Institute of Molecular Oncology

Milano, Italy

Institute of Molecular Oncology

Milano, Italy
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Makhija E.,National University of Singapore | Jokhun D.S.,National University of Singapore | Shivashankar G.V.,National University of Singapore | Shivashankar G.V.,Institute of Molecular Oncology
Proceedings of the National Academy of Sciences of the United States of America | Year: 2016

Forces generated by the cytoskeleton can be transmitted to the nucleus and chromatin via physical links on the nuclear envelope and the lamin meshwork. Although the role of these active forces in modulating prestressed nuclear morphology has been well studied, the effect on nuclear and chromatin dynamics remains to be explored. To understand the regulation of nuclear deformability by these active forces, we created different cytoskeletal states in mouse fibroblasts using micropatterned substrates. We observed that constrained and isotropic cells, which lack long actin stress fibers, have more deformable nuclei than elongated and polarized cells. This nuclear deformability altered in response to actin, myosin, formin perturbations, or a transcriptional down-regulation of lamin A/C levels in the constrained and isotropic geometry. Furthermore, to probe the effect of active cytoskeletal forces on chromatin dynamics, we tracked the spatiotemporal dynamics of heterochromatin foci and telomeres. We observed increased dynamics and decreased correlation of the heterochromatin foci and telomere trajectories in constrained and isotropic cell geometry. The observed enhanced dynamics upon treatment with actin depolymerizing reagents in elongated and polarized geometry were regained once the reagent was washed off, suggesting an inherent structural memory in chromatin organization. We conclude that active forces from the cytoskeleton and rigidity from lamin A/C nucleoskeleton can together regulate nuclear and chromatin dynamics. Because chromatin remodeling is a necessary step in transcription control and its memory, genome integrity, and cellular deformability during migration, our results highlight the importance of cell geometric constraints as critical regulators in cell behavior.

Bottos A.,Friedrich Miescher Institute for Biomedical Research | Bardelli A.,University of Turin | Bardelli A.,Institute for Cancer Research and Treatment | Bardelli A.,Institute of Molecular Oncology
Cellular and Molecular Life Sciences | Year: 2013

The acquisition of oncogenic mutations and promotion of angiogenesis are key hallmarks of cancer. These features are often thought of as separate events in tumor progression and the two fields of research have frequently been considered as independent. However, as we highlight in this review, activated oncogenes and deregulated angiogenesis are tightly associated, as mutations in cancer cells can lead to perturbation of the pro- and anti-angiogenic balance thereby causing aberrant angiogenesis. We propose that normalization of the vascular network by targeting oncogenes in the tumor cells might lead to more efficient and sustained therapeutic effects compared to therapies targeting tumor vessels. We discuss how pharmacological inhibition of oncogenes in tumor cells restores a functional vasculature by bystander anti-angiogenic effect. As genetic alterations are tumor-specific, targeted therapy, which potentially blocks the angiogenic program activated by individual oncogenes may lead to personalized anti-angiogenic therapy. © 2013 Springer Basel.

Reczko M.,Institute of Molecular Oncology | Reczko M.,Synaptic Ltd | Maragkakis M.,Institute of Molecular Oncology | Maragkakis M.,Martin Luther University of Halle Wittenberg | And 5 more authors.
Bioinformatics | Year: 2012

Motivation: Experimental evidence has accumulated showing that microRNA (miRNA) binding sites within protein coding sequences (CDSs) are functional in controlling gene expression. Results: Here we report a computational analysis of such miRNA target sites, based on features extracted from existing mammalian high-throughput immunoprecipitation and sequencing data. The analysis is performed independently for the CDS and the 3 '-untranslated regions (3 '-UTRs) and reveals different sets of features and models for the two regions. The two models are combined into a novel computational model for miRNA target genes, DIANA-microT-CDS, which achieves higher sensitivity compared with other popular programs and the model that uses only the 3 '-UTR target sites. Further analysis indicates that genes with shorter 3 '-UTRs are preferentially targeted in the CDS, suggesting that evolutionary selection might favor additional sites on the CDS in cases where there is restricted space on the 3′-UTR. © The Author 2012. Published by Oxford University Press. All rights reserved.

