijimolles, Victoria Campuzano, Lucía Pérez* & Mariano Barbacid. Molecular Oncology Programme and Comparative Pathology Unit*, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid We have targeted a K-ras allele in mouse embryonic stem cells by incorporating the oncogenic mutation in codon 12 (K-rasV12). To control expression of this allele, we inserted a floxed transcriptional STOP cassette within the first intron. Finally, to monitor its expression, we inserted an IRES-b-geo cassette between the stop codon and polyA signal. Activation of this targeted K-rasV12 allele is carried out by a resident inducible Cre-ERT2 recombinase after exposure to 4-OHT (1). Systemic and postnatal treatment of P10 mice with 4-OHT for 6 months results in K-rasV12 oncogene expression in a significant percentage of cells ranging from 15% in lung to 85% in colon. Seven months after treatment, these mice develop multiple bronchiolo-alveolar adenomas in their lungs. No significant additional pathologies were observed. Surprisingly, pancreas and colon, two organs in which human tumours display a high frequency of K-ras mutations (90% and 50% respectively), were completely normal. These observations indicate that tumour development induced by an endogenous K-ras oncogene is highly dependent upon cellular context. Moreover, the timing of activation also appears to be critical. Expression of the targeted K-rasV12 oncogene in the developing pancreas of mid-gestation embryos (E14.5) results in the development of pancreatic intraepithelial neoplasias (PanIN) that closely recapitulate the lesions observed in human patients. These observations indicate that the existence of cells permissive for K-rasV12 transformation not only depends on the tissue of origin but on their developmental stage. These permissive cells are likely to correspond with particular cell types, most likely stem cells. The molecular bases for the permissiveness and/or resistance to KrasV12 oncogenic transformation are currently being investigated. (1) Guerra, C., Mijimolle, N., Dhawahir, A., Dubus, P., Barradas, M., Serrano, M., Campuzano, V., & Barbacid, M. (2003). Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4, 111-120. 26 Workshop Modelos Animales en Cáncer 27 Workshop Animal Models for Cancer MANUEL GUZMAN [email protected] Cannabinoides: ¿Posibles agentes antitumorales? Manuel Guzmán Universidad Complutense de Madrid Los cannabinoides de Cannabis sativa y sus derivados sintéticos pueden controlar la decisión supervivencia/muerte celular a través de sus receptores específicos CB1 (“receptor central”) y CB2 (“receptor periférico”). La administración intratumoral de cannabinoides, tanto psicoactivos (agonistas CB1) como no psicoactivos (agonistas CB2), conduce a la regresión de tumores cerebrales malignos en ratas y ratones sin efectos colaterales importantes. Ello parece estar mediado tanto por la inducción directa de apoptosis de células tumorales como por la inhibición de la angiogénesis tumoral. En el primer caso, los cannabinoides producen apoptosis de células de glioma induciendo la síntesis de novo del segundo mensajero lipídico ceramida, lo que conlleva la activación sostenida de ERK y la inhibición de Akt. En el segundo caso, los cannabinoides parecen afectar a la vía del VEGF. En biopsias de astrocitomas humanos la expresión del receptor CB2 es proporcional al grado de malignidad, lo que podría hacer a estos tumores susceptibles al tratamiento con ligandos no psicoactivos y a este receptor un posible marcador de malignidad. Además, los cannabinoides parecen ser compuestos antitumorales selectivos, ya que no suelen afectar significativamente a la viabilidad de las células normales, a las que pueden incluso proteger frente a estímulos tóxicos. Así, los cannabinoides estimulan tanto in vitro como en el cerebro in vivo la proliferación y diferenciación glial de precursores neurales, y protegen de muerte a los astrocitos mediante la activación de Akt. Aunque estos hallazgos pueden ser esperanzadores, se precisa sin duda más investigación preclínica y clínica para dilucidar si los cannabinoides podrían emplearse algún día (aparte de como paliativos) como agentes terapéuticos en oncología. 28 Workshop Modelos Animales en Cáncer 29 Workshop Animal Models for Cancer LUIS HERRÁEZ-BARANDA [email protected] Zebrafish as an experimental model in cancer Luis A. Herráez Baranda ZF Biolabs, Tres Cantos, Madrid Zebrafish is a teleost fish of 3-4 cm. long original from tropical waters. This organism has several advantages for its use as an animal model in biomedical research, of which the most relevant are its small size, a very high reproductive rate, fast embryonic development that enables high scale studies at low costs, the embryo transparency, that allows a direct observation of alterations in the phenotype, and its genomic and proteomic similarity with other vertebrates, the human being included. These characteristics make the zebrafish an excellent experimental model to decipher the genetic basis of cancer. Studies with zebrafish have been carried out introducing random mutations and localizing the mutated gene that is responsible for the observed phenotype (forward genetics), as well as blocking the expression of the genetic content and studying the resulting phenotype (reversal genetics). On the other hand, the zebrafish embryo is becoming an increasingly popular model to perform highthroughput screenings of small molecules with a putative antitumoral activity, due to its lower ethical constraints and the possibility to test the compound in a whole organism. The last advances in genomics, such as development of DNA microarrays and the zebrafish genome sequencing project, along with the efforts to obtain stem cell lines or the development of the chemical genomics approach will contribute to the growing importance of this organism in the modelling of human diseases in general and cancer in particular. 30 Workshop Modelos Animales en Cáncer 31 Workshop Animal Models for Cancer MICHEL HERRANZ [email protected] MRI and PET follow-up of the anti-tumoral effect of Histone Deacetylases Inhibitors (HDACIs) in a gamma-irradiated mouse lymphoma model Michel Herranz1, Juan Martín Caballero2,Jesús Ruíz-Cabello3, Manuel Desco4 & Manel Esteller1 1 Cancer Epigenetics Laboratory, Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029, Madrid 2 Animal Facility, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029, Madrid 3 Instituto de Estudios Biofuncionales, Universidad Complutense de Madrid, 28040 Madrid 4 Medical Imaging Laboratory. Hospital Gregorio Marañón. E-28007. Madrid Pharmacological manipulation of chromatin remodelling process by histone deacetylase inhibitors (HDACis) might develop into a potent and specific strategy for the treatment of cancer. Alterations in histone acetylation may lead to changes in chromatin structure and transcriptional deregulation of genes. We used a mouse model of radiation-induced lymphomagenesis. In this model we performed a complete and exhaustive non-invasive imaging follow-up of HDACis treatment compare to PBS treatment as a control group. Our goal is to analyse the effect of histone deacetylase inhibitors in tumor initiation and progression. One hundred animals were irradiated and nine histone deacetylases inhibitors assays were evaluated. Intraperitoneal inoculation of six drugs (Valproate, Butyrate (2 different doses), TSA, SAHA, LAQ (2 different doses) and MS275) was performed, 10 animals each (5 males and 5 females) and one control assay: PBS (Phophate Buffer Saline) inoculation. Each mouse was imaged with a periodicity of 2 weeks after the establishment of the treatment using conventional T2-weighted fast spin echo sequences and 18F-FDG-PET (Possitron Emission Tomography) follow up of tumor metabolism. Significant decrease of tumor size has been found with several HDAC inhibitors and a protective and palliative effect of HDACi in the development of thymic lymphomas was found. Nuclear magnetic resonance imaging results confirm that surviving animals present a thymus structure and volume similar to controls one. Moreover, imaging analysis by Nuclear Magnetic Resonance and PET demonstrate that non-irradiated but HDACIs treated animals, and one-year surviving animals show no differences in thymus volume, structure, density or location compare to control animals. Detailed pathological analysis discard toxicity of these compounds. HDAC inhibitors remain one of the most promising classes of new anticancer agents. Further studies are currently in progress to delineate the optimal dosage and the duration of therapy. 