Publications de l’unité
Année de publication : 2017
A common intronic variant of PARP1 confers melanoma risk and mediates melanocyte growth via regulation of MITF.Nature genetics : 49 : 1326-1335 : DOI : 10.1038/ng.3927 En savoir plus
Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a ∼100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (-/GGGCCC; r(2) = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAF(V600E)-induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT-immortalized melanocytes expressing BRAF(V600E). PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, highlighting a new role for PARP1 in melanomagenesis.Replier
Chemical Imaging by NanoSIMS Provides High- Resolution Localization of the G-Quadruplex Interactive Drug (Br)-PhenDC3 on Human ChromosomesJournal of Molecular Biology and Molecular Imaging : 4 : 1029 En savoir plus
Determining the distribution of biologically active compounds within cells is a major issue to understand their mechanism of action and to optimize their properties. Over the past decade DNA secondary structures called G-quadruplexes (G4) have been identified as key modulators of genomic functions. This very active research field has led to the development of G4-targeted molecular probes that are used to track quadruplex forming domains in cells, which is achieved, in most cases, by conventional fluorescence microcopy. However, the intrinsic low resolution of fluorescence microcopy as well as the necessity to tag the drugs with fluorophores represent strong limitations. Here we present the use of secondary ion mass spectroscopy imaging (nanoSIMS) for mapping within metaphase human chromosomes the distribution of a bromobisquinolinium phenanthroline derivative (Br- PhenDC3) used as G-quadruplex probe. In addition a statistical approach to increase the accuracy and the spatial resolution of the nanoSIMS imaging was implemented as a plug in for the image analysis software ImageJ. The results demonstrate the presence of Br-PhenDC3both at terminal and interstitial regions of chromosomes and constitute a demonstration of the effectiveness of nanoSIMS imaging as an alternative method for accurate genome-wide mapping of DNA interactive drugs.Replier
Copper-alkyne complexation is responsible for the Nucleolar Localisation of Quadruplex Nucleic Acid Drugs Labelled by Click Chemistry.Angewandte Chemie (International ed. in English) : 56 : 11365-11369 : DOI : 10.1002/anie.201703783 En savoir plus
G-quadruplex(es) (G4) are non-canonical nucleic acid structures found in guanine-rich sequences. They can be targeted with small molecules (G4-ligands) acting as reporters, for tracking both in vitro and in cells. We explored the cellular localisation of PhenDC3, one of the most powerful G4 ligands, by synthesising two clickable azide and alkyne derivatives (PhenDC3-alk, PhenDC3-az) and labelling them in situ with the corresponding Cy5 click partners. A careful comparison of the results obtained for the copper-based CuAAC and copper-free SPAAC methodologies in fixed cells implicated Cu(I) /alkyne intermediates in the non-specific localisation of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labelling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localise drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.Replier
Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA.Nature communications : 8 : 16043 : DOI : 10.1038/ncomms16043 En savoir plus
The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1, which is essential for viral genome maintenance but highly antigenic. EBV has seemingly evolved a system in which the mRNA sequence encoding the glycine-alanine repeats (GAr) of the EBNA1 protein limits its expression to the minimal level necessary for function while minimizing immune recognition. Here, we identify nucleolin (NCL) as a host factor required for this process via a direct interaction with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL enhances GAr-based inhibition of EBNA1 protein expression, whereas its downregulation relieves the suppression of both expression and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.Replier
Predictive biomarkers of resistance to hypofractionated radiotherapy in high grade gliomaRADIATION ONCOLOGY : 12 : 123 : DOI : 10.1186/s13014-017-0858-0 En savoir plus
Background: Radiotherapy plays a major role in the management of high grade glioma. However, the radioresistance of glioma cells limits its efficiency and drives recurrence inside the irradiated tumor volume leading to poor outcome for patients. Stereotactic hypofractionated radiotherapy is one option for recurrent high grade gliomas. Optimization of hypofractionated radiotherapy with new radiosensitizing agents requires the identification of robust druggable targets involved in radioresistance.
Methods: We generated 11 xenografted glioma models: 6 were derived from cell lines (1 WHO grade III and 5 grade IV) and 5 were patient derived xenografts (2 WHO grade III and 3 grade IV). Xenografts were treated by hypofractionated radiotherapy (6x5Gy). We searched for 89 biomarkers of radioresistance (39 total proteins, 26 phosphoproteins and 24 ratios of phosphoproteins on total proteins) using Reverse Phase Protein Array.
