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Currently, effective therapeutic modalities for pancreatic ductal adenocarcinoma (PDAC) are quite limited, leading to gloomy prognosis and â¼6-months median patient survival. Recent advances showed the promise of photodynamic therapy (PDT) for PDAC patients. Next generation photosensitizers (PS) are based on "organelle-targeted-PDT" and provide new paradigm in the field of precision medicines to address the current challenge for treating PDAC. In this investigation, we have constructed a novel PS, named as N b B, for precise and simultaneous targeting of endoplasmic reticulum (ER) and lipid droplets (LDs) in PDAC, based on the fact that malignant PDAC cells are heavily relying on ER for hormone synthesis. Our live cell imaging and fluorescence recovery after photobleaching (FRAP) experiments revealed that N b B is quickly targeted to ER and subsequently to LDs and shows simultaneous dual fluorescence color due to polar and nonpolar milieu of ER and LDs. Interestingly, the same molecule generates triplet state and singlet oxygen efficiently and causes robust ER stress and cellular lipid peroxidation, leading to apoptosis in two different PDAC cells in the presence of light. Together, we present, for the first time, a potential next generation precision medicine for ER-LD organelle specific imaging and PDT of pancreatic cancer.
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Designing heavy-atom-free triplet photosensitizers (PSs) is a challenge for the efficient photodynamic therapy (PDT) of cancer. Helicenes are twisted polycyclic aromatic hydrocarbons (PAHs) with an efficient intersystem crossing (ISC) that is proportional to their twisting angle. But their difficult syntheses and weak absorption profile in the visible spectral region restrict their use as heavy-atom-free triplet PSs for PDT. On the other hand, boron-containing PAHs, BODIPYs are highly recognized for their outstanding optical properties. However, planar BODIPY dyes has low ISC and thus they are not very effective as PDT agents. We have designed and synthesized fused compounds containing both BODIPY and hetero[5]helicene structures to develop red-shifted chromophores with efficient ISC. One of the pyrrole units of the BODIPY core was also replaced by a thiazole unit to further enhance the triplet conversion. All the fused compounds have helical structure, and their twisting angles are also increased by substitutions at the boron centre. The helical structures of the BODIPY-hetero[5]helicenes were confirmed by X-ray crystallography and DFT structure optimization. The designed BODIPY-hetero[5]helicenes showed superior optical properties and high ISC with respect to [5]helicene. Interestingly their ISC efficiencies increase proportionally with their twisting angles. This is the first report on the relationship between the twisting angle and the ISC efficiency in twisted BODIPY-based compounds. Theoretical calculations showed that energy gap of the S1 and T1 states decreases in BODIPY-hetero[5]helicene as compared to planar BODIPY. This enhances the ISC rate in BODIPY-hetero[5]helicene, which is responsible for their high generation of singlet oxygen. Finally, their potential applications as PDT agents were investigated, and one BODIPY-hetero[5]helicene showed efficient cancer cell killing upon photo-exposure. This new design strategy will be very useful for the future development of heavy-atom-free PDT agents.
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We report the photophysical properties, self-assembly and biological evaluation of an isothiazolanthrone-based dye, 7-amino-6H-anthra[9,1-cd]isothiazol-6-one (AAT), which reveals anticancer properties and can be potentially used as dye for monitoring cell viability. The solvent-dependent photophysical studies suggest that the emission of AAT is sensitive to environment polarity due to which interesting changes in the colored emission may be observed owing to the charge transfer (CT) processes. AAT also self-assembles to tree-like branched morphologies and produce, a greenish emission inside the cells when imaged after short interval (15 mins) of incubation while a red fluorescence could be noted after 24â h. Interestingly, AAT also produce differential emission inside mouse normal cells as compared to its cancer cell lines since it possess anticancer activity. The experimental observations were also validated theoretically via computational modeling.
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Espectrometría de Fluorescencia , Animales , Ratones , Espectrometría de Fluorescencia/métodos , Supervivencia Celular , Línea Celular , SolventesRESUMEN
STING mediates innate immune responses that are triggered by the presence of cytosolic DNA. Activation of STING to boost antigen recognition is a therapeutic modality that is currently being tested in cancer patients using nucleic-acid based macrocyclic STING ligands. We describe here the discovery of 3,4-dihydroquinazolin-2(1H)-one based 6,6-bicyclic heterocyclic agonists of human STING that activate all known human variants of STING with high potency.
