The Vast Complexity of the Epigenetic Landscape during Neurodevelopment: An Open Frame to Understanding Brain Function.

Development is a well-defined stage-to-stage process that allows the coordination and maintenance of the structure and function of cells and their progenitors, in a complete organism embedded in an environment that, in turn, will shape cellular responses to external stimuli. Epigenetic mechanisms comprise a group of process that regulate genetic expression without changing the DNA sequence, and they contribute to the necessary plasticity of individuals to face a constantly changing medium. These mechanisms act in conjunction with genetic pools and their correct interactions will be crucial to zygote formation, embryo development, and brain tissue organization. In this work, we will summarize the main findings related to DNA methylation and histone modifications in embryonic stem cells and throughout early development phases. Furthermore, we will critically outline some key observations on how epigenetic mechanisms influence the rest of the developmental process and how long its footprint is extended from fecundation to adulthood.

Rethinking the Epigenetic Framework to Unravel the Molecular Pathology of Schizophrenia.

Schizophrenia is a complex mental disorder whose causes are still far from being known. Although researchers have focused on genetic or environmental contributions to the disease, we still lack a scientific framework that joins molecular and clinical findings. Epigenetic can explain how environmental variables may affect gene expression without modifying the DNA sequence. In fact, neuroepigenomics represents an effort to unify the research available on the molecular pathology of mental diseases, which has been carried out through several approaches ranging from interrogating single DNA methylation events and hydroxymethylation patterns, to epigenome-wide association studies, as well as studying post-translational modifications of histones, or nucleosomal positioning. The high dependence on tissues with epigenetic marks compels scientists to refine their sampling procedures, and in this review, we will focus on findings obtained from brain tissue. Despite our efforts, we still need to refine our hypothesis generation process to obtain real knowledge from a neuroepigenomic framework, to avoid the creation of more noise on this innovative point of view; this may help us to definitively unravel the molecular pathology of severe mental illnesses, such as schizophrenia.

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kß 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 PI3Kß, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kß kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kß depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kß inhibition. A small proportion of these tumors was resistant to PI3Kß 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 PI3Kß dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kß 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.

Targeted depletion of PIK3R2 induces regression of lung squamous cell carcinoma.

Oncogenic mutations in the PI3K/AKT pathway are present in nearly half of human tumors. Nonetheless, inhibitory compounds of the pathway often induce pathway rebound and tumor resistance. We find that lung squamous cell carcinoma (SQCC), which accounts for ~20% of lung cancer, exhibits increased expression of the PI3K subunit PIK3R2, which is at low expression levels in normal tissues. We tested a new approach to interfere with PI3K/AKT pathway activation in lung SQCC. We generated tumor xenografts of SQCC cell lines and examined the consequences of targeting PIK3R2 expression. In tumors with high PIK3R2 expression, and independently of PIK3CA, KRAS, or PTEN mutations, PIK3R2 depletion induced lung SQCC xenograft regression without triggering PI3K/AKT pathway rebound. These results validate the use PIK3R2 interfering tools for the treatment of lung squamous cell carcinoma.

Epigenetics in Schizophrenia: A Pilot Study of Global DNA Methylation in Different Brain Regions Associated with Higher Cognitive Functions.

Attempts to discover genes that are involved in the pathogenesis of major psychiatric disorders have been frustrating and often fruitless. Concern is building about the need to understand the complex ways in which nature and nurture interact to produce mental illness. We analyze the epigenome in several brain regions from schizophrenic patients with severe cognitive impairment using high-resolution (450K) DNA methylation array. We identified 139 differentially methylated CpG sites included in known and novel candidate genes sequences as well as in and intergenic sequences which functions remain unknown. We found that altered DNA methylation is not restricted to a particular region, but includes others such as CpG shelves and gene bodies, indicating the presence of different DNA methylation signatures depending on the brain area analyzed. Our findings suggest that epimutations are not relatables between different tissues or even between tissues' regions, highlighting the need to adequately study brain samples to obtain reliable data concerning the epigenetics of schizophrenia.

From Linkage Studies to Epigenetics: What We Know and What We Need to Know in the Neurobiology of Schizophrenia.

Schizophrenia is a complex psychiatric disorder characterized by the presence of positive, negative, and cognitive symptoms that lacks a unifying neuropathology. In the present paper, we will review the current understanding of molecular dysregulation in schizophrenia, including genetic and epigenetic studies. In relation to the latter, basic research suggests that normal cognition is regulated by epigenetic mechanisms and its dysfunction occurs upon epigenetic misregulation, providing new insights into missing heritability of complex psychiatric diseases, referring to the discrepancy between epidemiological heritability and the proportion of phenotypic variation explained by DNA sequence difference. In schizophrenia the absence of consistently replicated genetic effects together with evidence for lasting changes in gene expression after environmental exposures suggest a role of epigenetic mechanisms. In this review we will focus on epigenetic modifications as a key mechanism through which environmental factors interact with individual's genetic constitution to affect risk of psychotic conditions throughout life.

