Galbiati A.,The FIRC Institute of Molecular Oncology Foundation |
Beausejour C.,CHU Ste Justine |
d'Adda di Fagagna F.,The FIRC Institute of Molecular Oncology Foundation |
d'Adda di Fagagna F.,CNR Institute of Molecular Genetics
Aging Cell | Year: 2017
The DNA damage response (DDR) arrests cell cycle progression until DNA lesions, like DNA double-strand breaks (DSBs), are repaired. The presence of DSBs in cells is usually detected by indirect techniques that rely on the accumulation of proteins at DSBs, as part of the DDR. Such detection may be biased, as some factors and their modifications may not reflect physical DNA damage. The dependency on DDR markers of DSB detection tools has left questions unanswered. In particular, it is known that senescent cells display persistent DDR foci, that we and others have proposed to be persistent DSBs, resistant to endogenous DNA repair activities. Others have proposed that these peculiar DDR foci might not be sites of damaged DNA per se but instead stable chromatin modifications, termed DNA-SCARS. Here, we developed a method, named ‘DNA damage in situ ligation followed by proximity ligation assay’ (DI-PLA) for the detection and imaging of DSBs in cells. DI-PLA is based on the capture of free DNA ends in fixed cells in situ, by ligation to biotinylated double-stranded DNA oligonucleotides, which are next recognized by antibiotin anti-bodies. Detection is enhanced by PLA with a partner DDR marker at the DSB. We validated DI-PLA by demonstrating its ability to detect DSBs induced by various genotoxic insults in cultured cells and tissues. Most importantly, by DI-PLA, we demonstrated that both senescent cells in culture and tissues from aged mammals retain true unrepaired DSBs associated with DDR markers. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
Lechouane F.,University of Limoges |
Bonaud A.,University of Limoges |
Delpy L.,University of Limoges |
Casola S.,The FIRC Institute of Molecular Oncology Foundation |
And 5 more authors.
European Journal of Immunology | Year: 2013
B-cell terminal differentiation into antibody secreting plasma cells (PCs) features a trans-criptional shift driven by the activation of plasma cell lineage determinants such as Blimp-1 and Xbp-1, together with the extinction of Pax5. Little is known about the signals inducing this change in transcriptional networks and the role of the B-cell receptor (BCR) in terminal differentiation remains especially controversial. Here, we show that tonic BCR signal strength influences PC commitment in vivo. Using immuno-globulin light chain transgenic mice expressing suboptimal surface BCR levels and latent membrane protein 2A knock-in animals with defined BCR-like signal strengths, we show that weak, antigen-independent constitutive BCR signaling facilitates spontaneous PC differentiation in vivo and in vitro in response to TLR agonists or CD40/IL-4. Conversely, increasing tonic signaling completely prevents this process that is rescued by lowering surface BCR expression or through the inhibition of Syk phosphorylation. These findings provide new insights into the role of basal BCR signaling in PC differentiation and point to the need to resolve a strong BCR signal in order to guarantee terminal differentiation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Penco A.,University of Genoa |
Malabarba M.G.,The FIRC Institute of Molecular Oncology Foundation |
Malabarba M.G.,University of Milan |
Relini A.,University of Genoa |
And 2 more authors.
Human molecular genetics | Year: 2014
The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid β-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in β-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org.
Kumar R.,IFOM p53Lab Joint Research Laboratory |
Kumar R.,Nanyang Technological University |
Cheok C.F.,IFOM p53Lab Joint Research Laboratory |
Cheok C.F.,The FIRC Institute of Molecular Oncology Foundation |
And 2 more authors.
DNA Repair | Year: 2014
DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation of DSBs results in several detrimental effects in various cellular processes which can lead to neurological, immunological and developmental disorders. Failure of the repair of DSBs spurs mutagenesis and is a driver of tumorigenesis, thus underscoring the importance of the accurate repair of DSBs. Two major canonical DSB repair pathways are the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key mediator proteins which coordinate with other components of the DNA repair machinery in the NHEJ and HR pathways respectively, and their exclusive recruitment to DNA breaks/ends potentially decides the choice of repair by either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an important component of the DNA repair pathway which acts downstream of the ATM/53BP1 to inhibit the 5'-3' end resection of broken DNA ends, in-turn facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is conserved from yeast to humans but its function has evolved from telomere length regulation in yeast to the maintenance of genome integrity in mammalian cells. Recently its role in the maintenance of genomic integrity has been expanded to include the regulation of chromatin structure, replication timing and intra-S phase checkpoint. We present a summary of these important findings highlighting the various aspects of Rif1 functions and discuss the key implications for genomic integrity. © 2013 The Authors.