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Antineoplásicos/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Inmunidad Innata/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/síntesis química , Animales , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citosol/química , ADN/química , Haplorrinos , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Unión Proteica , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
The adaptor protein STING plays a major role in innate immune sensing of cytosolic nucleic acids, by triggering a robust interferon response. Despite the importance of this protein as a potential therapeutic target for serious unmet medical conditions including cancer and infectious disease there remains a paucity of STING ligands. Starting with a benzothiazinone series of weak STING activators (human EC50 â¼10 µM) we identified several chemotypes with sub-micromolar STING activity across all the major protein polymorphs. An example compound 53 based on an oxindole core structure demonstrated robust on-target functional activation of STING (human EC50 185 nM) in immortalised and primary cells and a cytokine induction fingerprint consistent with STING activation. Our study has identified several related series of potent small molecule human STING activators with potential to be developed as immunomodulatory therapeutics.
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Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Proteínas de la Membrana/agonistas , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Células Cultivadas , Citocinas/metabolismo , Descubrimiento de Drogas , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Oxindoles/química , Oxindoles/farmacología , Tiazinas/química , Tiazinas/farmacologíaRESUMEN
The cGAS/STING pathway initiates an innate immune response when DNA is detected in the cytosol. DNA bound cGAS synthesizes cyclic dinucleotides which bind and activate the adaptor STING, leading to downstream secretion of Type I interferons and other pro-inflammatory NFκB pathway cytokines. In the mouse, the STING driven innate immune response is central to immune based clearance of various tumors and this has triggered a significant effort focused on the discovery of human STING agonists for the treatment of cancer. This report uses an in vitro kinase assay to show that G10, a previously identified STING pathway activator is actually a weak but direct STING agonist and identifies other more potent leads.
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Proteínas de la Membrana/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/metabolismo , Proteínas de la Membrana/química , Ratones , Fosforilación , Dominios Proteicos , Estabilidad Proteica , Transducción de Señal , Células THP-1RESUMEN
An efficient turn-on fluorescence probe for biomolecules not only helps in its sensitive detection but is also useful to understand the different interactions that are operating between biomolecules and probes. Polycyclic aromatic molecules are known to be strong interacting ligand for DNA and extensively studied as a model cancer drug. However, these molecules show large decrease in their emission intensity, i.e., a turn-off probe for DNA. In the present work, we have synthesized a benzothiazole-based anthracene derivative and studied its interaction with a natural DNA with the aim of having a turn-on DNA probe with a polycyclic aromatic moiety. Our detailed spectroscopic studies show that the new probe strongly interacts with DNA molecules and results in a significant increase in its emission yield. Time-resolved studies show a large increase in probe's excited-state lifetime in DNA solution. Detailed experiments have been performed to understand its mode of interaction with DNA molecules. The mode of interaction has also been supported by the blind molecular docking studies. Further, the viscosity-dependent photophysical properties and detailed quantum chemical calculations confirm that the new probe belongs to molecular rotor class of molecules. Association with DNA molecules results in a significant retardation in the nonradiative deactivation process due to the torsional motion in the excited state of the probe and leads to a significant increase in its emission yield. Thus, due its molecular rotor nature, despite with a turn-off fluorophore unit, anthracene, the new probe, acts as a sensitive turn-on fluorescence probe for DNA molecules.
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Antracenos/química , Benzotiazoles/química , Sondas de ADN/química , ADN/química , Teoría Funcional de la Densidad , Estructura MolecularRESUMEN
Cancer stem cells (CSCs) are a key driver of tumor formation and metastasis, but how they are affected by nanomaterials is largely unknown. The present study investigated the effects of different carbon-based nanomaterials (CNMs) on neoplastic and CSC-like transformation of human small airway epithelial cells and determined the underlying mechanisms. Using a physiologically relevant exposure model (long-term/low-dose) with system validation using a human carcinogen, asbestos, we demonstrated that single-walled carbon nanotubes, multi-walled carbon nanotubes, ultrafine carbon black, and crocidolite asbestos induced particle-specific anchorage-independent colony formation, DNA-strand break, and p53 downregulation, indicating genotoxicity and carcinogenic potential of CNMs. The chronic CNM-exposed cells exhibited CSC-like properties as indicated by 3D spheroid formation, anoikis resistance, and CSC markers expression. Mechanistic studies revealed specific self-renewal and epithelial-mesenchymal transition (EMT)-related transcription factors that are involved in the cellular transformation process. Pathway analysis of gene signaling networks supports the role of SOX2 and SNAI1 signaling in CNM-mediated transformation. These findings support the potential carcinogenicity of high aspect ratio CNMs and identified molecular targets and signaling pathways that may contribute to the disease development.
