RESUMEN
G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag. However, this approach is limited by a low-labeling degree impeding precise visualization in specific subcellular regions. Herein, we present a new visualization strategy based on the immuno-recognition of 5-bromo-2'-deoxyuridine (5-BrdU) modified G4 ligands, functionalized prior- or post-G4-target binding by CuAAC. Remarkably, recognition of the tag by antibodies leads to the detection of the modified ligands exclusively when bound to a G4 target both in vitro, as shown by ELISA, and in cells, thereby providing a highly efficient G4-ligand Guided Immunofluorescence Staining (G4-GIS) approach. The obtained signal amplification revealed well-defined fluorescent foci located in the perinuclear space and RNase treatment revealed the preferential binding to G4-RNA. Furthermore, ligand treatment affected significantly BG4 foci formation in cells. Our work headed to the development of a new imaging approach combining the advantages of immunostaining and G4-recognition by G4 ligands leading to visualization of G4/ligands species in cells with unrivaled precision and sensitivity.
Asunto(s)
Bromodesoxiuridina , Técnica del Anticuerpo Fluorescente/métodos , G-Cuádruplex , Células A549 , Línea Celular , Química Clic , Ensayo de Inmunoadsorción Enzimática , Transferencia Resonante de Energía de Fluorescencia , Humanos , LigandosRESUMEN
Guanine-rich DNA can form four-stranded structures called G-quadruplexes (G4s) that can regulate many biological processes. Metal complexes have shown high affinity and selectivity toward the quadruplex structure. Here, we report the comparison of a panel of platinum (II) complexes for quadruplex DNA selective recognition by exploring the aromatic core around terpyridine derivatives. Their affinity and selectivity towards G4 structures of various topologies have been evaluated by FRET-melting (Fluorescence Resonance Energy Transfert-melting) and Fluorescent Intercalator Displacement (FID) assays, the latter performed by using three different fluorescent probes (Thiazole Orange (TO), TO-PRO-3, and PhenDV). Their ability to bind covalently to the c-myc G4 structure in vitro and their cytotoxicity potential in two ovarian cancerous cell lines were established. Our results show that the aromatic surface of the metallic ligands governs, in vitro, their affinity, their selectivity for the G4 over the duplex structures, and platination efficiency. However, the structural modifications do not allow significant discrimination among the different G4 topologies. Moreover, all compounds were tested on ovarian cancer cell lines and normal cell lines and were all able to overcome cisplatin resistance highlighting their interest as new anticancer drugs.
Asunto(s)
G-Cuádruplex/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Platino (Metal)/química , Proteínas Proto-Oncogénicas c-myc/química , Anticarcinógenos/química , Anticarcinógenos/uso terapéutico , Cisplatino/efectos adversos , Cisplatino/química , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Humanos , Ligandos , Conformación de Ácido Nucleico/efectos de los fármacos , Piridinas/químicaRESUMEN
We report a new turn-off fluorescent probe, PhenDV, for the identification of high affinity quadruplex (G4) DNA ligands. This push-pull fluorophore displays a high fluorescence quantum yield in water (ΦF = 0.21) and is a selective and strong quadruplex DNA binder. We describe its use as a fluorescent indicator for the G4 Fluorescent Intercalator Displacement (FID) assay as its fluorescence is strongly quenched when bound to G4 DNA and fully restored when displaced by ligand. This probe improves the sensitivity of the G4-FID assay, as the read out relies on increased fluorescence instead of quenching observed with classical on/off probes.
Asunto(s)
ADN/química , Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/química , G-Cuádruplex , Ligandos , Límite de DetecciónRESUMEN
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.
Asunto(s)
Linfocitos B/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Interacciones Huésped-Patógeno , Evasión Inmune/genética , Fosfoproteínas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Aminoquinolinas/farmacología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/virología , Línea Celular Tumoral , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , G-Cuádruplex , Células HCT116 , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/inmunología , Humanos , Leontopithecus , Ligandos , Fosfoproteínas/inmunología , Ácidos Picolínicos/farmacología , Quinolinas/farmacología , ARN Mensajero/inmunología , Proteínas de Unión al ARN/inmunología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , NucleolinaRESUMEN
G-Quadruplex(es) (G4) are noncanonical 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 localization of PhenDC3 , one of the most powerful G4 ligands, by synthesizing two clickable azide and alkyne derivatives (PhenDC3 -alk, PhenDC3 -az) and labeling 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 CuI /alkyne intermediates in the nonspecific localization of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labeling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localize drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.
