The effect of the proteasome inhibitor bortezomib on acute myeloid leukemia cells and drug resistance associated with the CD34+ immature phenotype

[Background]: Proteasome inhibition represents a promising novel anticancer therapy, and bortezomib is a highly selective reversible inhibitor of the proteasome complex. Acute myeloid leukemia (AML) is an immnunophenotypically heterogeneous group of diseases, with CD34+ cases being associated with drug resistance and poor outcome. We investigated the effects of bortezomib on the growth and survival of AML cells. [Design and Methods]: We studied the in vitro activity and mechanism of action of bortezomib on both cell lines and fresh cells from 28 AML patients including CD34+ and CD34- cases. [Results]: Bortezomib showed potent anti-AML activity (IC50 < 50 nM), which was greater than that of conventional agents (doxorubicin, cytarabine and fludarabine). Moreover, synergistic effects were observed when bortezomib was adminstered in combination with doxorubicin and cytarabine. Mechanistically, bortezomib induced accumulation of cells in the G2/M phase, with up-regulation of p27, together with cell death through an increase in the mitochondrial outer membrane permeability involving caspase-dependent and -independent pathways. The apoptotic activity of bortezomib on fresh CD34 + blast cells from patients was similar to that observed on CD34 - blast cells. Importantly, bortezomib was significantly more active than doxorubicin in the immature CD34+ cells, while there were no differences in its action on CD34- cells. [Conclusions]: Bortezomib induces apoptosis in acute myeloid leukemia cells in vitro. Whether this drug might be useful in the treatment of patients with acute myeloid leukemia can be established only in ad hoc clinical trials. ©2008 Ferrata Storti Foundation.We thank Johnson and Johnson Pharmaceutical Research and Development (JJPRD).Peer Reviewe

Deficient Spindle Assembly Checkpoint in Multiple Myeloma

Multiple myeloma (MM) is a hematological disease characterized by an abnormal accumulation of plasma cells in the bone marrow. These cells have frequent cytogenetic abnormalities including translocations of the immunoglobulin heavy chain gene and chromosomal gains and losses. In fact, a singular characteristic differentiating MM from other hematological malignancies is the presence of a high degree of aneuploidies. As chromosomal abnormalities can be generated by alterations in the spindle assembly checkpoint (SAC), the functionality of such checkpoint was tested in MM. When SAC components were analyzed in MM cell lines, the RNA levels of most of them were conserved. Nevertheless, the protein content of some key constituents was very low in several cell lines, as was the case of MAD2 or CDC20 in RPMI-8226 or RPMI-LR5 cells. The recovery of their cellular content did not substantially affect cell growth, but improved their ability to segregate chromosomes. Finally, SAC functionality was tested by challenging cells with agents disrupting microtubule dynamics. Most of the cell lines analyzed exhibited functional defects in this checkpoint. Based on the data obtained, alterations both in SAC components and their functionality have been detected in MM, pointing to this pathway as a potential target in MM treatment

Decreased breadth of the antibody response to the spike protein of SARS-CoV-2 after repeated vaccination

IntroductionThe rapid development of vaccines to prevent COVID-19 has raised the need to compare the capacity of different vaccines in terms of developing a protective humoral response. Previous studies have shown inconsistent results in this area, highlighting the importance of further research to evaluate the efficacy of different vaccines.MethodsThis study utilized a highly sensitive and reliable flow cytometry method to measure the titers of IgG1 isotype antibodies in the blood of healthy volunteers after receiving one or two doses of various vaccines administered in Spain. The method was also used to simultaneously measure the reactivity of antibodies to the S protein of the original Wuhan strain and variants B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.617.1 (Kappa).ResultsSignificant differences were observed in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech, and Ad26.COV.S/Janssen. Furthermore, a relative reduction in the reactivity of the sera with the Alpha, Delta, and Kappa variants, compared to the Wuhan strain, was observed after the second boosting immunization.DiscussionThe findings of this study provide a comparison of different vaccines in terms of anti-S antibody generation and cast doubts on the convenience of repeated immunization with the same S protein sequence. The multiplexed capacity of the flow cytometry method utilized in this study allowed for a comprehensive evaluation of the efficacy of various vaccines in generating a protective humoral response. Future research could focus on the implications of these findings for the development of effective COVID-19 vaccination strategies

La vía de ERK5-MEF2 en mieloma múltiple: implicaciones terapéuticas. Resumen de tesis