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An acyclic tetrapodal receptor (L4+-I)(4PF6)4- comprised of four 2-iodo-imidazolium motifs showed moderate to strong binding of halides through halogen bonding interactions in organic and aqueous media, with these binding levels established by performing isothermal titration calorimetry studies. Importantly, single crystals suitable for X-ray diffraction studies were obtained from both water and an acetonitrile-water binary solvent mixture, and exhibited halogen bonding interactions in the solid state.
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In response to DNA double strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that coordinate their detection, signaling and repair. Central to this network is the ATM kinase and its substrates at chromatin surrounding DSBs H2AX, MDC1 and 53BP1. In humans, germline inactivation of ATM causes Ataxia Telangiectasia (A-T), an autosomal recessive syndrome of increased proneness to hematological malignancies driven by clonal chromosomal translocations. Studies of cancers arising in A-T patients and in genetically engineered mouse models (GEMM) deficient for ATM and its substrates have revealed complex, multilayered roles for ATM in translocation suppression and identified functional redundancies between ATM and its substrates in this context. "Programmed" DSBs at antigen receptor loci in developing lymphocytes employ ubiquitous DDR factors for signaling and repair and have been particularly useful for mechanistic studies because they are region-specific and can be monitored in vitro and in vivo. In this context, murine thymocytes deficient for ATM recapitulate the molecular events that lead to transformation in T cells from A-T patients and provide a widely used model to study the mechanisms that suppress RAG recombinase-dependent translocations. Similarly, analyses of the fate of Activation induced Cytidine Deaminase (AID)-dependent DSBs during mature B cell Class Switch Recombination (CSR) have defined the genetic requirements for end-joining and translocation suppression in this setting. Moreover, a unique role for 53BP1 in the promotion of synapsis of distant DSBs has emerged from these studies.
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Roturas del ADN de Doble Cadena , Translocación Genética , Animales , Ataxia Telangiectasia/genética , Citidina Desaminasa/fisiología , Reparación del ADN , Ingeniería Genética , Humanos , Cambio de Clase de Inmunoglobulina/genética , Ratones , Recombinación GenéticaRESUMEN
DNA Damage Response (DDR) and DNA repair pathways are emerging as potent, ubiquitous suppressors of innate immune signaling in human cells. Here, we show that human cells surviving depletion of the Single Strand Break (SSB) repair protein PARP1 undergo p21-dependent senescence or cell cycle checkpoint activation in the context of activation of innate immune signaling, or viral mimicry. Specifically, we observe induction of a large number of interferon-stimulated genes (ISGs) and multiple pattern recognition receptors (PRRs; including RIG-I, MDA-5, MAVS, TLR3 and STING) and increased nuclear IRF3 staining. Mechanistically, depletion of the double-stranded RNA (dsRNA) helicase RIG-I or its downstream effector MAVS specifically rescues ISG induction in PARP1-depleted cells, suggesting that the RIG-I/MAVS pathway is required for sustained ISG expression in this context. Experiments with conditioned media or a neutralizing antibody to the α/ß-IFN receptor revealed that persistent ISG expression additionally requires an autocrine/paracrine loop. Finally, loss of PARP1 and radiation-induced DNA damage strongly synergize in the induction of p21 and ISGs. Overall, these findings increase our understanding of how PARP1 may suppress deleterious phenotypes associated to aging, inflammation and cancer in humans.
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Proliferación Celular , Neoplasias del Colon/patología , Factor 3 Regulador del Interferón/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Receptores de Ácido Retinoico/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Humanos , Interferones/metabolismo , ARN Bicatenario , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Peripheral blood was obtained from a 12-year old male carrying bialleleic inactivating mutations at the ATM locus, causing Ataxia-Telangiectasia (A-T). Blood erythroid cells were briefly expanded in vitro and induced pluripotent stem cells (iPSCs) were generated via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF4, hMYC and hBCL2L1. SF-003 iPSCs were free of genomically integrated reprogramming genes, had the specific compound heterozygous mutations, stable karyotype, expressed pluripotency markers and formed teratomas in immunodeficient (NOD scid gamma; NGS) mice. The SF-003 iPSC line may be a useful resource for in vitro modeling of A-T.
