Animales en Cáncer Workshop Modelos - Animal Models for Cancer

 
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          Coffee break
17:30          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
jose.aramburu@upf.edu

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
    Departament de Ciències Experimentals i de la Salut. Universitat Pompeu Fabra, Barcelona.
2
    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
mblasco@cnio.es

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. K5-
TRF2 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 XP-
like 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
dbuscher@genetrix.es

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
mcespedes@hsp.santpau.es

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
    Lab. d’Investigació Gastrointestinal, Institut de Recerca, Hospital Sant Pau, Barcelona
2
    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
gfresneda@cnio.es

Proteolysis as a target for cancer therapy: Cdc20 and the Anaphase Promoting
Complex
Gardenia Fresneda1, Irene García-Higuera2, Sergio Moreno2 & Marcos Malumbres1
1
    Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas, Madrid
2
    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
jgalan@cnio.es

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
Carme.gallego@cmb.udl.es

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
cgonzalez@pcb.ub.es

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
mcguerra@cnio.es

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 K-
rasV12 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
mpg@solea.quim.ucm.es

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
laherraez@zfbiolabs.com

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 high-
throughput 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
mherranz@cnio.es

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
cristina.lopez.rodriguez@crg.es

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 DNA-
binding 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 pro-
metastatic 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 NFAT5-
null 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
rmangues@hsp.santpau.es

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
2
 Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid

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 E-
cadherina y transducidas igualmente con Snail (GFP-Snail + E-cadherina). Las células GFP y GFP-
Snail 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
amartin@cnio.es

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
amp@correo.uniovi.es

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
jmartinalbo@cnio.es

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
raul.mendez@crg.es

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
omillan@cnic.es

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
imoreno@cnb.uam.es

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.muñoz@crg.es

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 ATM-
deficient 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
anebreda@cnio.es

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
pordonez@iib.uam.es

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,25-
dihydroxyvitamin 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
You can also read
Next slide ... Cancel