RESUMO
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.
Assuntos
Bromodesoxiuridina , Imunofluorescência/métodos , Quadruplex G , Células A549 , Linhagem Celular , Química Click , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência , Humanos , LigantesRESUMO
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.
Assuntos
Quadruplex G/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Platina/química , Proteínas Proto-Oncogênicas c-myc/química , Anticarcinógenos/química , Anticarcinógenos/uso terapêutico , Cisplatino/efeitos adversos , Cisplatino/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Ligantes , Conformação de Ácido Nucleico/efeitos dos fármacos , Piridinas/químicaRESUMO
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.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Feminino , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hematopoese/genética , Humanos , Lentivirus/genética , Leucemia Mieloide Aguda/patologia , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Células A549 , Aptâmeros de Nucleotídeos/efeitos adversos , Aptâmeros de Nucleotídeos/química , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Quadruplex G , Humanos , Neoplasias/genética , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/químicaRESUMO
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.