Sensitivity of hematopoietic stem cells to mitochondrial dysfunction by SdhD gene deletion

It is established that hematopoietic stem cells (HSC) in the hypoxic bone marrow have adapted their metabolism to oxygen-limiting conditions. This adaptation includes suppression of mitochondrial activity, induction of anerobic glycolysis, and activation of hypoxia-inducible transcription factor 1α (Hif1α)-dependent gene expression. During progression of hematopoiesis, a metabolic switch towards mitochondrial oxidative phosphorylation is observed, making this organelle essential for determining cell fate choice in bone marrow. However, given that HSC metabolism is essentially oxygen-independent, it is still unclear whether functional mitochondria are absolutely required for their survival. To assess the actual dependency of these undifferentiated cells on mitochondrial function, we have performed an analysis of the hematopoiesis in a mouse mutant, named SDHD-ESR, with inducible deletion of the mitochondrial protein-encoding SdhD gene. This gene encodes one of the subunits of the mitochondrial complex II (MCII). In this study, we demonstrate that, in contrast to what has been previously established, survival of HSC, and also myeloid and B-lymphoid progenitors, depends on proper mitochondrial activity. In addition, gene expression analysis of these hematopoietic lineages in SDHD-ESR mutants calls into question the proposed activation of Hif1α in response to MCII dysfunction.Ministerio de Ciencia e Innovación SAF2009-06970Junta de Andalucía CTS-4589Instituto de Salud Carlos III PI-0355-201

Mecanismos Moleculares de Formación de Tumores en Modelos Genéticos Animales con Defectos Mitocondriales por Deleción del Gen SdhD

La disfunción mitocondrial está ampliamente vinculada con la tumorogénesis. Una de las características principales de las células cancerosas es el cambio desde un metabolismo oxidativo mitocondrial dependiente de oxígeno hacia un metabolismo anaeróbico e

Estudio metodológico sobre la incorporación de las TIC en la enseñanza de las ciencias experimentales en bachillerato en centros educativos de Sevilla

A raíz de la llegada del ordenador e Internet al mercado público, comenzó la expansión de la Web convirtiéndose ésta en uno de los factores que más influyeron en la evolución hacia una sociedad con nuevos sectores laborales basados en entornos virtuales y en la que el sistema educativo ha tenido que adaptarse. El objetivo general de este trabajo fue obtener información acerca del proceso de adaptación de la metodología educativa a las tecnologías del mundo digital y detectar los posibles beneficios del uso de las Tecnologías de la Información y la Comunicación (TIC) en el proceso de enseñanza-aprendizaje. La metodología de investigación seguida fue mixta, basada en búsqueda bibliográfica y un trabajo de campo llevado a cabo en los centros educativos de Sevilla. Los resultados mostraron un alto índice de aceptación de los recursos TIC por parte del profesorado, a la vez que una alta predisposición a integrar a los alumnos en su manejo. Asimismo el nivel de uso y aprovechamiento que realizan los alumnos de estas tecnologías es frecuente tanto como fuente de entretenimiento como herramienta para el estudio. La opinión y actitud de profesores y alumnos sobre la integración de estas tecnologías en el centro es positiva en lo relativo a la disponibilidad de las mismas en los centros y en su gestión. Finalmente el uso de TIC durante las clases incrementa el nivel de atención y la calificación promedio obtenida por los alumnos. En el presente estudio se incluye una propuesta metodológica sobre una unidad didáctica de la materia de Biología y Geología de bachillerato a través de una metodología basada en las TIC

Genetically Modeled Mice with Mutations in Mitochondrial Metabolic Enzymes for the Study of Cancer