Extracto de tesis.[ES] Las vías de MAPKs juegan un papel fundamental en una gran variedad de procesos biológicos, entre los que se encuentran el crecimiento y la proliferación celular, la migración, la diferenciación y la supervivencia. Por estar involucradas en tantos procesos, las alteraciones de estas vías se han relacionado de manera muy relevante con el desarrollo y progresión de diferentes tipos de tumores. La vía de ERK1/2 es la más estudiada y su funcionamiento es importante para una correcta hematopoyesis. Otra vía de MAPKs menos estudiada pero que también juega un papel fundamental en una gran variedad de procesos biológicos es la vía de ERK5. Esta quinasa también se ha relacionado con la hematopoyesis y estudios previos de nuestro grupo han demostrado también su importancia en el mieloma múltiple. Uno de los sustratos más conocidos de ERK5 son los factores de transcripción MEF2. Estos factores tienen un importante papel en el desarrollo cardiovascular, neuronal y músculo-esquelético. En vertebrados existen cuatro factores MEF2: MEF2A, B, C, D. Analizamos la expresión de estos factores en diferentes líneas celulares de mieloma múltiple (MM) y en muestras de pacientes. Los factores MEF2A, MEF2C y MEF2D, están sobreexpresados en muestras de pacientes de MM. MEF2C se encuentra, además, fosforilado de manera basal. Para investigar la función biológica de estos factores en las células de MM utilizamos por un lado una forma dominante negativa que impide la unión al ADN de estos factores pero mantiene la capacidad de homo/heterodimerizar, y por otro lado silenciamos la expresión de los factores MEF2C y MEF2D mediante horquillas de ARN. Observamos que la inhibición de estos factores en las células de MM inhibe la proliferación celular. Además, el tratamiento de líneas celulares de MM con bortezomib provoca la defosforilación de MEF2C y la disminución de los niveles proteicos de MEF2D, lo que sugiere que, estos factores, pueden jugar un papel en el mecanismo de acción de este fármaco en MM. Pese a los avances en el tratamiento del MM que han conseguido alargar la supervivencia de los pacientes, esta patología continúa siendo incurable, por lo que es necesario el desarrollo de nuevas terapias y la investigación de su efectividad en el tratamiento de los pacientes. En base a la importancia demostrada de ERK5 en MM y a la sobreexpresión de sus sustratos, los factores MEF2 tanto en líneas celulares como en muestras de pacientes, consideramos interesante el estudio de TG02, un fármaco inhibidor de quinasas entre las que se incluye ERK5, como estrategia terapéutica en MM. TG02 muestra una potente eficacia in vitro en la inhibición de un amplio rango de CDKs y de otras quinasas, además de ERK5. En MM, la desregulación de las quinasas dependientes de ciclinas (CDKs) es un evento común, la mayoría de los pacientes presentan sobreexpresión o desregulación de alguna de las tres ciclinas tipo D que regulan estas CDKs, pero el MM es una patología compleja que implica la desregulación de diferentes vías además de las dependientes de CDKs. TG02 ejerció actividad antitumoral en varios modelos preclínicos de MM. Este fármaco inhibió de manera potente la proliferación de las células de MM incluso en un ambiente protector (tanto bajo la presencia de células estromales como tras la estimulación con factores de crecimiento). Además, indujo, ex vivo, la apoptosis de células plasmáticas de pacientes de MM sin afectar de manera drástica a los progenitores hematopoyéticos. El mecanismo de acción de TG02 sobre las células de mieloma fue principalmente la inducción de apoptosis, siendo la inhibición de la proteína MCL-1 importante en este proceso. Por último, TG02 presentó un efecto antitumoral in vivo en un modelo murino de xenoinjerto de MM humano: el tratamiento con TG02, tanto en monoterapia como en combinación con bortezomib o lenalidomida, indujo una inhibición significativa del crecimiento del tumor. TG02 es un nuevo inhibidor multiquinasa oral con una potente actividad antimieloma tanto in vitro como in vivo lo que indica que el espectro inhibitorio de este compuesto cubre vías oncogénicas importantes en el MM. Actualmente este compuesto se encuentra en ensayo clínico fase I en pacientes con MM refractario en EEUU[EN] MAPK pathways (Mitogen-activated protein kinases) play a key role in a variety of biological processes, including growth and include cell proliferation, migration, differentiation and survival. To be involved in many processes, alterations of these pathways have been implicated in a very important to the development and progression of different types of tumors. The route of ERK1 / 2 is the most studied and its performance is important for proper hematopoiesis. Another way of MAPKs less studied but it also plays a role in a variety of biological processes is the path ERK5. This kinase also been associated with hematopoiesis and previous studies by our group have also shown their importance in multiple myeloma. One of the most popular substrates are ERK5 MEF2 transcription factors. These factors have an important role in cardiovascular development, neural and musculoskeletal. In vertebrates MEF2 four factors: MEF2A, B, C, D. We analyzed the expression of these factors in different cell lines of multiple myeloma (MM) and in patient samples. The factors MEF2A, MEF2C and MEF2D, are overexpressed in MM patient samples. MEF2C is also so basal phosphorylated. To investigate the biological role of these factors in MM cells use one hand a dominant negative form which prevents DNA binding of these factors but maintains the ability to homo / heterodimerize, and secondly the expression silenced factor MEF2C and MEF2D by RNA hairpins. Observed that the inhibition of these factors on MM cells inhibits cell proliferation. Furthermore, treatment of MM cell lines with bortezomib causes MEF2C dephosphorylation and decreased MEF2D protein levels, suggesting that these factors may play a role in the mechanism of action of this drug in MM. Despite advances in the treatment of MM have gotten extend patient survival, this disease remains incurable, making it necessary to develop new therapeutics and research of their effectiveness in the treatment of patients. Based on the demonstrated importance of ERK5 in MM and overexpression of their substrates, MEF2 factors in both cell lines and patient samples, considered interesting study TG02, kinase inhibitor drug between ERK5 included as therapeutic strategy in MM. TG02 shows potent in vitro efficacy in inhibiting a broad range of CDKs and other kinases, and ERK5. In MM, deregulation of cyclin-dependent kinases (CDKs) is a common event, most patients show overexpression or deregulation of any of the three D-type cyclins that regulate these CDKs, but the MM is a complex disease that involves deregulation of different ways besides the CDKs dependent. TG02 exerted antitumor activity in several preclinical models of MM. This drug potently inhibited proliferation of MM cells even in a protective environment (either in the presence of stromal cells and after stimulation with growth factors). Also induced, ex vivo, cell apoptosis MM patients plasma without drastically affecting the hematopoietic progenitors. The mechanism of action of TG02 on myeloma cells was primarily the induction of apoptosis, inhibition being MCL-1 protein in this process. Finally, TG02 showed an antitumor effect in vivo in a xenograft murine model of human MM: TG02 treatment, both as monotherapy and in combination with bortezomib or lenalidomide, induced a significant inhibition of tumor growth. TG02 is a new oral multi-kinase inhibitor with potent activity antimyeloma both in vitro and in vivo indicating that the inhibitory spectrum of this compound covers major oncogenic pathways in MM. This compound is currently in phase I clinical trial in patients with refractory MM US

La vía de ERK5-MEF2 en Mieloma Múltiple: Implicaciones terapéuticas

Tesis Doctoral presentada por la licenciada Dña. Stela Álvarez Fernández para optar al grado de Doctora por la Universidad de Salamanca y realizada en el Instituto de Biología Molecular y Celular del Cáncer.[ES]: Las vías de MAPKs juegan un papel fundamental en una gran variedad de procesos biológicos, entre los que se encuentran el crecimiento y la proliferación celular, la migración, la diferenciación y la supervivencia. Por estar involucradas en tantos procesos, las alteraciones de estas vías se han relacionado de manera muy relevante con el desarrollo y progresión de diferentes tipos de tumores. La vía de ERK1/2 es la más estudiada y su funcionamiento es importante para una correcta hematopoyesis. Otra vía de MAPKs menos estudiada pero que también juega un papel fundamental en una gran variedad de procesos biológicos es la vía de ERK5. Esta quinasa también se ha relacionado con la hematopoyesis y estudios previos de nuestro grupo han demostrado también su importancia en el mieloma múltiple. Uno de los sustratos más conocidos de ERK5 son los factores de transcripción MEF2. Estos factores tienen un importante papel en el desarrollo cardiovascular, neuronal y músculo-esquelético. En vertebrados existen cuatro factores MEF2: MEF2A, B, C, D. Analizamos la expresión de estos factores en diferentes líneas celulares de mieloma múltiple (MM) y en muestras de pacientes. Los factores MEF2A, MEF2C y MEF2D, están sobreexpresados en muestras de pacientes de MM. MEF2C se encuentra, además, fosforilado de manera basal. Para investigar la función biológica de estos factores en las células de MM utilizamos por un lado una forma dominante negativa que impide la unión al ADN de estos factores pero mantiene la capacidad de homo/heterodimerizar, y por otro lado silenciamos la expresión de los factores MEF2C y MEF2D mediante horquillas de ARN. Observamos que la inhibición de estos factores en las células de MM inhibe la proliferación celular. Además, el tratamiento de líneas celulares de MM con bortezomib provoca la defosforilación de MEF2C y la disminución de los niveles proteicos de MEF2D, lo que sugiere que, estos factores, pueden jugar un papel en el mecanismo de acción de este fármaco en MM. Pese a los avances en el tratamiento del MM que han conseguido alargar la supervivencia de los pacientes, esta patología continúa siendo incurable, por lo que es necesario el desarrollo de nuevas terapias y la investigación de su efectividad en el tratamiento de los pacientes.En base a la importancia demostrada de ERK5 en MM y a la sobreexpresión de sus sustratos, los factores MEF2 tanto en líneas celulares como en muestras de pacientes, consideramos interesante el estudio de TG02, un fármaco inhibidor de quinasas entre las que se incluye ERK5, como estrategia terapéutica en MM. TG02 muestra una potente eficacia in vitro en la inhibición de un amplio rango de CDKs y de otras quinasas, además de ERK5. En MM, la desregulación de las quinasas dependientes de ciclinas (CDKs) es un evento común, la mayoría de los pacientes presentan sobreexpresión o desregulación de alguna de las tres ciclinas tipo D que regulan estas CDKs, pero el MM es una patología compleja que implica la desregulación de diferentes vías además de las dependientes de CDKs. TG02 ejerció actividad antitumoral en varios modelos preclínicos de MM. Este fármaco inhibió de manera potente la proliferación de las células de MM incluso en un ambiente protector (tanto bajo la presencia de células estromales como tras la estimulación con factores de crecimiento). Además, indujo, ex vivo, la apoptosis de células plasmáticas de pacientes de MM sin afectar de manera drástica a los progenitores hematopoyéticos. El mecanismo de acción de TG02 sobre las células de mieloma fue principalmente la inducción de apoptosis, siendo la inhibición de la proteína MCL-1 importante en este proceso. Por último, TG02 presentó un efecto antitumoral in vivo en un modelo murino de xenoinjerto de MM humano: el tratamiento con TG02, tanto en monoterapia como en combinación con bortezomib o lenalidomida, indujo una inhibición significativa del crecimiento del tumor. TG02 es un nuevo inhibidor multiquinasa oral con una potente actividad antimieloma tanto in vitro como in vivo lo que indica que el espectro inhibitorio de este compuesto cubre vías oncogénicas importantes en el MM. Actualmente este compuesto se encuentra en ensayo clínico fase I en pacientes con MM refractario en EEUU.[EN]: MAPK pathways play a key role in a variety of biological processes, including growth and include cell proliferation, migration, differentiation and survival. To be involved in many processes, alterations of these pathways have been implicated in a very important to the development and progression of different types of tumors. The route of ERK1 / 2 is the most studied and its performance is important for proper hematopoiesis. Another way of MAPKs less studied but it also plays a role in a variety of biological processes is the path ERK5. This kinase also been associated with hematopoiesis and previous studies by our group have also shown their importance in multiple myeloma. One of the most popular substrates are ERK5 MEF2 transcription factors. These factors have an important role in cardiovascular development, neural and musculoskeletal. In vertebrates MEF2 four factors: MEF2A, B, C, D. We analyzed the expression of these factors in different cell lines of multiple myeloma (MM) and in patient samples. The factors MEF2A, MEF2C and MEF2D, are overexpressed in MM patient samples. MEF2C is also so basal phosphorylated. To investigate the biological role of these factors in MM cells use one hand a dominant negative form which prevents DNA binding of these factors but maintains the ability to homo / heterodimerize, and secondly the expression silenced factor MEF2C and MEF2D by RNA hairpins. Observed that the inhibition of these factors on MM cells inhibits cell proliferation. Furthermore, treatment of MM cell lines with bortezomib causes MEF2C dephosphorylation and decreased MEF2D protein levels, suggesting that these factors may play a role in the mechanism of action of this drug in MM.Despite advances in the treatment of MM have gotten extend patient survival, this disease remains incurable, making it necessary to develop new therapeutics and research of their effectiveness in the treatment of patients. Based on the demonstrated importance of ERK5 in MM and overexpression of their substrates, MEF2 factors in both cell lines and patient samples, considered interesting study TG02, kinase inhibitor drug between ERK5 included as therapeutic strategy in MM. TG02 shows potent in vitro efficacy in inhibiting a broad range of CDKs and other kinases, and ERK5. In MM, deregulation of cyclin-dependent kinases (CDKs) is a common event, most patients show overexpression or deregulation of any of the three D-type cyclins that regulate these CDKs, but the MM is a complex disease that involves deregulation of different ways besides the CDKs dependent. TG02 exerted antitumor activity in several preclinical models of MM. This drug potently inhibited proliferation of MM cells even in a protective environment (either in the presence of stromal cells and after stimulation with growth factors). Also induced, ex vivo, cell apoptosis MM patients plasma without drastically affecting the hematopoietic progenitors. The mechanism of action of TG02 on myeloma cells was primarily the induction of apoptosis, inhibition being Mcl-1 protein in this process. Finally, TG02 showed an antitumor effect in vivo in a xenograft murine model of human MM: TG02 treatment, both as monotherapy and in combination with bortezomib or lenalidomide, induced a significant inhibition of tumor growth. TG02 is a new oral multi-kinase inhibitor with potent activity antimyeloma both in vitro and in vivo indicating that the inhibitory spectrum of this compound covers major oncogenic pathways in MM. This compound is currently in phase I clinical trial in patients with refractory MM in USA.Peer Reviewe