Vlachos I.S.,Institute of Molecular Oncology | Vlachos I.S.,National and Kapodistrian University of Athens | Hatzigeorgiou A.G.,Institute of Molecular Oncology | Hatzigeorgiou A.G.,University of Thessaly
Clinical Biochemistry | Year: 2013

Objective: This review aims to provide a brief introduction to each major category of available tools and algorithms for microRNA (miRNA) research, as well as to present some of the most widely used or promising representative applications. Methods: Only tools offering a fully functional web interface have been included, excluding implementations requiring deployment in local servers or workstations. Furthermore, we have specifically evaluated implementations focusing on Homo sapiens or on mammals used extensively in in vivo research, such as mice and rats. Results: We present an overview of databases and repositories of miRNA sequences and expression, a commentary on miRNA target prediction algorithms, tools for miRNA functional investigation, and online pipelines for the analysis of high throughput experiments. Examples and case studies are provided at the end of the manuscript, which can hopefully contribute in elucidating the utility of these implementations to basic and applied research. Conclusions: Computational tools and algorithms play a significant role in miRNA-related research, supporting equally basic and applied research efforts. However, numerous challenges still remain to be answered by the relevant research community. © 2013 The Canadian Society of Clinical Chemists.

Vergoulis T.,IMIS Institute | Vlachos I.S.,Institute of Molecular Oncology | Vlachos I.S.,National and Kapodistrian University of Athens | Alexiou P.,Institute of Molecular Oncology | And 9 more authors.
Nucleic Acids Research | Year: 2012

As the relevant literature and the number of experiments increase at a super linear rate, databases that curate and collect experimentally verified microRNA (miRNA) targets have gradually emerged. These databases attempt to provide efficient access to this wealth of experimental data, which is scattered in thousands of manuscripts. Aim of TarBase 6.0 ( tarbase) is to face this challenge by providing a significant increase of available miRNA targets derived from all contemporary experimental techniques (gene specific and high-throughput), while incorporating a powerful set of tools in a user-friendly interface. TarBase 6.0 hosts detailed information for each miRNA-gene interaction, ranging from miRNA- and gene-related facts to information specific to their interaction, the experimental validation methodologies and their outcomes. All database entries are enriched with function-related data, as well as general information derived from external databases such as UniProt, Ensembl and RefSeq. DIANA microT miRNA target prediction scores and the relevant prediction details are available for each interaction. TarBase 6.0 hosts the largest collection of manually curated experimentally validated miRNA-gene interactions (more than 65 000 targets), presenting a 16.5-175-fold increase over other available manually curated databases. © The Author(s) 2011. Published by Oxford University Press.

Manzoni R.,Institute of Molecular Oncology | Montani F.,Italian National Cancer Institute | Visintin C.,Italian National Cancer Institute | Caudron F.,ETH Zurich | And 2 more authors.
Journal of Cell Biology | Year: 2010

In budding yeast, the phosphatase Cdc14 orchestrates progress through anaphase and mitotic exit, thereby resetting the cell cycle for a new round of cell division. Two consecutive pathways, Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN), contribute to the progressive activation of Cdc14 by regulating its release from the nucleolus, where it is kept inactive by Cfi1. In this study, we show that Cdc14 activation requires the polo-like kinase Cdc5 together with either Clb-cyclin-dependent kinase (Cdk) or the MEN kinase Dbf2. Once active, Cdc14 triggers a negative feedback loop that, in the presence of stable levels of mitotic cyclins, generates periodic cycles of Cdc14 release and sequestration. Similar phenotypes have been described for yeast bud formation and centrosome duplication. A common theme emerges where events that must happen only once per cycle, although intrinsically capable of oscillations, are limited to one occurrence by the cyclin-Cdk cell cycle engine. © 2010 Manzoni et al.

Roubelakis M.G.,National and Kapodistrian University of Athens | Roubelakis M.G.,University of Oxford | Tsaknakis G.,University of Oxford | Tsaknakis G.,Institute of Molecular Oncology | And 3 more authors.
PLoS ONE | Year: 2013

Human amniotic fluid obtained at amniocentesis, when cultured, generates at least two morphologically distinct mesenchymal stem/stromal cell (MSC) subsets. Of these, the spindle shaped amniotic fluid MSCs (SS-AF-MSCs) contain multipotent cells with enhanced adipogenic, osteogenic and chondrogenic capacity. Here, we demonstrate, for the first time, the capacity of these SS-AF-MSCs to support neovascularization by umbilical cord blood (UCB) endothelial colony forming cell (ECFC) derived cells in both in vitro and in vivo models. Interestingly, although the kinetics of vascular tubule formation in vitro were similar when the supporting SS-AF-MSCs were compared with the best vasculogenic supportive batches of bone marrow MSCs (BMSCs) or human dermal fibroblasts (hDFs), SS-AF-MSCs supported vascular tubule formation in vivo more effectively than BMSCs. In NOD/SCID mice, the human vessels inosculated with murine vessels demonstrating their functionality. Proteome profiler array analyses revealed both common and distinct secretion profiles of angiogenic factors by the SS-AF-MSCs as opposed to the hDFs and BMSCs. Thus, SS-AF-MSCs, which are considered to be less mature developmentally than adult BMSCs, and intermediate between adult and embryonic stem cells in their potentiality, have the additional and very interesting potential of supporting increased neovascularisation, further enhancing their promise as vehicles for tissue repair and regeneration. © 2013 Roubelakis et al.