Phosphoinositide 3-kinase p85beta regulates invadopodium formation.

The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85ß, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85ß might facilitate cell invasion. We show that p85ß localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85ß induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85ß lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85ß function in invadopodium-like formation, p85ß levels increased in metastatic melanoma and p85ß depletion reduced invadopodium formation and invasion. These results show that p85ß enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85ß levels.

Distinct and specific roles of AKT1 and AKT2 in androgen-sensitive and androgen-independent prostate cancer cells.

AKT isoforms are expressed in prostate cancer and their expression and localization have different associations with clinical characteristics. However, the distinct roles of the AKT isoforms in prostate cancer cells are largely unknown. In the present study, we demonstrate distinct roles for AKT1 and AKT2 in cell growth and migration. Ablation of AKT1 and AKT2 decreased the proliferation of the androgen-independent cell line PC-3, although by different mechanisms. AKT1 ablation induced loss of cell adhesion and subsequent apoptosis. AKT2 (but not AKT1) ablation promoted cell cycle arrest at G0/G1, associated with downregulation of cyclin D, CDK6 and CDK2, and upregulation and cytoplasmic-to-nuclear redistribution of p27. The increase of p27 protein levels was due to more gene transcription and an increase in protein stability. The increased stability of p27 was induced by delocalisation of Skp2 and a lower level of p27 phosphorylation at Thr187. AKT1 and AKT2 ablation inhibited and stimulated PC-3 cell migration, respectively. An AKT isoform-specific function could be associated with its subcellular localization. We found that AKT1 and AKT2 were mainly localised in the cytoplasm and nucleus, respectively. In androgen-sensitive cell line LNCaP, the ablation of AKT1 or AKT2 caused apoptosis but in androgen-independent LNCaP sublines, the effect of AKT1 ablation was lower; whereas no changes were observed after AKT2 ablation. Taken together, our data show that AKT1 and AKT2 have non-redundant roles in the regulation of PC-3 cell proliferation and migration. These could be explained by their subcellular localization and/or the specific regulation of downstream effectors. Furthermore, contribution of AKT isoforms to the progression of prostate cancer may change from an androgen-sensitive to a hormone-refractory stage. These findings may help design new targeted strategies for inhibiting AKT isoforms in prostate cancer.

Estandarización de marcadores moleculares microsatélites para su uso en la industria forestal de Misiones, Argentina / Standarization of microsatellite molecular markers for Misiones (Argentina) forest industry

Provincia de Misiones posee actualmente una actividad forestal en pujante crecimiento ubicóndose entre las primeras del pais. Este marco de desarrollo productivo permite predecir un ámbito de crecimiento favorecido por las nuevas condiciones del mercado internacional. Por otro lado a pesar del avance de la tecnologi­a industrial, no se ha alcanzado el nivel de desarrollo biotecnológico óptimo que conjugue la calidad genética con caracterí­sticas fenotópicas de excelencia en las especies maderables de mayor demanda en la Provincia basó¡ndose la selección en criterios netamente fenotópicos y en la experiencia del productor, sin contarse con métodos moleculares desarrollados en la región. Este trabajo presenta los resultados del Proyecto Federal de Innovacíon Productiva (PFIP Mi09) cuyo objetivo principal fue estandarizar y transferir al sector productivo un conjunto de marcadores moleculares microsalites para ser aplicado al análisis de poblaciones y forestaciones de Araucaria angustifolia y Pinus taeda provenientes de la Provincia de Misiones (Argentina). Esto permitirá conocer el perfil genético de plantaciones y poblaciones de estas especies forestales, pudiendo aplicarse a la certificación de calidad en la producción forestal o a la selección de ejemplares de especies nativas.

Misiones Province currently has the first intensive forestry activity of Argentine. This framework of productive development allows predict an area of growth favored by the new conditions of the international market. On the other side despite the progress of industrial technology, has not been reached the optimal level of biotechnological development that combining quality with genomic and phenotypic characteristics of forest species. This work presents the results of Federal Project of Productive Innovation (PFIP Mi09) whose main objective was standardize and transfer to the productive sector a set of microsatellites molecular markers to be applied to the populations analysis of Araucaria angustifolia and Pinus taeda forestation from the Misiones (Argentine). This will reveal the plantations and forest genetic profile and may be applied to genetic certification of forest production quality.

EGF promotes neuroendocrine-like differentiation of prostate cancer cells in the presence of LY294002 through increased ErbB2 expression independent of the phosphatidylinositol 3-kinase-AKT pathway.