32 Workshop Modelos Animales en Cáncer 33 Workshop Animal Models for Cancer CRISTINA LÓPEZ-RODRÍGUEZ [email protected] Loss of NFAT5 in mice causes growth deficiency and lack of expression of an osmoprotective transcription program in kidney Cristina López-Rodríguez1,2 & Anjana Rao2 1 Centro de Regulación Genómica (CRG), Barcelona, 2The Center for Bood Research and Pathology Department, Harvard Medicval School, Boston, USA NFAT5 is a transcription factor that belongs to the Rel (NFATc/ NF- B) family of proteins. Its DNAbinding domain has features of both NF- B and NFATc proteins. Like NF- B, NFAT5 binds DNA as an obligate dimer, but recognizes the prototypical DNA sequences bound by NFATc proteins. Out of the DNA binding domain, NFAT5 differs substantially from NFATc and NF- B. NFAT5 is restricted to vertebrates. However, Drosophila, that lacks NFATc proteins, expresses dNFAT, a transcription factor whose DNA-binding domain is highly homologous to NFAT5. dNFAT has been shown to genetically interact with KSR and the Ras pathway, Ras85D or pannier, and participate in growth-related processes. Moreover, overexpression of dNFAT in the nervous system alters synapse formation and axon guidance during fly development. NFAT5 does not seem to be regulated by the same signaling pathways that control the activity of NFATc or NF- B proteins. NFAT5 is activated by hypertonicity, induced in response to mitogens in lymphocytes, and upregulated and activated through the prometastatic integrin 6/ 4 in carcinoma cells. These responses are possibly mediated by different mechanisms, which at present are poorly understood. We have analyzed the function of NFAT5 by generating a mouse model that does not express NFAT5. In the mouse, NFAT5 protein is expressed in most organs during embryonic development but its levels are very low or undetectable in most adult tissues except for abundant expression at sites with high cellular proliferation, such as thymus and testis. Deletion of NFAT5 affected both embryonic and perinatal survival. A fraction of the NFAT5-null mice survived into adulthood and revealed a striking growth phenotype with an overall reduction of body and organ size to about half of wild-type littermates. In addition, the life-span of adult NFAT5null mice is severely compromised, in part due to progressive renal atrophy as mice age, caused by the lack of an osmoprotective gene expression program at the kidney medulla and defective regeneration of a functional medullary region. The kidney phenotype of NFAT5-null mice provides a demonstration of its role in the osmoprotective response. However, it does not explain the embryonic mortality and growth defects observed, indicating that NFAT5 has additional functions connected to cell growth. 34 Workshop Modelos Animales en Cáncer 35 Workshop Animal Models for Cancer RAMÓN MANGUES [email protected] La expresión ectópica de E-cadherina favorece el crecimiento de metástasis retroperitoneales en un modelo ortotópico de carcinoma de colon Mª Virtudes Céspedes1, María Jesús Larriba2, M. Parreño1, Paloma Ordóñez-Morán2, Isolda Casanova1, Miguel Angel Pavón1, F. Joseph Sancho1, Manuel Trias1, Alberto Muñoz2 & Ramón Mangues1 1 Laboratori d’Investigació Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, Barcelona Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid 2 Hemos estudiado el efecto de la expresión ectópica de Snail y E-cadherina sobre la invasividad y la aparición de metástasis en un modelo de xenotransplante ortotópico en ratón atímico de células de carcinoma de colon humano SW480-ADH. Se han inyectado en la pared colónica tres tipos de células SW480-ADH en sendos grupos de 8 animales: a) células infectadas con un retrovirus control expresando la proteína GFP (células GFP), b) células transducidas con el gen Snail de ratón mediante infección retroviral (GFP-Snail); y c) células transfectadas establemente con el gen Ecadherina y transducidas igualmente con Snail (GFP-Snail + E-cadherina). Las células GFP y GFPSnail muestran similar capacidad invasiva y patrón de metástasis y de muerte de los animales; ello indica que Snail no potencia la capacidad metastásica en este modelo. Por el contrario, la coexpresión de Snail y E-cadherina (células GFP-Snail + E-cadherina) induce un dramático aumento del número y de la tasa de crecimiento de las metástasis retroperitoneales (pero no de las hepáticas n i ganglionares), reduciendo a la mitad la supervivencia de los animales. Estamos investigando las bases moleculares de este efecto. Una hipótesis es que la Ecadherina sea proteolizada en el contexto de las metástasis retroperitoneales, lo que podría causar la disrupción de los contactos célula-célula y la acumulación y translocación nuclear de la beta-catenina, con inducción de su actividad transcripcional. 36 Workshop Modelos Animales en Cáncer 37 Workshop Animal Models for Cancer ALBERTO MARTÍN [email protected] Cell cycle inhibition and tumor suppression by p27Kip1 and p21Cip1 are independent of Cdk2 Alberto Martín,1 Junko Odajima,1 Sarah L. Hunt,1 Pierre Dubus,2 Sagrario Ortega,1 Marcos Malumbres1 & Mariano Barbacid1 1 Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain. 2 E.A. 2406, Histologie et Pathologie Moléculaire, University of Bordeaux 2, 33076 Bordeaux, France. Genetic studies have indicated that Cdk2, a kinase thought to be essential for cell cycle progression, is dispensable for mitotic cell division (Ortega et al. Nat. Genet. 2003, Berthet et al. Curr. Biol. 2003) These observations have raised questions regarding other proposed roles for Cdk2 including mediating the tumor suppressor activities of p27Kip1 and p21Cip1 cell cycle inhibitors. Ablation of Cdk2 in p27Kip1 deficient mice does not revert organomegalia, retinal dysplasia or pituitary tumor development. Cdk2 is also dispensable for p21Cip1-induced cell cycle arrest after DNA damage. Finally, ectopic expression of p27Kip1 and p21Cip1 induces cell cycle arrest in cells lacking Cdk2. These results indicate that Cdk2 is not an essential target for either p27Kip1 or p21Cip1. Moreover, they raise a note of caution regarding the suitability of Cdk2 as a target for therapeutic intervention, at least in those tumors lacking Cip/Kip tumor suppressors. 38 Workshop Modelos Animales en Cáncer 39 Workshop Animal Models for Cancer ALBERTO MARTÍN-PENDÁS [email protected] Dual roles of MMPs in cancer progression Alberto M Pendas, Alicia Folgueras, Milagros Balbín & Carlos López-Otín Instituto Universitario de Oncología Principado de Asturias, Oviedo Over the last years, the relevance of the matrix metalloproteinase (MMP) family in cancer research has grown considerably. These enzymes were initially associated with the invasive properties of tumour cells, owing to their ability to degrade all major protein components of the extracellular matrix (ECM) and basement membranes. However, further studies have demonstrated the implication of MMPs in early steps of tumour evolution, including stimulation of cell proliferation and modulation of angiogenesis. The establishment of causal relationships between MMP overproduction in tumour or stromal cells and cancer progression has prompted the development of clinical trials with a series of inhibitors designed to block the proteolytic activity of these enzymes. Unfortunately, the results derived from using broad-spectrum MMP inhibitors (MMPIs) for treating patients with advanced cancer have been disappointing in most cases. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8(-/-)mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. On the other hand, we have generated mice deficient in MMP-19, an MMP widely expressed in human tissues which has been associated with ovulation and angiogenic processes and is deregulated in diverse pathological conditions such as rheumatoid arthritis and cancer. These mice are viable and fertile and do not display any obvious abnormalities. However, Mmp19-null mice exhibit decreased susceptibility to skin tumors induced by chemical carcinogens. Based on these results, we suggest that this enzyme plays an in vivo role in some of the tissue remodeling events associated with tumor progression. 40 Workshop Modelos Animales en Cáncer 41 Workshop Animal Models for Cancer JORGE MARTINALBO [email protected] Modelling Polo kinase function and degradation in the mouse Jorge Martinalbo & Marcos Malumbres Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas, Madrid The maintenance of genomic integrity in eukaryotic organisms depends on the error-free segregation of chromosomes during mitotic cell division. This is ensured by several checkpoint mechanisms, the inactivation of which could eventually contribute to carcinogenesis. Entry into mitosis in vertebrates is guarded by a G2/M checkpoint that can be activated by certain insults, such as chromosomal and microtubule damage. The key regulator of this prometaphase checkpoint – Chfr (Checkpoint with FHA and RING domains) – is one of the most frequently inactivated mitotic genes in human cancer, mainly via an epigenetic pathway involving promoter methylation and suggesting a role as a putative tumor suppressor. The direct target of the ubiquitin-ligase Chfr is Polo-like kinase 1 (Plk1), one of the master regulators of cell division, which also shows a deregulated expression pattern in a variety of tumors. To further investigate the in vivo roles of the CHFR and Plk1 in mammalian cell division, we are generating conditional knockout mouse models for both Plk1 and Chfr, as well as a gain-of-function knock-in model for Plk1. 42 Workshop Modelos Animales en Cáncer 43 Workshop Animal Models for Cancer RAÚL MÉNDEZ [email protected] Xenopus oocytes as a model to study translational control of cell cycle Raúl Méndez Centre de Regulació Genòmica, Barcelona Fully-grown oocytes are arrested at Prophase-I until progesterone induces resumption of meiosis. The expression of new gene products required for the orderly progression through cell cycle takes place in the absence of transcription and is driven by a complex network of translational regulation of stored maternal mRNAs. These mRNAs encode for proteins that regulate chromosome segregation and M-Phase Checkpoints, including several proto-oncogene. Therefore, the Xenopus oocyte constitutes an ideal system to study potential mRNAs targets whose translation is de-regulated during cell transformation. Mos, cyclin B1, and several other dormant mRNAs in oocytes contain short poly(A) tails (~20-40 nts), and it is only when these tails are elongated (to ~150 nts) that translation takes place. Cytoplasmic polyadenylation requires two elements in the 3’-UTR, the hexanucleotide AAUAAA, which is also necessary for nuclear pre-mRNA cleavage and polyadenylation, and the nearby cytoplasmic polyadenylation element (CPE). The CPE is bound by CPEB, a highly conserved zinc finger and RRM type RNA-binding protein. The CPE is not only necessary for cytoplasmic polyadenylation-induced translation in maturing oocytes, it also mediates translational repression (masking) in unstimulated oocytes. This event is mediated by Maskin, a protein that interacts with CPEB as well as the cap binding protein eIF-4E. The detailed analysis of the cis-acting elements present in the cyclin B family of mRNAs has allowed us to propose a global model of CPE-mediated translational regulation that can be extrapolated to explain the differential translational control of all known cytoplasmically polyadenylated mRNAs and even to predict the translational regulation of mRNAs with putative CPEs. This model is based in a combinatorial distribution of three cis-acting elements (i.e., NRE, CPE and Hexanucleotide), which recruit three trans-acting factors (i.e., Pumilio, CPEB and CPSF). The number, relative position and exact sequence of these elements determine the specific time and amount of polyadenylation, as well as the active repression of the mRNA, allowing for a very accurate control of gene expression. This approach combined with a functional screening for cytoplasmically polyadenylated mRNAs has allowed us to identify new CPE-regulated mRNAs that encode for factors controlling chromosome segregation and M-phase exit. 44 Workshop Modelos Animales en Cáncer 45 Workshop Animal Models for Cancer OLGA MILLÁN [email protected] Imagen molecular in vivo: abordajes multimodales para el seguimiento de tumores Olga Millán Instituto de Alta Tecnología, PRBB, Barcelona. La imagen molecular es una nueva disciplina en biomedicina que consiste en la representación visual, caracterización y cuantificación de los procesos biológicos que ocurren a nivel celular en un organismo vivo. Las imágenes que se obtienen reflejan mecanismos moleculares y celulares que ocurren en un entorno fisiológico auténtico. La aparición de la imagen molecular se debe al avance de la biología molecular y celular, al uso de animales transgénicos, a la creación de drogas y moléculas altamente específicas, y al rápido desarrollo de instrumentación para la imagen animal. Dentro de la imagen molecular existen distintas modalidades de detección que varían en función de las propiedades físico-químicas de los trazadores: las tecnologías PET y SPECT, respectivamente, reconstruyen las imágenes a partir de la emisión de positrones y de partículas gamma por parte del sujeto de estudio; la tecnología CT obtiene imágenes tridimensionales a partir de disparos secuenciales de Rayos X en un radio de 360º en torno al paciente; y los sistemas ópticos de imagen in vivo detectan la emisión de bioluminiscencia y/o fluorescencia, generando imágenes bidimensionales. En oncología, las nuevas tecnologías de imagen no invasiva combinadas con el uso de genes reportadores se han usado con éxito para el seguimiento de mecanismos de transducción de señales, expresión génica y terapias celulares adoptivas. No obstante, cada combinación de técnica de imagen y gen reportador tiene sus propias ventajas e inconvenientes. Los abordajes multimodales permiten la combinación de diferentes técnicas de imagen durante el transcurso de un mismo estudio, aprovechando las características y utilidades más óptimas de cada modalidad. En este trabajo se describe la construcción de una herramienta que reúne tres genes reportadores – GFP, Luciferasa y HSV-TK– y que una vez expresada de forma estable en las células tumorales, posibilita su seguimiento por diversas técnicas de imagen. También se presentan ejemplos de su funcionalidad tanto in vitro como in vivo, en un modelo experimental de cáncer de próstata. 46 Workshop Modelos Animales en Cáncer 47 Workshop Animal Models for Cancer IGNACIO MORENO DE ALBORÁN [email protected] C-Myc regulates cell size and ploidy but is not essential for postnatal proliferation in liver Esther Baena1, Alberto Gandarillas2, Mireia Vallespinós1, Jennifer Zanet2, Oriol Bachs3, Clara Redondo4, Isabel Fabregat5, Carlos Martinez-A1 and Ignacio Moreno de Alborán1 1 Department of Immunology and Oncology (DIO), Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid E-2804, Spain 2 Institut Universitaire de Recherche Clinique. Laboratoire de Dermatologie Moléculaire UPRES EA3754. F-34093, Montpellier, France 3 Departament de Biologia Cel.lular i Anatomia Patologica, Institut d'Investigacions Biomediques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, E-08036, Barcelona, Spain 4 Dept. Anatomía Patológica, Hospital Ramón y Cajal, Carretera de Colmenar Km 9, Madrid E-28034, Spain 5 Departamento de Bioquímica y Biología Molecular, Instituto de Bioquímica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain The c-Myc protein is a transcription factor implicated in the regulation of multiple biological processes including cell proliferation, cell growth, apoptosis, and metabolism. In vivo overexpression of c-myc is linked to tumor development in a number of mouse models. More recently, several studies have addressed the role of dMyc in cell and organ growth in Drosophila3-5. Whether c-Myc plays a similar role in vertebrates remains to be elucidated. Here we show that perinatal inactivation of c-Myc in liver causes disorganized organ architecture, decreased hepatocyte size, and endoreplication. Furthermore, c-Myc appears to have distinct roles in proliferation in liver. Thus, postnatal hepatocyte proliferation does not require c-Myc, whereas it is necessary for liver regeneration in adult mice. Interestingly, free radicals content is augmented and apoptosis is increased in the liver. These results show novel physiological functions of c-myc in liver development and hepatocyte proliferation and growth. 