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Ataxia Telangiectasia/patología , Reprogramación Celular , Eritroblastos/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Secuencia de Bases , Diferenciación Celular , Línea Celular , Niño , Análisis Mutacional de ADN , Eritroblastos/metabolismo , Exones , Eliminación de Gen , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Cariotipo , Masculino , Ratones , Ratones Endogámicos NOD , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
Biallelic mutations in ATM result in the neurodegenerative syndrome Ataxia-Telangiectasia, while ATM haploinsufficiency increases the risk of cancer and other diseases. Previous studies revealed low reprogramming efficiency from A-T and carrier fibroblasts, a barrier to iPS cell-based modeling and regeneration. Here, we tested the feasibility of employing circulating erythroid cells, a compartment no or minimally affected in A-T, for the generation of A-T and carrier iPS cells. Our results indicate that episomal expression of Yamanaka factors plus BCL-xL in erythroid cells results in highly efficient iPS cell production in feeder-free, xeno-free conditions. Moreover, A-T iPS cells generated with this protocol maintain long-term replicative potential, stable karyotypes, re-elongated telomeres and capability to differentiate along the neural lineage in vitro and to form teratomas in vivo. Finally, we find that haploinsufficiency for ATM does not limit reprogramming from human erythroid cells or in vivo teratoma formation in the mouse.
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Ataxia Telangiectasia/patología , Reprogramación Celular , Células Eritroides/citología , Células Madre Pluripotentes Inducidas/citología , Adolescente , Animales , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Secuencia de Bases , Diferenciación Celular , Línea Celular , Linaje de la Célula , Análisis Mutacional de ADN , Células Eritroides/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Cariotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Telómero/metabolismo , Acortamiento del Telómero , Teratoma/metabolismo , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.
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Reparación del ADN por Unión de Extremidades , ADN/genética , Histonas/genética , Linfoma/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasas/genética , Neoplasias del Timo/genética , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/patología , Bencimidazoles/farmacología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Cromatina/química , Cromatina/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Proteína Quinasa Activada por ADN/deficiencia , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Puntos de Control de la Fase G2 del Ciclo Celular , Histonas/deficiencia , Humanos , Autoantígeno Ku/deficiencia , Autoantígeno Ku/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/deficiencia , Cultivo Primario de Células , Neoplasias del Timo/metabolismo , Neoplasias del Timo/patologíaRESUMEN
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Genes Supresores de Tumor , Proteínas de la Membrana/metabolismo , Neuroblastoma/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Niño , Femenino , Humanos , Proteínas de la Membrana/genética , Ratones Desnudos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Telomere dysfunction promotes genomic instability and carcinogenesis via inappropriate end-to-end chromosomal rearrangements, or telomere fusions. Previous work indicates that the DNA Damage Response (DDR) factor 53BP1 promotes the fusion of telomeres rendered dysfunctional by loss of TRF2, but is dispensable for the fusion of telomeres lacking Pot1 or critically shortened (in telomerase-deficient mice). Here, we examine a role for 53BP1 at telomeres rendered dysfunctional by loss or catalytic inhibition of DNA-PKcs. Using mouse embryonic fibroblasts lacking 53BP1 and/or DNA-PKcs, we show that 53BP1 deficiency suppresses G1-generated telomere fusions that normally accumulate in DNA-PKcs-deficient fibroblasts with passage. Likewise, we find that 53BP1 promotes telomere fusions during the replicative phases of the cell cycle in cells treated with the specific DNA-PKcs inhibitor NU7026. However, telomere fusions are not fully abrogated in DNA-PKcs-inhibited 53BP1-deficient cells, but occur with a frequency approximately 10-fold lower than in control 53BP1-proficient cells. Treatment with PARP inhibitors or PARP1 depletion abrogates residual fusions, while Ligase IV depletion has no measurable effect, suggesting that PARP1-dependent alternative end-joining operates at low efficiency at 53BP1-deficient, DNA-PKcs-inhibited telomeres. Finally, we have also examined the requirement for DDR factors ATM, MDC1 or H2AX in this context. We find that ATM loss or inhibition has no measurable effect on the frequency of NU7026-induced fusions in wild-type MEFs. Moreover, analysis of MEFs lacking both ATM and 53BP1 indicates that ATM is also dispensable for telomere fusions via PARP-dependent end-joining. In contrast, loss of either MDC1 or H2AX abrogates telomere fusions in response to DNA-PKcs inhibition, suggesting that these factors operate upstream of both 53BP1-dependent and -independent telomere rejoining. Together, these experiments define a novel requirement for 53BP1 in the fusions of DNA-PKcs-deficient telomeres throughout the cell cycle and uncover a Ligase IV-independent, PARP1-dependent pathway that fuses telomeres at reduced efficiency in the absence of 53BP1.