Results: Both type of xenografted models showed equivalent spectrum of sensitivity and profile of response to hypofractionated radiotherapy. We report that Phospho-EGFR/EGFR, Phospho-Chk1/Chk1 and VCP were associated to resistance to hypofractionated radiotherapy.
Conclusions: Several compounds targeting EGFR or CHK1 are already in clinical use and combining them with stereotactic hypofractionated radiotherapy for recurrent high grade gliomas might be of particular interest.Replier
Translational research in radiobiology in the framework of France HADRON national collaborationTranslational Cancer Research : 6 : S795-S806 : DOI : 10.21037/tcr.2017.06.33 En savoir plus
France HADRON is a French national collaboration that brings together 26 teams in nine different French cities (Lyon, Caen, Clermont-Ferrand, Paris-Orsay, Nice, Toulouse, Marseille, Strasbourg and Grenoble). This infrastructure was created in 2013 and follows the ETOILE and ARCHADE projects; it is presently funded by public money. Ten teams, from different scientific background, using a wide range of approaches, participate in radiobiology researches upon protons and carbon ion therapy in comparison with photons used in classical radiation oncology. These approaches combine the use of multiple biological models of radioresistant tumors and healthy tissue critical for treatment tolerance. Thus, the determinants of the radioresistance of head and neck squamous cell carcinoma (HNSSC), high-grade gliomas, sarcomas including chondrosarcomas and the ubiquitous endothelial cells and fibroblasts are studied. Multiple methodological approaches are implemented. In particular, these include: the extraction of tumor stem cell populations and their radiobiological comparison with the differentiated tumor cells; the studies in hypoxic and normoxic conditions; the development and the use of models of 3D cell cultures that highlight important differences in cell behaviors; the cell biology with the analysis of typical neoplastic mechanisms such as invasive and cell migration processes, as well as late recovery of irradiated tumor cells that reenter proliferation; the molecular biochemistry with analysis of resistance signaling pathways, telomerase regulation and genomic analysis of predictors of radioresistance in retrospective and prospective series of patients (gliomas, HNSSC, sarcomas) and finally the oxidative stress, genotoxicity and inflammatory measurements in normal human cells at early and late times after irradiation. This work allows a better understanding of the mechanisms of cellular and tumor radioresistance, normal tissue toxicity, but they also enable pre-clinical and clinical approaches. Thus, experimental approaches of combinations of pharmacological treatments acting on the identified resistance or toxicity mechanisms with the different types of radiation studied are developed. The comparison between stem cells and differentiated cells is very interesting in this respect. This information, combined with phenomenological data on survival and relative biological effectiveness (RBE), contributes to the adjustment and parameterization of bio-mathematical predictive models of the cellular response such as the Nanox® model. This work enables, on the one hand, to suggest hypotheses of clinical trials aimed at acting on tumor radioresistance; and, on the other hand, to integrate into studies associated with prospective transnational clinical research protocols, comparing carbontherapy vs. photon or proton radiotherapy, in collaboration with the European carbontherapy centers, in particular the Heidelberg Ion Therapy (HIT) in Germany and Centro Nazionale di Adroterapia Oncologica (CNAO) in Italy. The potential of France HADRON in radiobiology is important and can make a useful contribution to this field of research, in particular by pursuing a convergence approach of the methods, the models and the topics investigated.Replier
Association of a Platinum Complex to a G-Quadruplex Ligand Enhances Telomere DisruptionCheminal Research in Toxicology : 30 : 1629-1640 : DOI : 10.1021/acs.chemrestox.7b00131 En savoir plus
Telomeres protect the ends of chromosomes against illegitimate recombination and repair. They can be targets for G-quadruplex ligands and platinum complexes due to their repeated G-rich sequences. Protection of telomeres is ensured by a complex of six proteins, including TRF2, which inhibits the DNA damage response pathway. We analyzed telomere modifications induced in cancer cells by the experimental hybrid platinum complex, Pt-MPQ, comprising both an ethylene diamine monofunctional platinum complex and a G-quadruplex recognition moiety (MPQ). Pt-MPQ promotes the displacement of two telomeric proteins (TRF2 and TRF1) from telomeres, as well as the formation of telomere damage and telomere sister losses, whereas the control compound MPQ does not. This suggests that the platinum moiety potentiates the targeting of the G-quadruplex ligand to telomeres, opening a new perspective for telomere biology and anticancer therapy. Interestingly, the chemotherapy drug cisplatin, which has no specific affinity for G-quadruplex structures, partially induces the TRF2 delocalization from telomeres but produces less telomeric DNA damage, suggesting that this TRF2 displacement could be independent of G-quadruplex recognition.