RESUMEN
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.
Asunto(s)
Aminoquinolinas/metabolismo , G-Cuádruplex , Guanosina/metabolismo , Oligonucleótidos/metabolismo , Ácidos Picolínicos/metabolismo , Piridinas/metabolismo , Quinolinas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Aminoquinolinas/química , Sitios de Unión , Dicroismo Circular , Guanosina/química , Ligandos , Modelos Moleculares , Oligonucleótidos/química , Ácidos Picolínicos/química , Potasio/química , Potasio/metabolismo , Piridinas/química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
CD45 is a pan-leukocyte protein with tyrosine phosphatase activity involved in the regulation of signal transduction in hematopoiesis. Exploiting CD45 KO mice and lentiviral shRNA, we prove the crucial role that CD45 plays in acute myeloid leukemia (AML) development and maintenance. We discovered that CD45 does not colocalize with lipid rafts on murine and human non-transformed hematopoietic cells. Using a mouse model, we proved that CD45 positioning within lipid rafts is modified during their oncogenic transformation to AML. CD45 colocalized with lipid rafts on AML cells, which contributes to elevated GM-CSF signal intensity involved in proliferation of leukemic cells. We furthermore proved that the GM-CSF/Lyn/Stat3 pathway that contributes to growth of leukemic cells could be profoundly affected, by using a new plasma membrane disrupting agent, which rapidly delocalized CD45 away from lipid rafts. We provide evidence that this mechanism is also effective on human primary AML samples and xenograft transplantation. In conclusion, this study highlights the emerging evidence of the involvement of lipid rafts in oncogenic development of AML and the targeting of CD45 positioning among lipid rafts as a new strategy in the treatment of AML.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Microdominios de Membrana/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Femenino , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Hematopoyesis/genética , Humanos , Lentivirus/genética , Leucemia Mieloide Aguda/patología , Antígenos Comunes de Leucocito/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AS1411 is a g-quadruplex-forming aptamer capable of selectively entering cancer cells by nucleolin receptor-mediated uptake. In this study, we investigated the cell internalization properties and plasticity of AS1411 carrying different locked nucleic acid-containing cargo oligonucleotides (ONs) for delivery into A549 and U2OS cells. We found that internalization efficiency is highly governed by ON cargo chemistry and composition since the inherent antitumor properties of AS1411 were lost when attached to a nontoxic ON, noTox. However, a toxic ON, Tox, demonstrated potent cytotoxicity after aptamer-mediated uptake in A549 cells. We also examined the effect of unlocked nucleic acid (UNA) modifications in the loop region of the aptamer, and how the cargo ONs and UNA incorporation affect the secondary structure of AS1411, in the presence or absence of two novel ellipticine derivatives. These findings add new insights to the design and future applications of aptamer-guided delivery of ON cargo to cancer cells.
Asunto(s)
Aptámeros de Nucleótidos/administración & dosificación , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Oligodesoxirribonucleótidos/administración & dosificación , Células A549 , Aptámeros de Nucleótidos/efectos adversos , Aptámeros de Nucleótidos/química , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , G-Cuádruplex , Humanos , Neoplasias/genética , Oligodesoxirribonucleótidos/genética , Oligonucleótidos/administración & dosificación , Oligonucleótidos/químicaRESUMEN
Telomeres are nucleoprotein structures at the end of chromosomes which stabilize and protect them from nucleotidic degradation and end-to-end fusions. The G-rich telomeric single-stranded DNA overhang can adopt a four-stranded G-quadruplex DNA structure (G4). Stabilization of the G4 structure by binding of small molecule ligands enhances radiosensitivity of tumor cells, and this combined treatment represents a novel anticancer approach. We studied the effect of the platinum-derived G4-ligand, Pt-ctpy, in association with radiation on human glioblastoma (SF763 and SF767) and non-small cell lung cancer (A549 and H1299) cells in vitro and in vivo. Treatments with submicromolar concentrations of Pt-ctpy inhibited tumor proliferation in vitro with cell cycle alterations and induction of apoptosis. Non-toxic concentrations of the ligand were then combined with ionizing radiation. Pt-ctpy radiosensitized all cell lines with dose-enhancement factors between 1.32 and 1.77. The combined treatment led to increased DNA breaks. Furthermore, a significant radiosensitizing effect of Pt-ctpy in mice xenografted with glioblastoma SF763 cells was shown by delayed tumor growth and improved survival. Pt-ctpy can act in synergy with radiation for efficient killing of cancer cells at concentrations at which it has no obvious toxicity per se, opening perspectives for future therapeutic applications.