Mitochondrial dysfunction has long been implicated in progression of cancer. As a paradigm, the “Warburg effect,” which by means of a switch toward anaerobic metabolism enables cancer cells to proliferate in oxygen limiting conditions, is well established. Besides this metabolic transformation of tumors, it has been discovered that mutations in genes encoding mitochondrial proteins are the etiological factors in different types of cancer. This confers to mitochondrial dysfunction a causative role, rather than resultant, in tumor genesis beyond its role in tumor progression and development. Mitochondrial proteins encoded by tumor-suppressor genes are part of the succinate-dehydrogenase, the fumarate-hydratase, and the mitochondrial isocitrate-dehydrogenase enzymes, all of them participating in the Krebs cycle. The spectrum of tumors associated with mutations in these genes is becoming larger and varies between each enzyme. Several mechanisms of tumorigenesis have been proposed for the different enzymatic defects, most of them based on studies using cellular and animal models. Regarding the molecular pathways implicated in the oncogenic transformation, one of the first accepted theories was based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as “pseudo-hypoxic drive.” This mechanism has been linked to the three types of mutations, thus suggesting a central role in cancer. However, other alternative molecular processes, such as oxidative stress or altered chromatin remodeling, have been also proposed to play an onco-pathogenic role. In the recent years, the role of oncometabolites, a new concept emerged from biochemical studies upon these tumors, has acquired relevance as responsible for tumor formation. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. In this review, we summarize the results obtained from mouse strains genetically modified in the three different enzymes.Peer reviewedPeer Reviewe

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kβ silencing

Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kβ, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kβ kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kβ depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kβ inhibition. A small proportion of these tumors was resistant to PI3Kβ downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kβ dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kβ associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN-mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN.This work was financed by grants from the Spanish Ministry of Science and Innovation (SAF2011-29530 to FXR, SAF2013-48657 to ACC; Consolider ONCOBIO to FXR, Network of Cooperative Research in Cancer cofinanced by the European Regional Development Fund (RTICC RD12/0036/0059 to ACC and RD12/0036/0034 to FXR), the Madrid regional government (BMD2502 to ACC), and an AECC (Spanish Association against Cancer) grant to FXR

Most strongly up- and down-regulated genes in SDHD-ESR tissues.

a<p>; Values >0 indicate up-regulated genes. Values <0 indicate down-regulated genes. FDR: False discovery rate.</p

Microarray analysis.

<p><b>A. </b><i>SdhD</i> mRNA levels in heterozygous (+/−) and SDHD-ESR adrenal medulla and kidney relative to wild-type (+/+) tissues 7 days after the start of the tamoxifen treatment, as obtained from the corresponding microarray feature (Gene Bank accession n° NM_025848). *, <i>P</i>≤0.05; ***, <i>P</i>≤0.001. The number of samples is 8 per group. <b>B.</b> Supervised hierarchical clustering of adrenal medulla (AM) and kidney samples based on genes that showed significant differences in their expression level. The heatmap and the hierarchical tree are shown for 8 samples, grouped in pairs, per genotype.</p

“Pseudo-hypoxic” response in SDHD-ESR mouse tissues.

<p><b>A.</b> Succinate-ubiquinone oxidoreductase activity (SQR) in kidney 7 days after the start of the tamoxifen treatment. <b>B.</b> Relative mRNA level of <i>Vegf</i>, <i>Glut1</i>, and <i>Phd3</i> genes in kidney at 7 and 21 days after the start of the tamoxifen treatment. n.d.: non-determined. <b>C.</b> Western blot of Hif1α with protein extracts from kidney after tamoxifen treatment. Protein extracts from the pancreas of a β-cell-specific von Hippel-Lindau gene knock-out (VHL-KO) mouse and a wild-type littermate (VHL-wt) are loaded as controls. n.a.: non-applicable. Between 3 and 8 individuals per group were analyzed in each experiment. *, <i>P</i>≤0.05; ***, <i>P</i>≤0.001.</p

Expression levels of HIF1α-mediated hypoxia responsive genes.

<p>Date are expressed as the log ratio ± SEM between either the homozygous (+/−) or the inducible SDHD-ESR mutant and the wild type (+/+) expression levels for each gene in each tissue as obtained from the microarray analysis. <i>Glut1</i>: glucosyltransferase 1 (NM_172380), <i>HK2</i>: Hexokinase 2 (NM_013820), <i>LDHA</i>: Lactate dehydrogenase A (NM_001136069), <i>PDK1</i>: Pyruvate dehydrogenase kinase 1 (NM_172665), <i>Vegf</i>: Vascular endothelial growth factor (NM_001025257).</p