Triphenylamines Induce Cell Death Upon 2-Photon ExcitationMolecular Imaging : 16 : 1-4 : DOI : 10.1177/1536012117714164 En savoir plus
Photodynamic therapy (PDT) is a promising therapeutic method for several diseases, in particular for cancer. This approach uses a photosensitizer, oxygen, and an external light source to produce reactive oxygen species (ROS) at lethal doses to induce cell death. One drawback of current PDT is the use of visible light which has poor penetration in tissues. Such a limitation could be overcome by the use of novel organic compounds compatible with photoactivation under near-infrared light excitation. Triphenylamines (TPAs) are highly fluorescent compounds that are efficient to induce cell death upon visible light excitation (458 nm), but outside the biological spectral window. Interestingly, we recently showed that TPAs target cytoplasmic organelles of living cells, mainly mitochondria, and induce a high ROS production upon 2-photon excitation (in the 760-860 nm range), leading to a fast apoptosis process. However, we observed significant differences among the tested TPA compounds in terms of cell distribution and time courses of cell death-related events (apoptosis vs necrosis). In summary, TPAs represent serious candidates as photosensitizers that are compatible with 2-photon excitation to simultaneously trigger and imaging cell death although the relationship between their subcellular localization and the cell death mechanism involved is still a matter of debate.Replier
Looking for the Most Suitable Orthotopic Retinoblastoma Mouse Model in Order to Characterize the Tumoral Development.Investigative ophthalmology & visual science : 58 : 3055-3064 : DOI : 10.1167/iovs.17-21760 En savoir plus
Purpose: Because retinoblastoma therapies have many adverse effects, new approaches must be developed and evaluated on animal models. We describe orthotopic xenograft models of retinoblastoma using different strains of mice, suitable for this purpose.
Methods: Human retinoblastoma tumors were established on immunodeficient mice by subcutaneous engraftment of tumors from enucleated eyes. The orthotopic model was obtained by subretinal injections of suspension cells into the right eye of immunodeficient (Swiss-nude, severe combined immunodeficiency [SCID]) and immunocompetent mice (C57BL/6N, B6Albino). In vivo tumor growth was monitored by fundus and spectral-domain optical coherence tomography (SD-OCT) imaging and compared with histology.
Results: Retinal and vitreal tumor growth was achieved both in immunocompetent and immunodeficient strains after the subretinal injection of tumor cells. The best tumor engraftment rate was obtained in the SCID mice (68.8%). No tumor growth was observed in the C57BL/6N strain. Chronic retinal detachment may occur in most strains after the subretinal injection, in particular the Swiss-nude strain, which exhibits retinal degeneration.
Conclusions: The setting up of an orthotopic mouse model depends mainly on the choice of the engrafted cells (cell lines or patient-derived xenografts) but it can also depend on the xenografted mouse strain. Severe combined immunodeficiency mice (an immunodeficient strain) achieved the best tumor engraftment rate (68.8%). However, intraocular tumor growth was also satisfactory (50%) in the immunocompetent strain B6Albino, and this strain will allow to exploit the immune response after a tumor treatment. Both of these strains may therefore be recommended when setting up orthotopic retinoblastoma xenografts.Replier
Efficient conformational sampling and weak scoring in docking programs? Strategy of the wisdom of crowdsJournal of Cheminformatics : 9 : 37 : DOI : 10.1186/s13321-017-0227-x En savoir plus
Background: In drug design, an efficient structure-based optimization of a ligand needs the precise knowledge of the protein-ligand interactions. In the absence of experimental information, docking programs are necessary for ligand positioning, and the choice of a reliable program is essential for the success of such an optimization. The performances of four popular docking programs, Gold, Glide, Surflex and FlexX, were investigated using 100 crystal structures of complexes taken from the Directory of Useful Decoys-Enhanced database.
Results: The ligand conformational sampling was rather efficient, with a correct pose found for a maximum of 84 complexes, obtained by Surflex. However, the ranking of the correct poses was not as efficient, with a maximum of 68 top-rank or 75 top-4 rank correct poses given by Glidescore. No relationship was found between either the sampling or the scoring performance of the four programs and the properties of either the targets or the small molecules, except for the number of ligand rotatable bonds. As well, no exploitable relationship was found between each program performance in docking and in virtual screening; a wrong top-rank pose may obtain a good score that allows it to be ranked among the most active compounds and vice versa. Also, to improve the results of docking, the strengths of the programs were combined either by using a rescoring procedure or the United Subset Consensus (USC). Oddly, positioning with Surflex and rescoring with Glidescore did not improve the results. However, USC based on docking allowed us to obtain a correct pose in the top-4 rank for 87 complexes. Finally, nine complexes were scrutinized, because a correct pose was found by at least one program but poorly ranked by all four programs. Contrarily to what was expected, except for one case, this was not due to weaknesses of the scoring functions.