Asunto(s)
Neoplasias Encefálicas/terapia , Carcinoma de Pulmón de Células no Pequeñas/terapia , ADN de Neoplasias/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Glioblastoma/terapia , Neoplasias Pulmonares/terapia , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/radioterapia , Xenoinjertos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Ratones , Telomerasa/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
G-quadruplex (G4) structures in intron 3 of the p53 pre-mRNA modulate intron 2 splicing, altering the balance between the fully spliced p53 transcript (FSp53, encoding full-length p53) and an incompletely spliced transcript retaining intron 2 (p53I2 encoding the N-terminally truncated Δ40p53 isoform). The nucleotides forming G4s overlap the polymorphism rs17878362 (A1 wild-type allele, A2 16-base pair insertion) which is in linkage disequilibrium with rs1642785 in intron 2 (c.74+38 G>C). Biophysical and biochemical analyses show rs17878362 A2 alleles form similar G4 structures as A1 alleles although their position is shifted with respect to the intron 2 splice acceptor site. In addition basal FSp53 and p53I2 levels showed allele specific differences in both p53-null cells transfected with reporter constructs or lymphoblastoid cell lines. The highest FSp53 and p53I2 levels were associated with combined rs1642785-GG/rs17878362-A1A1 alleles, whereas the presence of rs1642785-C with either rs17878362 allele was associated with lower p53 pre-mRNA, total TP53, FSp53 and p53I2 levels, due to the lower stability of transcripts containing rs1642785-C. Treatment of lymphoblastoid cell with the G4 binding ligands 360A or PhenDC3 or with ionizing radiation increased FSp53 levels only in cells with rs17878362 A1 alleles, suggesting that under this G4 configuration full splicing is favoured. These results demonstrate the complex effects of intronic TP53 polymorphisms on G4 formation and identify a new role for rs1642785 on mRNA splicing and stability, and thus on the differential expression of isoform-specific transcripts of the TP53 gene.
Asunto(s)
Adenocarcinoma/genética , Empalme Alternativo/genética , Neoplasias de la Mama/genética , G-Cuádruplex/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Intrones/genética , Polimorfismo Genético/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/patología , Adenocarcinoma/radioterapia , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Exones , Femenino , Genotipo , Humanos , Isoformas de Proteínas , ARN Mensajero/genética , Radiación Ionizante , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
A number of small molecules demonstrate selective recognition of G-quadruplexes and are able to stabilize their formation. In this work, we performed the synthesis of two biotin-tagged G4 ligands and analyzed their interactions with DNA by two complementary techniques, FRET and FID. The compound that exhibited the best characteristics (a biotin pyridocarboxamide derivative with high stabilization of an intramolecular quadruplex and excellent duplex-quadruplex specificity) was used as bait for in vitro selection (SELEX). Among 80 DNA aptamer sequences selected, only a small minority (5/80) exhibited G4-prone motifs. Binding of consensus candidates was confirmed by SPR. These results indicate that G4 ligands that appear highly specific when comparing affinities or stabilization for one quadruplex against one duplex, do not only bind quadruplex sequences but may also recognize other nucleic motifs as well. This observation may be relevant when whole genome or transcriptome analysis of binding sites is seeked for, as unexpected binding sites may also be present.