Impaired expression of DICER, DROSHA, SBDS and some microRNAs in mesenchymal stromal cells from myelodysplastic syndrome patients

This is an open-access paper.[Background]: Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cells-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. [Design and Methods]: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. [Results]: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR- 222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. [Conclusions]: This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.Grants from Consejeria de Educacion de la Junta de Castilla y León (HUS03A09) and Instituto de Salud Carlos III (FIS09/01530).Peer reviewe

Potent antimyeloma activity of a novel ERK5/CDK inhibitor

[Purpose]: To analyze the antimyeloma potential of TG02, an ERK5/CDK inhibitory drug. [Experimental Design]: Utilizing different multiple myeloma cell lines we determined the effect of TG02 over viability by MTT assays. The apoptotic effect over multiple myeloma patient samples was studied ex vivo by cytometry. The mechanism of action of TG02 was analyzed in the cell line MM1S, studying its effect on the cell cycle, the induction of apoptosis, and the loss of mitochondrial membrane potential by cytometry and Western blot. Two models of multiple myeloma xenograft were utilized to study the in vivo action of TG02. [Results]: TG02 potently inhibited proliferation and survival of multiple myeloma cell lines, even under protective bone marrow niche conditions, and selectively induced apoptosis of primary patient-derived malignant plasma cells. TG02 displayed significant single-agent activity in two multiple myeloma xenograft models, and enhanced the in vivo activity of bortezomib and lenalidomide. Signaling analyses revealed that the drug simultaneously blocked the activity of CDKs 1, 2, and 9 as well as the MAP kinase ERK5 in MM1S cells, leading to cell-cycle arrest and rapid commitment to apoptosis. TG02 induced robust activation of both the intrinsic and extrinsic pathways of apoptosis, and depletion of XIAP and the key multiple myeloma survival protein Mcl-1. [Conclusions]: TG02 is a promising new antimyeloma agent that is currently in phase I clinical trials in leukemia and multiple myeloma patients.This work was supported by grants from the Ministry of Science and Innovation of Spain (BFU2009-07728/BMC to A. Pandiella) and Instituto de Salud Carlos III (PS09/00868 to A. Esparís-Ogando). S. Álvarez-Fernández is supported by the Cancer Center Network Program from the ISCIII (RD06/0020/0041). Our Cancer Research Institute, and the work carried out at our laboratory, receive support from the European Community through the regional development funding program (FEDER), and from the Fundación Ramón Areces.Peer Reviewe

Zalypsis has in vitro activity in acute myeloid blasts and leukemic progenitor cells through the induction of a DNA damage response

This is an open-access paper.-- et al.[Background]: Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin. [Design and Methods]: The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid. [Results]: Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34+ CD38− Lin−) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX). [Conclusions]: The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients’ samples provides the rationale for the investigation of this compound in clinical trials.This work was supported by a grant from the Ministry of Science and Innovation of Spain (BFU2006-01813/BMC and RD06/0020/ 0041). The CIC receives support from the European Community through the regional development funding program (FEDER). This work was also supported by the ‘Acción Transversal del Cáncer’ project, through an agreement between the Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Science and Innovation, and the Cancer Research Foundation of Salamanca University. Our group also receives support from the Junta de Castilla y Léon through ‘Ayudas destinadas a financiar programas de actividad investigadora a realizar por grupos de investigación de excelencia de Castilla y León’.Peer reviewe