48 Workshop Modelos Animales en Cáncer 49 Workshop Animal Models for Cancer PURA MUÑOZ-CÁNOVES pura.muñ[email protected] Sequential phosphorylation of p53 by ATM and ATR kinases in response to MNNG-induced DNA damage: activation of PAI-1 gene expression by MNNG Berta Vidal, Maribel Parra, Mercè Jardí & Pura Muñoz-Cánoves Centre de Regulació Gen`mica (CRG), Barcelona-Spain The alkylating agent MNNG is an environmental carcinogen that causes DNA lesions leading to cell death. We previously demonstrated that MNNG induced the transcriptional activity of the plasminogen activator inhibitor-1 (PAI-1) gene in a p53-dependent manner. However, the mechanism(s) linking external MNNG stimulation and PAI-1 gene induction remained to be elucidated. Here, we show that ATM and ATR kinases, but not DNA-PK, which participate in DNA damage-activated checkpoints, regulate the phosphorylation of p53 at serine 15 in response to MNNG cell treatment. Using ATMdeficient cells, ATM was shown to be required for early phosphorylation of serine 15 in response to MNNG, whereas catalytically inactive ATR selectively interfered with late phase serine 15 phosphorylation. In contrast, DNA-PK-deficient cells showed no change in the MNNG-induced serine 15 phosphorylation pattern. In agreement with this, sequential activation of ATM and ATR kinases was also required for adequate induction of the endogenous PAI-1 gene by MNNG. Finally, we showed that cells derived from PAI-1-deficient mice were more resistant to MNNG-induced cell death than normal cells, suggesting that p53-dependent PAI-1 expression partially mediated this effect. Since PAI-1 is involved in the control of tumor invasiveness, our finding that MNNG induces PAI-1 gene expression via ATM/ATR-mediated phosphorylation of p53 sheds new insight on the role of these DNA damage-induced cell cycle checkpoint kinases. 50 Workshop Modelos Animales en Cáncer 51 Workshop Animal Models for Cancer ÁNGEL R. NEBREDA [email protected] Signal transduction and cell cycle regulation Ángel R. Nebreda Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid Xenopus oocytes are cells naturally arrested at the G2/M border of the first meiotic division that can enter into M-phase of meiosis upon progesterone stimulation. This process of meiotic maturation involves a number of changes in the oocyte including the dissolution of the nuclear membrane, chromosome condensation and formation of the metaphase spindle. Protein phosphorylation plays a major role in the meiotic maturation of oocytes and many signalling pathways activated during this process are also involved in somatic cell proliferation and differentiation. The process of maturation is independent of transcription, but requires translation of specific maternal mRNAs stored in the oocyte. The final steps in oocyte maturation involve the activation of maturation-promoting factor (MPF), a complex of cyclin B and the Cdk1 protein kinase that triggers entry into M-phase of the cell cycle (both in mitosis and meiosis) in all eukaryotic cells. We are interested in how signalling pathways regulate the cell cycle machinery that controls the G2/M transition and M phase progression. Our work focuses on the regulation of the Cdc25C phosphatase and the Wee1/Myt1 protein kinases, which directly control the phosphorylation and activation of Cdk1. We are investigating the interplay between MAP kinase pathways, which stimulate G2/M progression, and the cAMP-dependent protein kinase (PKA) that is critical to maintain the G2 arrest. We are also using the Xenopus oocyte system to identify new cell cycle regulators. 52 Workshop Modelos Animales en Cáncer 53 Workshop Animal Models for Cancer PALOMA ORDOÑEZ-MORÁN [email protected] 1alpha,25-Dihydroxyvitamin D3 have profound effects on the gene expression profile, differentiation and proliferation of human colon cancer cells in vitro and in vivo Ordóñez-Morán, P.; Larriba, M. J.; Pálmer, H. G.; González-Sancho, J. M. & Muñoz, A. Instituto de Investigaciones Biomédicas, Madrid Epidemiological and preclinical data indicate that vitamin D3 and its most active metabolite 1alpha,25dihydroxyvitamin D3 (1alpha,25(OH)2D3) have anticancer activity. Accordingly, clinical trials are underway using non-hypercalcemic 1alpha,25(OH)2D3 analogs (EB1089, MC903,…) against various neoplasms including colon cancer. These compounds inhibit the proliferation and promote the differentiation of human colon cancer SW480-ADH cells. Their pro-differentiation effects are linked to the induction of the expression of E-cadherin and other adhesion proteins. In addition, 1alpha,25(OH)2D3 promotes the translocation of beta-catenin from the nucleus to the plasma membrane, leading to beta–catenin-TCF-4 transcriptional activity inhibition (J. Cell Biol., 154, 369-388, 2001). We have investigated also the gene expression profiles associated with 1alpha,25(OH)2D3 treatment of SW480-ADH cells using oligonucleotide microarrays. 1alpha,25(OH)2D3 changed the expression levels of numerous genes involved in transcription, cell adhesion, DNA synthesis, apoptosis and intracellular signaling (Cancer Res., 63, 7799-7806, 2003). We performed an in vivo study in which severe immune-deficient scid mice were injected subcutaneously with SW480-ADH cells. Half the animals were treated with EB1089 and the other half with placebo. In agreement with the results obtained in cultured cells, tumour growth was inhibited by EB1089 (30%). Presently, we are analizing the effect of EB1089 on the expression of some 1alpha,25(OH)2D3-target genes in the xenografted tumors. In addition, we are studying these genes in the colon of VDR(-/-) mice, donated by Dr. Marie Demay (Massachusetts General Hospital, Boston). Our results contribute to understand the effect of 1alpha,25(OH)2D3 action in colon cancer and explain its effects on cell proliferation and phenotype, supporting the therapeutic and prevention role of the non-hypercalcemic derivatives of 1alpha,25(OH)2D3 in colon cancer. 54 Workshop Modelos Animales en Cáncer 55 Workshop Animal Models for Cancer SAGRARIO ORTEGA [email protected] Developing tools to study tumor angiogenesis in the mouse Javier Martín, Inés Martínez, Jaime Muñoz, Marta Riffo, Carmen Gómez & Sagrario Ortega Biotechnology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid One of the interests of our lab is to generate tools for the study of cancer in vivo using the mouse as a genetic model. In the past few years, conditional gene targeting in murine embryonic stem (ES) cells has proven to be extremely valuable to reproduce in the mouse the same genetic lessions associated with cancer in humans, and therefore to create genetic models of this disease. Moreover, this technology has also been used to express a reporter protein under the control of the endogenous regulatory sequences of a gene of choice, for the purpose of monitoring its expression, and as a consequence, monitoring a biological process associated with it. We are combining both approaches to study tumor angiogenesis in vivo using the mouse as a model system. It is well documented that neovascularization plays a role in the growth, invasion and metastatic spread of solid tumors, and antiangiogenic drugs can potentially be used as anticancer therapies. We have chosen genes that are expressed especifically in endothelial cells to co-express fluorescent/bioluminescent proteins using a gene targeting strategy to create bicistronic mRNAs. These knockin reporter lines are then crossed with gene targeted mice that are predisposed to develop solid tumors, in order to monitor the angiogenesis associated to the growth and expansion of these tumors in vivo and their response to anti-angiogenic drugs. We are also generating knockin lines to induce mutations (gene activation or inactivation) specifically in endothelial cells by expressing a CreERT2 fusion protein under the control of endogenous regulatory sequences of endothelial cell specific genes, and study the efect of these mutations in tumor angiogenesis and growth. 56 Workshop Modelos Animales en Cáncer 57 Workshop Animal Models for Cancer HECTOR PEINADO [email protected] A key role for ß-catenin in mouse skin carcinogenesis Héctor Peinado, Joerg Huelsken, Walter Birchmeier & Amparo Cano Instituto de Investigaciones Biomédicas, Madrid ß-catenin plays a key role in vertebrates in cadherin-mediated cell adhesion and is also implicated in diverse signaling pathways both in development and in adult tissues (1). Multiple reports have shed light into the mechanisms of ß-catenin regulation and function and have illustrated that this protein is a central player in Wnt signaling pathway (2). Importantly, deregulation of the Wnt signaling and/or the loss of cell-cell adhesion have been involved during tumour progression. In consequence, alterations in adhesion and migration are characteristics of tumour cells that ignore normal regulatory signals from their environment (3). In mouse epidermis stabilized ß-catenin overexpression has been related to tumour formation (4), indicating the importance of ßcatenin control during epidermal keratynocyte differentiation. To get further insights into the implication of ß-catenin in tumorigenesis we applied twostage carcinogenesis protocol knockout mice bearing conditional mutation of the ß-catenin gene in the epidermis and hair follicles of the mouse using keratin14-driven Cre/loxP technology (5). We have shown that ß-catenin deletion in stem epidermis cells completely blocks tumour formation. Moreover, ß-catenin signalling pathway is active in papillomas formed in control mice, indicating a putative collaboration between H-ras mutation and ß-catenin in mouse epidermis tumour progression. All this data, suggest a key role ß-catenin in mouse epithelial carcinogenesis although further studies must be done to understand the molecular mechanisms involved. 1. Nelson, W. J., R. Nusse, Science 303, 1483 (2004). 2. Huelsken, J., W. Birchmeier, Curr. Opin. Genet. Dev. 11, 547 (2001). 3. Polakis, P., Curr. Opin. Genet. Dev. 9, 15 (1999). 4. Gat, U. et al., Cell 95, 605 (1998). 5. Huelsken, J. et al., Cell 105, 533 (2001). 58 Workshop Modelos Animales en Cáncer 59 Workshop Animal Models for Cancer MARÍA PÉREZ CARO [email protected] Snail overexpression in development and cancer Manuel Sánchez-Martín 1,5, Pedro Antonio Pérez-Mancera1, María Pérez-Caro1, Inés GonzálezHerrero1, Teresa Flores2, Alberto Orfao3, Alfonso Gutiérrez-Adán4, Belén Pintado4, and Isidro Sánchez-García1 1 Laboratorio 13, Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Campus Unamuno, 37007 Salamanca , 2 Servicio de Anatomía Patológica, Universidad de Salamanca 3 Servicio de Citometría, Universidad de Salamanca, 4 Area de Reproducción Animal, Centro de Investigación y Tecnología, Ctra de la Coruña km 5.9, 28040-Madrid 5 Departamento de Medicina, Universidad de Salamanca The Snail zinc-finger transcription factor triggers epithelial-mesenchymal transitions (EMTs) during embryonic development and cancer progression(1-4). Indeed, Snail mutant mice die at gastrulation due to a defective EMT and maintained E-cadherin expression(4). However, relatively little is known about the consequences of Snail overexpression in malignancy. To investigate the potential role of Snail overexpression in development and in cancer, we generated mice carrying a tetracyclinerepressible Snail transgene. These mice show morphological alterations and develop both epithelial and mesenchymal tumours (leukaemia and sarcomas) in almost all cases examined. Suppression of the Snail transgene did not rescue the malignant phenotype. Overall, the findings demonstrate a specific and critical role for Snail in the pathogenesis of cancer. 1. Cano, A., et al. The transcription factor Snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nature Cell Biol. 2, 76-83, 2000. 2. Batlle, E., et al The transition factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature Cell Biol. 2, 84-89, 2000. 3. Carver EA, et al._The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition. Mol Cell Biol. 2001 4. Palmer, H. et al The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer. Nature Med. 2004 60 Workshop Modelos Animales en Cáncer 61 Workshop Animal Models for Cancer IGNACIO PÉREZ DE CASTRO [email protected] Target validation in vivo of mitotic regulators in cancer Ignacio Pérez de Castro & Marcos Malumbres Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid During mitosis, correct chromosome segregation is an essential process which deregulation has recently been associated with cancer. We have started an in vivo approach to analyze the therapeutic potential in cancer treatment of some mitotic regulators such as Aurora kinases. These molecules are frequently overexpressed in a variety of human tumors and, more importantly, their deregulation seems to provoke mitotic catastrophe and apoptosis in vitro. In fact, that is the mechanism used by many of the current drugs such as paclitaxel. One of our goals will include the development of mouse models for the inactivation of the Aurora-A kinases by generating a conditional knock out for each one of the three Aurora genes. Our second objective consists on the generation of a knock in mouse that will express a non-degradable form of these proteins, and therefore resistant to its proteolytic degradation in mitosis. These new models are being generated using a modification of the standard conditional gene-targeting strategies. In both knock out and knock in models, expression of the modified allele will be detected in vitro and in vivo by the concomitant expression of a color marker through a bicistronic RNA. Conditional knock out models will express the lacZ gene instead of the targeted exon using Cre-mediated inversion (in collaboration with N.B. Ghyselinck and P. Chambon, Strasbourg). Similarly, knock in models will express the lacZ marker using an IRES sequence inserted in the 3'-UTR of the modify allele. These two modifications will allow us to label knock out and knock in cells after conditional inactivation or activation of the targeted gene; an essential tool to analyze in vivo the role of these alterations in cancer development. The information derived from this work will be crucial for a better knowledge of the function of Aurora kinases and their role in tumor progression. In addition, both mouse models for loss-of-function and gain-of-function will be a valuable tool to improve current therapeutic strategies in cancer, frequently directed against mitotic progression. 62 Workshop Modelos Animales en Cáncer 63 Workshop Animal Models for Cancer ISIDRO SÁNCHEZ-GARCÍA [email protected] Of man in mouse: modelling human cancer genotype-phenotype correlations in mice Isidro Sánchez-García Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Salamanca To date many genetic changes have been described and reported in the cancer process. Since the first mutations were described, several attempts to establish genotype-phenotype correlations for these genetic alterations have been reported. Moreover, in vitro data have suggested effects of mutant proteins in proliferation. Genotype-phenotype correlations are not only important for predicting the clinical course of the disease and to allow tailor-made surveillance of individuals at risk, but also have implications for the elucidation of the molecular genetic mechanisms underlying genesis of cancer and the development of gene-based therapies. Here, we discuss genotype-phenotype correlation of cancer in mouse and man, and the functional aspects that may account for these observations important to both understand and treat the human disease process. 64 Workshop Modelos Animales en Cáncer 65 Workshop Animal Models for Cancer MARÍA SÁNCHEZ-MARTÍN [email protected] Análisis del inicio y progresión de la leucemia mediante la expresión condicional del gen BCR-ABL en ratones 1 1 *, Manuel Sánchez-Martín2, Teresa Flores3, Alberto Orfao4, Gutierrez-Cianca María Pérez-Caro *, Noelia 5 5 1 Alfonso Gutiérrez-Adán , Belén Pintado & Isidro Sánchez-García 1 Laboratorio 13, Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Campus Unamuno, 37007-SALAMANCA (SPAIN). 2 Departamento de Medicina, Universidad de Salamanca 3 Servicio de Anatomía Patológica, Universidad de Salamanca 4 Servicio de Citometría, Universidad de Salamanca. 5 Area de Reproducción Animal, Centro de Investigación y Tecnología, Ctra de la Coruña km 5.9, 28040Madrid. La consecuencia más frecuente de las traslocaciones cromosómicas en el cáncer humano es la fusión génica. La traslocación t(9;22)(q34;q11) puede dar lugar a la proteína BCR-ABLp190 (p190) que se asocia a LLA-B. Los modelos de ratón clásicos han demostrado la capacidad tumorogénica de estos genes de fusión pero no han permitido estudiar cuestiones de gran relevancia en el cáncer humano, debido a que no permiten modular la expresión génica, como por ejemplo, el papel de estos genes de fusión en el inicio y el mantenimiento del tumor o la influencia de la cantidad de producto de fusión en el desarrollo tumoral. Por esta razón, hemos desarrollado un sistema de regulación de la expresión génica basado en el operón de resistencia a tetraciclina de E.coli que reúne en un única unidad transcripcional todos los elementos necesarios para modular la expresión del gen de fusión p190. Este sistema nos ha permitido desarrollar un modelo de ratón fisiológicamente relevante (CombitTAp190), que expresan el gen de fusión de manera dependiente de la adición de doxiciclina en el agua de bebida. Utilizando este modelo hemos podido responder cuestiones de enorme transcendencia para el entendimiento del cáncer y su tratamiento: i) la expresión del gen de fusión durante el desarrollo embrionario es capaz de iniciar la leucemia; ii) una vez iniciado el tumor, el mantenimiento del mismo es independiente del p190 ya que impone a la células un destino tumoral, cuestionando su utilización como dianas terapéuticas. Castellanos A, et al. A BCR-ABL(p190) fusion gene made by homologous recombination causes Bcell acute lymphoblastic leukemias in chimeric mice with independence of the endogenous bcr product. Blood. 1997 Huettner CS, et al Reversibility of acute B-cell leukaemia induced by BCR-ABL1.Nat Genet. 2000 Jan 66 Workshop Modelos Animales en Cáncer 67 Workshop Animal Models for Cancer MANUEL SÁNCHEZ-MARTÍN [email protected] Slug in cancer development Pedro Antonio Pérez-Mancera1*, Inés González-Herrero1*, María Pérez-Caro1, Noelia GutiérrezCianca1, Teresa Flores2, Alfonso Gutiérrez-Adán3, Belén Pintado3, Manuel Sánchez-Martín 1,4 and Isidro Sánchez-García1 1 Laboratorio 13, Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Campus Unamuno, 37007-SALAMANCA (SPAIN). 2 Servicio de Anatomía Patológica, Universidad de Salamanca. 3 Area de Reproducción Animal, Centro de Investigación y Tecnología, Ctra de la Coruña km 5.9, 28040-Madrid. 4 Departamento de Medicina, Universidad de Salamanca The SNAIL-related zinc-finger transcription factor, SLUG (SNAI2), is critical for the normal development of neural crest-derived cells and loss of-function SLUG mutations have been proven to contribute to piebaldism and Waardenburg syndrome type 2 in a dose-dependent fashion(1-5). While aberrant induction of SLUG has been documented in cancer cells, relatively little is known about the consequences of SLUG overexpression in malignancy(6). To investigate the potential role of SLUG overexpression in development and in cancer, we generated mice carrying a tetracycline-repressible Slug transgene. These mice were morphologically normal at birth, and developed mesenchymal tumours (leukaemia and sarcomas) in almost all cases examined. Suppression of the Slug transgene did not rescue the malignant phenotype. Furthermore, the BCR-ABL oncogene, which induces Slug expression in leukaemic cells, did not induce leukaemia in Slug-deficient mice, implicating Slug in BCR-ABL leukaemogenesis in vivo. Overall, the findings indicate that while Slug overexpression is not sufficient to cause overt morphogenetic defects in mice, they demonstrate a specific and critical role for Slug in the pathogenesis of mesenchymal tumours. 1. Nieto M.A., et al. Control of cell behavior during vertebrate development by Slug, a zinc finger gene. Science (1994). 2. Sefton M., et al. Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo. Development (1998). 3. Nieto, M.A. The Snail superfamily of zinc finger transcription factors. Nat. Rev. Mol. Cell. Biol. (2002). 4. Sanchez-Martin M., et al. SLUG (SNAI2) deletions in patients with Waardenburg disease. Hum Mol Genet. (2002). 5. Sanchez-Martin M et al. Deletion of the SLUG (SNAI2) gene results in human piebaldism. Am-JMed-Genet. (2003). 68 Workshop Modelos Animales en Cáncer 69 Workshop Animal Models for Cancer MIRENTXU SANTOS [email protected] Tumorigénesis epitelial y alteraciones en el desarrollo producidas por una forma activa de Akt Mirentxu Santos, Carmen Segrelles, Sergio Ruiz, Mª Fernanda Lara & Jesús M. Paramio Biología Molecular Celular, CIEMAT. Madrid La vía de señalización de PI3K/Pten/Akt tiene un papel preponderante en el cáncer, ya que su activación aumenta la proliferación y disminuye la apoptosis, convirtiéndose así en un atractivo blanco de intervención terapéutica. Sin embargo, se necesitan modelos animales para validar este aspecto. Nuestros datos demostraron que Akt desempeña un papel fundamental en la transducción de señales en la carcinogénesis de piel de ratón (Oncogene 21, 53-56, 2002), y la implicación de Akt en la angiogénesis tumoral (Carcinogénesis 25 1137-1147, 2004). Nuevos datos revelan que Akt transforma las células epiteliales por mecanismos específicos transcripcionales y post-traduccionales. Para conocer las funciones de Akt in vivo hemos generado un modelo transgénico en el que una forma permanentemente activa de Akt (myrAkt) se expresa en la capa basal de epitelios estratificados (K5myrAkt). En los animales obtenidos los niveles de expresión del transgén son de 0, 5 a 1 veces los niveles de Akt endógenos, y la actividad quinasa de 3 a 7 veces mayor. Se han observado además de alteraciones en el desarrollo del pelo, uñas y dientes, la aparición esporádica de tumores epidérmicos y de otros tejidos de origen epitelial. Con una penetrancia casi total, desarrollan lesiones en el epitelio oral (labios, paladar, lengua y mucosa oral) que desembocan en la malignización y aparición de carcinomas. Estos datos sugieren que Akt juega un papel en la tumorigénesis epitelial y en el desarrollo de derivados ectodérmicos. Los animales K5myrAkt pueden representar un modelo informativo para el conocimiento del proceso tumorigénico en la cavidad oral, pudiéndose emplear en el futuro como método de análisis preclínico para drogas o ensayos experimentales. 70 Workshop Modelos Animales en Cáncer 71 Workshop Animal Models for Cancer JORDI SURRALLÉS [email protected] Linking chromatin and chromosome fragility: involvement of histone H2AX in the Fanconi anemia/BRCA tumour supressor pathway Jordi Surrallés Universidad Autónoma de Barcelona, Barcelona Fanconi anemia (FA) is a rare genetic disease characterized by chromosome fragility, bone marrow failure, congenital abnormalities and high predisposition to cancer. To get further insights into the molecular biology of FA, here we studied the dynamics and genetic regulation of FANCD2 relocation to DNA damage in nuclei locally exposed to ultraviolet radiation (UVR). Here we show that UVR induces FANCD2 phosphorylation and monoubiquitination in a timing consistent with the dynamics of its relocation to the site of damage. This FANCD2 relocation strictly depends on FANCA, FANCD2 K561, BRCA1 and ATR but is independent of ATM, suggesting that the whole FA/BRCA pathway is activated in response to UVR. Interestingly, FANCD2 relocation to the UVR-induced damage requires one of the ATR kinase substrate, the histone variant H2AX, as shown in H2AX-/- MEFs, although H2AX is not required for FANCD2 activation. Thus, both posttranslational modifications of FANCD2 are necessary but not sufficient for FANCD2 functioning at the site of DNA damage. In addition, the dynamics of H2AX phosphorylation at the site of damage is identical to the dynamics of FANCD2 relocation to the same site and H2AX-/- MEFs show an excess of chromatid-type chromosomal aberrations after treatment with DNA cross-linkers. All these observations suggest an involvement of histone H2AX in the FA/BRCA pathway downstream FANCD2 activation and therefore provide evidence for a novel link between chromatin structure and chromosome stability in tumor suppression. Our observations are consistent with the notion that histone H2AX could be the organizer of a network of tumour suppression pathways (Surralles et al., 2004). Surrallés J, Jackson SP, Jasin M, Kastan MB, West SC and Joenje H (2004) Molecular cross talk among chromosome fragility syndromes. Genes Dev. 18, 1359-1370. 