An increased neuroendocrine (NE) cell population in prostate cancer is associated with more aggressive disease and recurrence after androgen-deprivation therapy, although the mechanism responsible is unknown. In this study, we report that the treatment of LNCaP cells with epidermal growth factor (EGF) in the presence of LY294002, an inhibitor of the phosphoinositol 3'-kinase (PI3K)-AKT pathway, induced an increase of levels and activity of ErbB2. Under these conditions, we also observed cell survival and NE differentiation. When we treated with wortmannin, another PI3K inhibitor, or we knocked down PI3K or AKT isoforms in the presence of EGF, ErbB2 up-regulation was not observed, suggesting that the increase of ErbB2 induced by EGF plus LY294002 is not mediated by the PI3K-Akt pathway. Other targets of LY294002 were also discounted. We also show that ErbB2 up-regulation is directly involved in neuroendocine differentiation but not in cell survival as ErbB2 levels increased in parallel with NE differentiation marker levels, whereas ErbB2 knockdown reduced them; other NE differentiation inducers also increased the ErbB2 levels and the immunohistochemical analysis of prostate cancer samples showed colocalization of ErbB2 and chromogranin A. We found that, in LNCaP cells, EGF in combination with LY294002 increased ErbB2 levels by a PI3K/AKT-independent mechanism and that this increase was associated with the acquisition of a NE phenotype. These results suggest that is worth reconsidering ErbB2 as a drug target in prostate cancer and this should be kept in mind when designing new clinical schedules for the treatment of this disease.

Tumoral prostate shows different expression pattern of somatostatin receptor 2 (SSTR2) and phosphotyrosine phosphatase SHP-1 (PTPN6) according to tumor progression.

Prostate proliferation is dependent of androgens and many peptide hormones. Recent reports suggest that SSTR2 and SHP-1 were two fundamental components on antiproliferative effect of somatostatin. Many studies on SHP-1 revealed that the expression of this protein was diminished or abolished in several of the cancer cell lines and tissues examined. However, it is necessary to confront the cell lines data with real situation in cancer cases. Our studies have shown that epithelial expressions of both proteins, SHP-1 and SSTR2, in normal and benign hyperplasia are localized in the luminal side of duct and acinar cells. Also, SSTR2 is expressed in stromal cells. In malignant prostate tissue, SHP-1 was diminished in 28/45 cases or absent in 12/45 cases, whereas SSTR2 epithelial was diminished in 38/45 cases or lost in only 2/45 cases. The intensity of immunostained was highly negative correlated with Gleason grade for two proteins.

Aplicación de un trabajo práctico autoguiado para la formación en el uso de herramientas bioinformáticas de alumnos de pregrado en Bioquímica Clínica / Self-guided training program in the use of bioinformatic tools for undergraduate students of clinical biochemistry

Durante las últimas décadas el gran avance científico y tecnológico llevó a la investigación biológica a las puertas de la era postgenómica. La disponibilidad de información crucial para el desarrollo de nuevos proyectos ha provocado un cambio de paradigma en la investigación biológica demandándole profesionales que cuenten con formación en Bioinformática. En este trabajo se muestran los resultados de la incorporación de un trabajo práctico autoguiado para introducir a alumnos que estudien Bioquímica al uso de recursos bioinformáticos aplicándolos a un ejemplo concreto. Las actividades consisten en la realización de un análisis genómico, transcriptómico y proteómico de un gen con implicaciones biomédicas. Además se plantea como aplicación tecnológica el diseño de cebadores específicos para la amplificación de un fragmento del gen. Como último punto se propone analizar la función biológica mediante el programa de visualización molecular RasMol versión 2.7.2 (”por Herbert Bernstein 1998-2000). La metodología incluye grupos de 3-4 alumnos que cursan Biología Celular y Molecular de la Carrera de Bioquímica de la Universidad Nacional de Misiones, solicitándoseles respuestas concretas que se obtienen a través del análisis bioinformático. Los resultados de la aplicación del trabajo práctico autoguiado demuestran que el 100% de los alumnos fueron capaces de responder las consignas. Sin embargo se necesita mayor manejo de programas de visualización molecular para futuras aplicaciones (AU)

The great scientific and technological advances of recent decades have brought biological investigation into the postgenomic age. The ready availability of crucial information for the development of new projects has caused a paradigm shift in biological investigation, in which a solid training in bioinformatics is now a basic requirement. This study presents the results of the incorporation of a self-guided practical program to introduce students of clinical biochemistry to the use of bioinformatic resources. The activities comprised genomic, transcriptomic and proteomic analysis of a gene with biomedical implications. The design of specific primers for the amplification of a fragment of the gene was proposed as a technological application. The RasMol program, version 2.7.2 (” by Herbert Bernstein 1998-2000) was used to analyses biological functions. In groups of three or four, students studying cellular and molecular biology as part of their degree in biochemistry at the National University of Misiones were asked to provide concrete answers to questions using bioinformatic analysis. The results showed that 100% of the students were able to answer all the questions. However, a wider-ranging treatment of molecular visualization programs is needed for future applications (AU)