Longo V.D.,University of Southern California | Longo V.D.,Institute of Molecular Oncology | Panda S.,Salk Institute for Biological Studies
Cell Metabolism | Year: 2016

Most animals alternate periods of feeding with periods of fasting often coinciding with sleep. Upon >24 hr of fasting, humans, rodents, and other mammals enter alternative metabolic phases, which rely less on glucose and more on ketone body-like carbon sources. Both intermittent and periodic fasting result in benefits ranging from the prevention to the enhanced treatment of diseases. Similarly, time-restricted feeding (TRF), in which food consumption is restricted to certain hours of the day, allows the daily fasting period to last >12 hr, thus imparting pleiotropic benefits. Understanding the mechanistic link between nutrients and the fasting benefits is leading to the identification of fasting-mimicking diets (FMDs) that achieve changes similar to those caused by fasting. Given the pleiotropic and sustained benefits of TRF and FMDs, both basic science and translational research are warranted to develop fasting-associated interventions into feasible, effective, and inexpensive treatments with the potential to improve healthspan. © 2016 Elsevier Inc.

Karkali K.,Institute of Molecular Oncology | Panayotou G.,Institute of Molecular Oncology
Biochemical and Biophysical Research Communications | Year: 2012

Dual-Specificity Phosphatases (DUSPs) are enzymes that remove phosphate groups from both phospho-tyrosine and phospho-serine/threonine residues. A subgroup of DUSPs specifically targets Mitogen-Activated Protein Kinases (MAPKs) and has been shown to participate in the regulation of differential cellular responses to the large variety of stimuli conveyed by MAPK-pathways. In Drosophila, Puckered has been identified as a DUSP, exhibiting specificity towards the c-Jun-N-terminal kinase (JNK). Recent studies have signified its role in regulating JNK-dependent processes, including immunity, stress tolerance and longevity. Puckered expression depends on the activation of the JNK pathway whereas it's degradation is mediated by the ubiquitin-proteasome system. In this study we show that Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress and that phosphorylation affects the interaction between Puckered and these MAPKs. In silico analysis of the Puckered amino acid sequence revealed several MAPK consensus phosphorylation motifs. Expression of Puckered in the heterologous system of HEK293 cells and subsequent stimulation with arsenite resulted in reduced mobility of Puckered in SDS-PAGE. Similar results were obtained when Puckered was co-expressed with the constitutively active forms of JNK and p38. This mobility shift was abolished by lambda-phosphatase treatment or by simultaneous inhibition of JNK and p38. Analysis by mass-spectrometry identified Puckered phosphorylation in Ser413, though phosphorylation on this site was found irrespective of stimulation. Finally, phosphorylation of Puckered enhanced its interaction both with JNK and p38. Our results suggest a possible functional role of Puckered phosphorylation by MAPKs. © 2012 Elsevier Inc.

Gkiafi Z.,Institute of Molecular Oncology | Panayotou G.,Institute of Molecular Oncology
Journal of Proteome Research | Year: 2011

Epstein-Barr virus (EBV) infection is a major health problem associated with a variety of diseases, including Burkitt's lymphoma. EBV promotes its effects through the activation of multiple signaling pathways, with NF-κB mediated transcription being a major target. We have undertaken a comparative proteomic approach using 2D-electrophoresis and mass spectrometry to identify EBV-regulated proteins in the BL41 Burkitt's lymphoma cell line. Many of the proteins differentially regulated were previously known mediators of EBV action. Among the novel proteins identified, three members of the conserved COMMD family were found to be down-regulated. Further analysis of this family at the mRNA level, using reverse-transcriptase or real-time PCR, showed that 7 out of 10 COMMD members were affected in EBV-transformed BL41 cells. Since COMMD family proteins have been implicated as negative regulators of the NF-κB transcription factor, our data are consistent with a hypothesis that EBV down-regulates COMMD proteins in order to enhance NF-κB mediated transcriptional events and B-lymphocyte transformation. © 2011 American Chemical Society.

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