72 Workshop Modelos Animales en Cáncer 73 Workshop Animal Models for Cancer JELENA UROSEVIC [email protected] Generation of a B-Raf conditional knock-in mouse tumor model Jelena Urosevic & Mariano Barbacid Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain The Raf serine/threonine kinases (B-Raf, c-Raf and A-Raf) are thought to a play key role in the conserved Ras/Raf/MEK/ERK signal transduction pathway integrating the mitogenic signals of Ras proteins into the ERK kinases, the key effectors of this pathway. In normal cells, Ras proteins activate B-Raf by binding to its Ras binding domain which in turn activates its kinase activity. Recently it has been shown that B-Raf is mutated in 66% of human malignant melanomas and in 15% of human colorectal cancers. The most common mutation (80%), V599E replaces a valine residue located within the kinase domain, by a glutamic acid. As a result, B-raf is thought to be constitutively activated, thus sending mitogenic signals to the ERK pathway regardless of any upstream signalling. We have decided to develop a conditional knock-in mouse model for the V599E mutation in order to study the role of B-raf in tumor development. To generate this mouse model we have been constructed two targeting vectors. One of them targets the B-Raf allele in mouse embryonic stem cells by replacing the normal exon 16 by sequences carrying the oncogenic V599E mutation. Expression of this oncogenic allele will be controlled by a floxed (loxP sites) STOP cassette inserted within intron 15. The mutated exon has also been flanked with Frt sites to allow the possibility to knock out this oncogenic allele once the tumor has developed by expressing the Flp recombinase. This strategy will allow us to study tumor dependence on B-Raf expression during different steps of tumor development. Finally, to monitor the expression of the oncogenic allele by a colour marker, a second targeting vector has been used to incorporate an IRES-beta-geo cassette between the translational stop codon and the polyA signal, a strategy that has been previouisly used in our laboratory to successfully express a conditional K-ras oncogene (Guerra et al., Cancer Cell, 4, 111-120, 2003). 74 Workshop Modelos Animales en Cáncer 75 Workshop Animal Models for Cancer MIREIA VALLESPINÓS [email protected] Identification of c-Myc target genes in vivo Mireia Vallespinós, Esther Baena & Ignacio Moreno de Alborán Departamento Inmunología y Oncología (Centro Nacional de Biotecnología), Madrid The c-Myc protein is a transcription factor implicated in the regulation of multiple biological processes including cell proliferation, cell growth, apoptosis, and metabolism. C-Myc binds to consensus sequences (E-boxes) found on target genes and regulate the transcription of these genes. Functional inactivation of c-Myc in the germline leads to embryonic lethality at day 9.5. To study cmyc function in the bone marrow, we bred our previously described c-myc flox/flox conditional mouse with mx-cre transgenic mouse (c-myc flox/flox; mx-cre+). These mice show a dramatic phenotype in bone marrow characterized by abnormal hematopoiesis and death of myeloid and lymphoid lineages. We used microarray technology to identify and characterize potential c-Myc target genes involved in the regulation of hematopoiesis of c-myc flox/flox; mx-cre+ mice. Total RNA from bone marrow cells of cmyc flox/flox; mx-cre+ and control mice, was hybridized into microarrays that contained about 12,000 genes and ESTs. A total of 104 genes showed differential expression in c-myc flox/flox; mx-cre+ mice compared to the expression in their control littermates. We are currently validating these results. 76 Workshop Modelos Animales en Cáncer 77 Workshop Animal Models for Cancer ANXO VIDAL [email protected] La deficiencia de p27Kip1 desenmascara la función antioncogénica de p130 y p107 Gloria Martínez1, Nancy Yeh2, David Shaffer2, Lawrence Frohman3, Andrew Koff2 & Anxo Vidal1,2 1 Dept. Fisioloxía, Univ. Santiago de Compostela, Santiago de Compostela Dept. Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York 3 Section of Endocrinology, University of Illinois, USA 2 En el presente trabajo examinamos genéticamente la redundancia funcional entre el inhibidor de CDK, p27Kip1, y los miembros de la familia de retinoblastoma, p130Rb2 y p107. Para ello generamos y caracterizamos cepas de ratones con combinaciones de mutaciones para estos loci. La ausencia de p130 aceleró la aparición y aumentó la frecuencia de adenomas hipofisarios y feocromocitomas característicos de la cepa p27(-/-). Este fenotipo se asoció con un aumento del índice proliferativo, lo que sugiere que p27 y p130 cooperan inhibiendo la proliferación, al menos en estos dos órganos. Significativamente, los ratones del genotipo p107(+/-);p130(-/-);p27(-/-) presentaron supervivencia reducida, con una elevada incidencia de neoplasias. Más aún, la heterozigosidad de p107 aceleró el desarrollo de los tumores hipofisarios, además de observarse un mayor espectro tumoral. E stos resultados ponen de manifiesto la función supresora de tumores de p130 y p107 in vivo, y sugieren un papel parcialmente redundante de estos inhibidores del ciclo celular en la prevención del desarrollo tumoral de diversos órganos. Trabajo financiado por Ministerio de Ciencia y Tecnología, Xunta de Galicia y Cultek S.L. 78 Workshop Modelos Animales en Cáncer 79 Workshop Animal Models for Cancer JOANA VISA [email protected] A standarized protocol applied in the animal facility (SE-IRO) for humane endpoints in mouse models for cancer research J. Visa, M. González & D. Solanes Servei d’Estabulari. Institut de Recerca Oncológica, Hospital Duran y Reynals The incidence of cancer in the world is projected to continue to increase. According to the World Health Organization, cancer causes 6 million deaths every year which represents 12% of worldwide deaths. In the research field, laboratory animals have been extensively used as experimental models to increase our understanding about the causes of cancer and to develop improved treatments (10% of all mice used for research purposes are related to oncological research). When these studies involve the intentional induction of cancer in the animal model, the potential for the animal to experience discomfort or distress exists, and thus, it justifies a special consideration from people responsible of their use and welfare. The heterogeneity of scientific objectives and experimental tumor models used in our Cancer Research Institute and the compliance with regional and European regulations, has risen the need to design a protocol to monitor the animals. The protocol has to be useful for all the experimental procedures, regardless of their nature, and has to be approved by the Animal Experimental Commission from the Generalitat de Catalunya. To establish appropriate management systems to monitor animals for the onset of tumor-associated disease we assess for paraneoplastic conditions (such as cachexia) and tumor development (such as tumor distension, ulceration, size). According to the ILAR Guidelines, we have developed a standardized protocol to monitor the animals which has led to a marked refinement of humane endpoints. This presentation will address some of the main issues associated with developing scoring systems and protocols to monitor the animals used in cancer research. 80 Workshop Modelos Animales en Cáncer 81 Workshop Animal Models for Cancer ANA M. ZUBIAGA [email protected] Dissecting the unique and shared functions of E2F transcription factors Iglesias, A.; Infante, A.; García-Aranaga, I.; Laresgoiti, U.; Fullaondo, A.; Bernales, I.; Vicario, A. & Zubiaga, A.M. Dept. Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Bilbao, Spain The significance of the Rb/E2F pathway in the control of cell proliferation is illustrated by the finding that the genes that control E2F activity are perturbed in the majority of human cancers. E2F transcription factors heterodimerize with DP proteins to form a DNA-binding transcriptional activator, and regulate the expression of genes involved in DNA replication and cell cycle control. Although much has been learned about the biological properties of the E2F transcription factors, the precise roles of each individual member, as well as the level of redundancy among them, need to be resolved. Our analyses with mouse strains carrying targeted mutations for E2F1 and E2F2 have demonstrated that these genes perform specific as well as overlapping biological functions, implying that each E2F regulates a distinct set of target genes. E2F1(-/-) mice display defective thymocyte apoptosis, increased tumor susceptibility, testicular atrophy and exocrine gland dysplasia. E2F2(-/-) mice exhibit increased proliferation of hematopoietic cells and frequently develop autoimmunity and tumors. E2F1/E2F2 compound-mutant mice develop non-autoimmune diabetes and exocrine pancreatic dysfunction, suggesting that E2F1 and E2F2 share a role in normal pancreatic function. To identify gene expression profiles that characterize the biochemical pathways regulated by each E2F member, we are carrying out genomic as well as proteomic analyses of gene expression, employing DNA microarray technology and mass spectrometry. As a complementary approach to these global studies, we are performing chromatin immunoprecipitation assays and promoter analyses of E2F-responsive genes, which allows us to examine the binding of each E2F to their target promoters, as well as the transcriptional activity of these promoters under several physiological conditions. 