PubMed | CNR Institute of Molecular Genetics, National and Kapodistrian University of Athens and The FIRC Institute of Molecular Oncology Foundation
Type: Journal Article | Journal: Cell death and differentiation | Year: 2014
Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.
Feng Y.,University of Bristol |
Santoriello C.,The FIRC Institute of Molecular Oncology Foundation |
Mione M.,The FIRC Institute of Molecular Oncology Foundation |
Hurlstone A.,University of Manchester |
Martin P.,University of Bristol
PLoS Biology | Year: 2010
It has not previously been possible to live image the earliest interactions between the host environment and oncogenetransformed cells as they initiate formation of cancers within an organism. Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin. Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden. Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages. We show that a major component of the signal drawing inflammatory cells to oncogenic HRASG12V-transformed cells is H2O2, which is also a key damage cue responsible for recruiting neutrophils to a wound. Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair. These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble nonhealing wounds. © 2010 Feng et al.
Cappellari O.,University College London |
Cappellari O.,University of Rome La Sapienza |
Cappellari O.,San Raffaele Hospital |
Benedetti S.,University College London |
And 12 more authors.
Developmental Cell | Year: 2013
Pericytes are endothelial-associated cells that contribute to vessel wall. Here, we report that pericytes may derive from direct conversion of committed skeletal myoblasts. When exposed to Dll4 and PDGF-BB, but not Dll1, skeletal myoblasts downregulate myogenic genes, except Myf5, and upregulate pericyte markers, whereas inhibition of Notch signaling restores myogenesis. Moreover, when cocultured with endothelial cells, skeletal myoblasts, previously treated with Dll4 and PDGF-BB, adopt a perithelial position stabilizing newly formed vessel-like networks in vitro and in vivo. In a transgenic mouse model in which cells expressing MyoD activate Notch, skeletal myogenesis is abolished and pericyte genes are activated. Even if overexpressed, Myf5 does not trigger myogenesis because Notch induces Id3, partially sequestering Myf5 and inhibiting MEF2 expression. Myf5-expressing cells adopt a perithelial position, as occasionally also observed in wild-type (WT) embryos. These data indicate that endothelium, via Dll4 and PDGF-BB, induces a fate switch in adjacent skeletal myoblasts. © 2013 Elsevier Inc.
Corada M.,The FIRC Institute of Molecular Oncology Foundation |
Nyqvist D.,The FIRC Institute of Molecular Oncology Foundation |
Orsenigo F.,The FIRC Institute of Molecular Oncology Foundation |
Caprini A.,The FIRC Institute of Molecular Oncology Foundation |
And 7 more authors.
Developmental Cell | Year: 2010
The Wnt/β-catenin pathway is evolutionary conserved signaling system that regulates cell differentiation and organogenesis. We show that endothelial specific stabilization of Wnt/β-catenin signaling alters early vascular development in the embryo. The phenotype resembles that induced by upregulation of Notch signaling, including lack of vascular remodeling, altered elongation of the intersomitic vessels, defects in branching, and loss of venous identity. Both in vivo and in vitro data showthat β-catenin upregulates Dll4 transcription and strongly increases Notch signaling in the endothelium, leading to functional and morphological alterations. The functional consequences of β-catenin signaling depend on the stage of vascular development and are lost when a gain-of-function mutation is induced at a late stage of development or postnatally. Our findings establish a link between Wnt and Notch signaling in vascular development. We propose that early and sustained β-catenin signaling prevents correct endothelial cell differentiation, altering vascular remodeling and arteriovenous specification. © 2010 Elsevier Inc.
Casola S.,The FIRC Institute of Molecular Oncology Foundation
Methods in molecular biology (Clifton, N.J.) | Year: 2010
In 1991, Soriano and coworkers isolated the ROSA26 locus in a gene-trap mutagenesis screening performed in mouse embryonic stem (ES) cells. The ubiquitous expression of ROSA26 in embryonic and adult tissues, together with the high frequency of gene-targeting events observed at this locus in murine ES cells has led to the establishment in the past 10 years of over 130 knock-in lines expressing successfully from the ROSA26 locus a variety of transgenes including reporters, site-specific recombinases and, recently, noncoding RNAs. Different strategies can be employed to drive transgene expression from the ROSA26 locus. This chapter provides an overview of the current methodologies used to generate ROSA26 knock-in lines and describes different approaches that exploit the ROSA26 gene to control expression of transgenes, including miRNAs, in a temporal, cell-type, and stage-specific fashion.
PubMed | The FIRC Institute of Molecular Oncology Foundation
Type: Journal Article | Journal: Nucleic acids research | Year: 2012
Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro, when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology.