1,721,054 research outputs found
Mechanistic roles of R-loops and micronuclei in the innate immune response induced by anticancer G-quadruplex binders
No full textAlmost 1000 compounds are known to bind specifically to G-quadruplexes (G4), a type of non-canonical DNA structure. They
were developed to discover new anticancer drugs, as non-canonical DNA structures are considered promising oncological
targets. Nevertheless, few entered into clinical trials and none showed efficacy in patients.
Recently, my group reported that PDS, a specific G4 ligand, stimulates micronuclei formation in human U2OS cells and that the
mechanism of micronuclei generation involves an accumulation of double-strand DNA breaks (DSB) due to increased levels of
R-loops, another type of non-canonical DNA/RNA structures. Micronuclei can be a source of cytoplasmatic DNAs that can
activate Interferon-stimulated genes (ISG) and innate immunity response. As we have data supporting that PDS can indeed
activate ISG in human cancer cells in a STING-dependent manner, my hypothesis is that ligand-stabilized G4s cause R-loopmediated
DSBs, triggering micronuclei formation and ISG activation, which is likely mediated by cytoplasmic DNA sensors
and the STING pathway. This may result in a significant stimulation of an immune response against the tumour.
Here, I pursue a line of investigation to discover new anticancer G4 ligands that is not based on improving the cytotoxic
potency, but rather on improving the ability to elicit immuno-stimulatory pathways in cancer cells. My specific aims are:
- to define the roles of R-loops and DNA repair factors in nuclear PDS-triggered DNA repair mechanisms leading to
micronuclei generation in cancer cells;
- to establish the role of the cytoplasmic DNA sensors and related-pathways in PDS-induced immune response in human
cancers and cell lines;
- to discover new G4 ligand hits with optimised activity in inducing micronuclei/immune response in cancer cells.
The project is divided into four work-packages, three addressing specific molecular and genetic aspects of the mechanism, and
one focused on the discovery of effective G4 ligands as immune stimulation and anti-tumor agents. By using NGS and CRISPR
technologies, we will investigate the mechanisms leading from DSBs to micronuclei, including the roles of replication and
repair pathways, and then ISG activation in several cancer cell types. By bioinformatics, we will investigate nucleic acid
immunity gene mutations in human cancers. Structure-activity studies will define structural determinants of immuno-stimulation
activity of G4 ligands. We will use a murine tumor model to demonstrate the stimulation of the immune system against the
tumor by G4 ligands.
The results will demonstrate the role of R-loops in DSB formation at functional genomic regions, and the DNA repair factors in
micronuclei generation. In particular, we will define the role played by BRCA1/2 gene in micronuclei formation. In addition, the
findings will establish the main cytoplasmatic pathway that leads to the activation of ISG in cancer cells. Investigating a new
pharmacophore, we expect to find more effective and potent G4 ligands in activating ISG in cancer cells
If successful, the project will discover new ligand hits with the potential to optimise clinical immunotherapeutic protocols in
unresponsive cancers. The results will establish a new rationale to discover G4 ligands, or other chemicals, as effective drugs in
human cancer patients
Highly Purified Top1-Bound DNA Fragments
No full textTopoisomerase 1–DNA cleavage complexes (Top1ccs) form at DNA sites of Top1 activity and are increased by a highly specific anticancer drug (camptothecin, CPT) in living cells. Various methods are available to detect Top1ccs in cultured cells, including protocols based on the use of specific antibodies. Here, we describe a protocol to isolate Top1ccs at high purity, which does not depend on antibodies
G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders
Full text linkGenomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs
Biotecnologie e modificazioni genetiche. Scienza, etica, diritto
No full textIl volume presenta una trattazione degli aspetti etico-filosofici e socio-giuridici delle biotecnologie. Il primo capitolo intende porre l’attenzione sugli aspetti scientifici che fanno riferimento alla teoria evolutiva, ai concetti di mutazione, diversità e norma, per arrivare a interpretare e comprendere l’attuale scenario degli effetti prodotti dalle innovazioni biotecnologiche in un’ottica sia scientifica che storica. Il secondo capitolo è centrato sulla illustrazione dei principali strumenti caratterizzanti il ragionamento bioetico e biogiuridico: si parte dalle teorie, i valori, le regole e le norme fondamentali che contraddistinguono l’evoluzione e l’applicazione della riflessione bioetica, per arrivare ai dilemmi e ai problemi emergenti dallo sviluppo scientifico, biomedico e biotecnologico. A seguire tre capitoli di taglio specifico, rispettivamente centrati sulle biotecnologie in ambito biomedico, animale e ambientale, dove la trattazione scientifica e la riflessione bioetico-giuridica, presentate nei primi due capitoli, sono riprese e applicate con la finalità di comprendere e interpretare le potenzialità, opportunità, criticità e gli interrogativi emergenti a livello di dibattito bioetico
Abstract 510: Topoisomerase IIβ silencing increases R loops at specific genomic loci associated with an increase of γH2AX and cell cycle progression delay in human cancer cells
No full textAbstract
DNA topoisomerases (Top) are important players in maintaining the genome stability of cells by removing negative and positive DNA supercoils during transcription, replication and other DNA transactions. Recent evidence demonstrated that Top 1 silencing and poisoning by camptothecin affect the formation of R-loops, which are RNA/DNA hybrid structures involved in genome instability and are favored by negative supercoils of the DNA. The findings overall demonstrate that Top 1 reduces R loop levels during transcription whereas it favors R loop formation at early origins of DNA replication. Here, we address the question of whether DNA topoisomerase IIβ can also contribute to steady-state levels of R loop structures in the genome of U2OS cancer cells. We have then investigated the effects of Top2β silencing and Top2 poisons, doxorubicin and etoposide, on R loops and DNA damage in human U2OS cancer cells. Similarly to published data with camptothecin (Marinello et al., Nucleic Acids Research, 2013), Top2 poisons increase cellular R loops by IF after short treatment times and reduce them after 1 hour of treatment. The bi-phasic effect of poisons is mainly dependent on transcription. Moreover, we have determined the changes of R-loop levels by IF after Top2β silencing and the findings demonstrate that the enzyme can strongly modulate the formation of R loops as a full Top2β depletion increases nuclear R-loop levels. In addition, Top2β depletion leads to a slight increase of phosphorylation of H2AX histone along with cell cycle delay and eventually cell death. Thus, Top2β depletion can trigger genomic DNA breakage through alterations of R-loops. In order to establish which are the genomic regions of altered R loop levels, we have mapped Top2β-dependent R loop alterations by the DRIP method showing that specific genomic sites are affected by Top2β. Bioinformatic analyses of R loop maps in U2OS cells will be presented and discussed at the meeting. Altogether the findings demonstrate a critical role of Top2β in governing R loop structures in human cancer cells indicating that DNA torsional tension is a main driving factor of R loop formation and hence genome instability in cancer cells.
Citation Format: Maria Delcuratolo, Jessica Marinello, Giovanni Capranico. Topoisomerase IIβ silencing increases R loops at specific genomic loci associated with an increase of γH2AX and cell cycle progression delay in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2017-510</jats:p
Genome instability by G-quadruplex ligands are mediated by R-loops in human cancer cells
No full text
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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