82 Workshop Modelos Animales en Cáncer 83 Workshop Animal Models for Cancer 84 Workshop Modelos Animales en Cáncer Workshop Modelos Animales en Cáncer Lista de Participantes List of Participants 22-23 Octubre/October 2004 Institut Municipal d’Investigació Mèdica (IMIM), Barcelona 85 Workshop Animal Models for Cancer 86 Workshop Modelos Animales en Cáncer Ibane Abasolo Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Maurici Brunet Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Jaume Adan LBI, Merck, Farma y Quimica,S.A. Barcelona [email protected] Dirk Buscher Genetrix S.L., Madrid [email protected] Maria Luisa Campos Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Andrea Anfosso Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Carmen Carneiro Dept. de Fisioloxia, Universidad de Santiago de Compostela, Santiago de Compostela [email protected] José Aramburu Universitat Pompeu Fabra, Barcelona [email protected] Víctor M. Arce Fisiología, Universidad de Santiago de Compostela, Santiago de Compostela [email protected] Claudia Cases LBI, Merck Farma y Quimica, Barcelona [email protected] Lydia Castro Fisiología, Universidad de Santiago de Compostela, Santiago de Compostela [email protected] Esther Baena Chaparro 411 Centro Nacional de Biotecnología (CNB)CSIC, Madrid [email protected] María Virtudes Céspedes Lab. Investigació gastrointestinal, Hospital de Sant Pau, Barcelona [email protected] Lucía Barrado Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Ester Civit Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Miguel Beato Expresión Génica, Centre de Regulació Genòmica, Barcelona [email protected] Indira Coronel Grupo de Aplicaciones Biomédicas de la RMN, Universidad Autónoma de Barcelona, Barcelona [email protected] Marina Benito-Vicente Biomolecules Structure, Instituto de Investigaciones Biomédicas "Alberto Sols", Madrid [email protected] Jose A. Costoya Fisiología, Universidad de Santiago de Compostela, Santiago de Compostela [email protected] Carmen Blanco-Aparicio Grupo de Diseño de Ensayos, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Natàlia Dave IMIM, Universitat Pompeu Fabra, Barcelona [email protected] María A. Blasco Telomerase and telomeres Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Guillermo de Cárcer Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] 87 Workshop Animal Models for Cancer Carme Gallego Cell cycle, Universitat de Lleida, Lleida [email protected] Maxy Bernard De los Santos Delgado Unidad de Regulación de la Expresión Génica , UAM-CSIC Madrid [email protected] Antonio García de Herreros Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Mireia Duñach Dept. de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, Bellaterra (Barcelona) [email protected] Gabriel Gil-Gómez Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Laura España LBI, Merck, Farma y Quimica S.A., Barcelona [email protected] Cayetano González ICREA i IRBB, Parc Científic de Barcelona, Barcelona [email protected] Lluis Espinosa Regulacio Transcripcional, Institut Recerca Oncologica, Barcelona [email protected] Carmen Guerra Experimental Oncology Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Myriam Fabre In Vitro Cell Technologies, Barcelona [email protected] Vanessa Fernandez Regulacio Transcripcional, Institut Recerca Oncologica, Barcelona [email protected] Manuel Guzmán Universidad Complutense de Madrid, Madrid [email protected] Luis A. Herráez Baranda R&D department, ZF Biolabs, Tres Cantos (Madrid) [email protected] Gonzalo Fernández-Miranda Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Michel Herranz Epigenética del Cáncer, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Cristina Fillat Centre de Regulació Genòmica, Barcelona [email protected] Marta Herreros Instituto Biomedicina, Universidad de Leon, León [email protected] Clara Francí Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Pilar López-Larrubia SIERMAC, Instituto de Investigaciones Biomédicas "Alberto Sols", Madrid [email protected] Gardenia Fresneda Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Cristina López-Rodríguez Centre de Regulació Genòmica, Barcelona [email protected] Javier Galán Experimental Oncology Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Marcos Malumbres Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] 88 Workshop Modelos Animales en Cáncer Ramón Mangues Bafalluy Lab. Investigació Gastrointestinal, Hospital de Sant Pau, Barcelona [email protected] Ángel R. Nebreda Cell Signalling and Cell Cycle Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Alberto Martín Experimental Oncology Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] M. Ángela Nieto Instituto Cajal, Madrid [email protected] Alberto Martín-Pendás Instituto Universitario de Oncología Principado de Asturias, Oviedo [email protected] David Olmeda 1.13, Instituto de Investigaciones Biomédicas, Madrid [email protected] Jorge Martinalbo Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Paloma Ordóñez-Morán Endocrinología Molecular, Instituto de Investigaciones Biomédicas, Madrid [email protected] Sagrario Ortega Transgenic Unit, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Isabel Martinez-Lacaci Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche (Alicante) [email protected] Hector Peinado 1.13, Instituto de Investigaciones Biomédicas, Madrid [email protected] Raúl Mendez Centre de Regulació Genòmica, Barcelona [email protected] María Pérez-Caro Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Salamanca [email protected] Anna Merlos Cellular Biology, Universitat Pompeu Fabra, Barcelona [email protected] Ignacio Pérez de Castro Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Ramon Messeguer LBI, Merck, Farma y Quimica, S.A., Barcelona [email protected] Ignacio Moreno de Alborán 411 Centro Nacional de Biotecnologia-CSIC, Madrid [email protected] José Antonio Pintor Mol. Biol., IRNA.CSIC, Sevilla [email protected] Olga Millán Instituto de Alta Tecnología, PRBB, Barcelona. [email protected] Francisco X. Real Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Pura Muñoz-Cánoves Differentiation, Centre de Regulació Genòmica, Barcelona [email protected] Tiago Rodrigues NMR Laboratory, Instituto Investigaciones Biomédicas "Alberto Sols", Madrid [email protected] Pilar Navarro Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] 89 Workshop Animal Models for Cancer Esther Rodríguez Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Gema Tarrason LBI, Merck, Farma y Quimica, S.A., Barcelona [email protected] Jelena Urosevic Experimental Oncology Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid [email protected] Ramon Roset Unitat de Biologia Cel·lular i Molecular, IMIM, Barcelona [email protected] Daniel Valverde Saubí Grup d'Aplicacions Biomèdiques de la RMN, Universitat Autònoma de Barcelona, Cerdanyola del Vallès [email protected] Isidro Sánchez-García Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Salamanca [email protected] Mireia Vallespinós 411, Centro Nacional de Biotecnología-CSIC, Madrid [email protected] Manuel Sánchez-Martín Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/ Universidad de Salamanca, Salamanca [email protected] Anxo Vidal Fisioloxia, Universidad de Santiago de Compostela, Santiago de Compostela, A Coruña [email protected] Mirentxu Santos Lafuente Biología Molecular y Celular, DIAE, CIEMAT, Madrid [email protected] Joana Visa Servei d'Estabulari, IRO- Hospital Duran y Reynals, Hospitalet de LLobregat [email protected] Marcos Seoane Fisiología, Universidad de Santiago de Compostela, Santiago de Compostela [email protected] Ana Zubiaga Genetics, University of the Basque Country, Bilbao [email protected] Marta Soler LBI, Merck, Farma y Quimica, S.A., Barcelona [email protected] Jordi Surralles Departament of Genetics and Microbiology, Universidad Autónoma de Barcelona, Bellaterra [email protected] 90 Workshop Modelos Animales en Cáncer 91 Workshop Animal Models for Cancer 92 Workshop Modelos Animales en Cáncer 93 Workshop Animal Models for Cancer 94 Workshop Modelos Animales en Cáncer 95 Workshop Animal Models for Cancer 96 Workshop Modelos Animales en Cáncer 97 Workshop Animal Models for Cancer 98
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