Conclusions: We conclude that the scoring functions should be improved to detect the correct poses, but sometimes their failure may be due to other varied considerations. To increase the chances of success, we recommend to use several programs and combine their results.Replier
What stoichiometries determined by mass spectrometry reveal about the ligand binding mode to G-quadruplex nucleic acids.Biochimica et Biophysica Acta : 1861 : 1353-1361 : DOI : 10.1016/j.bbagen.2017.01.010 En savoir plus
G-quadruplexes (G4s) have become important drug targets to regulate gene expression and telomere maintenance. Many studies on G4 ligand binding focus on determining the ligand binding affinities and selectivities. Ligands, however, can also affect the G4 conformation. Here we explain how to use electrospray ionization mass spectrometry (ESI-MS) to monitor simultaneously ligand binding and cation binding stoichiometries. The changes in potassium binding stoichiometry upon ligand binding hint at ligand-induced conformational changes involving a modification of the number of G-quartets. We investigated the interaction of three quadruplex ligands (PhenDC3, 360A and Pyridostatin) with a variety of G4s. Electrospray mass spectrometry makes it easy to detect K(+) displacement (interpreted as quartet disruption) upon ligand binding, and to determine how many ligand molecules must be bound for the quartet opening to occur. The reasons for ligand-induced conversion to antiparallel structures with fewer quartets are discussed. Conversely, K(+) intake (hence quartet formation) was detected upon ligand binding to G-rich sequences that did not form quadruplexes in 1mM K(+) alone. This demonstrates the value of mass spectrometry for assessing not only ligand binding, but also ligand-induced rearrangements in the target sequence. This article is part of a Special Issue entitled « G-quadruplex » Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.Replier
Targeting Conformational Activation of CDK2 Kinase.Biotechnology journal : 12 : 1600531 : DOI : 10.1002/biot.201600531 En savoir plus
Cyclin-dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP-binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T-loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP-competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T-loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T-loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.Replier
Copper(II)-Controlled Molecular Glue for Mismatched DNA.ChemBioChem : 18 : 618-622 : DOI : 10.1002/cbic.201600675 En savoir plus
Isothermal hybridization of two DNA strands bearing three thymine-thymine (T-T) mismatches can be brought about in the presence of a stoichiometric amount of a bis-naphthalene macrocycle, 2,7-BisNP-NH. This process can be reverted by addition of a Cu(II) salt due to formation of a dinuclear metal complex which does not bind to DNA. Subsequent sequestration of Cu(II) releases the macrocycle and restores the hybridization state of DNA strands, thus allowing implementation of a fast fluorescent two-state DNA switch.Replier
iGUVs: Preparing Giant Unilamellar Vesicles with a Smartphone and Lipids Easily Extracted from Chicken EggsJournal of Chemical Education : 94 : 644-649 : DOI : 10.1021/acs.jchemed.6b00951 En savoir plus
Since the first report of electroformed micrometer-sized liposomes in the 1980s, giant unilamellar vesicles (GUVs) have generated a lot of interest in the biophysical and biochemical communities. However, their penetration rate in high school or at the undergraduate level is still limited because of the requirement of specialized materials for their fabrication. The main objective of this article is to translate the manufacture of these interesting microsystems from highly specialized research laboratories to general chemistry or biology laboratories with the help of everyday objects. Vesicles are made of lipids, which can easily be extracted from chicken eggs. Once obtained, the lipids can be reassembled to form giant vesicular structures in a sugar/aqueous medium by using a do-it-yourself electroformation device. For that, the homemade electroformation chamber is plugged into the audio output of a smartphone or a tablet, which generates audio signals with variable amplitude and frequency. These GUVs prepared with a smart device (iGUVs) are then resuspended into a salt solution for their visualization under a simple microscope. iGUVs bring the opportunity to teachers to stimulate scientific discussion from a wide variety of scientific disciplines such as colloidal chemistry, biophysical chemistry, statistics and cell biology.Replier
The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein.Scientific reports : 7 : 43992 : DOI : 10.1038/srep43992 En savoir plus
The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria.Replier