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Proteínas Cromosómicas no Histona/metabolismo , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Mamíferos/metabolismo , Telómero/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular , Cromonas/farmacología , ADN Ligasas/metabolismo , Replicación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN/metabolismo , Embrión de Mamíferos/citología , Epistasis Genética/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fase G1 , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Morfolinas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Fase S/efectos de los fármacos , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Dynamics of metal to ligand charge transfer in the excited states of ruthenium polypyridyl complexes, which have shown promise as materials for artificial solar energy harvesting, has been of immense interest recently. Mixed ligand complexes are especially important for broader absorption in the visible region. Dynamics of ultrafast vibrational energy relaxation and inter-ligand charge transfer processes in the excited states of a heteroleptic ruthenium complex, [Ru(bpy)2(pap)](ClO4)2 (where bpy is 2,2'-bipyridine and pap is 2-(phenylazo)pyridine) have been investigated using femtosecond to nanosecond time-resolved transient absorption spectroscopic techniques. A good agreement between the TA spectrum of the lowest excited (3)MLCT state of [Ru(bpy)2(pap)](ClO4)2 complex and the anion radical spectrum of the pap ligand, which has been generated using the pulse radiolysis technique, confirmed the charge localization at the pap ligand. While the lifetime of the inter-ligand charge transfer from the bpy to the pap ligand in the (3)MLCT state is about 2.5 ps, vibrational cooling of the pap-localized(3)MLCT state occurs over a much longer time scale with a lifetime of about 35 ps. Ultrafast charge localization dynamics observed here may have important consequences in artificial solar energy harvesting systems, which employ heteroleptic ruthenium complexes.
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Lack of affordable technologies for delivering microRNAs and siRNAs into cells on a large scale has hindered our efforts to rapidly parse through hundreds of dysregulated genes/microRNAs in order to identify drivers of complex diseases. The instability and polyanionic nature of naked microRNAs impede efficient cellular uptake and reduce half-life. Viral delivery requires cloning, microRNA mimics/inhibitors require costly modifications, and both require toxic lipofection or electroporation. To address these challenges, we developed a robust method for delivering unmodified microRNAs into cells on cysteamine-functionalized gold nanoparticles (AuNPs). We validated our method in two different tumor models and found that the best formulation of miR(1)-AuNP(10)-S-PEG(0.5) had the highest payload (10-20 fold higher than lipofection), lowest toxicity (98% of cell viability following treatment), efficient uptake (96% of cells took it), fastest endosomal escape and increased half-lives (at least 5 days) impacting cell proliferation and patterns of target gene expression.
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Cisteamina/química , Oro/química , MicroARNs/administración & dosificación , Nanopartículas/química , Neoplasias/genética , Línea Celular Tumoral , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/farmacocinéticaRESUMEN
MYCN is a major driver of neuroblastoma tumorigenesis and MYCN amplification is the worst prognostic indicator of aggressive NB. To identify potentially therapeutic tumor suppressor microRNAs for aggressive NB, we utilized a conditional MYCN system to simulate MYCN-amplified and nonamplified tumor types and performed a genome-wide search for MYCN target microRNA promoters differentially repressed under high MYCN conditions. We identified 20 gene promoters hosting 30 microRNAs that were directly bound and differentially regulated by MYCN. Eleven of these genes showed significant clinical correlations for neuroblastoma with 4 genes linked with better survival and 7 genes linked with poor survival. Surprisingly, expression analysis of host genes and microRNAs demonstrated that 8 of 11 pairs were repressed by high levels of MYCN regardless of the clinical correlation of the host gene. We therefore predicted these intronic microRNAs would be tumor suppressors. In fact, detailed gain of function studies for two miRs, miR-591 and miR-558, confirmed potent tumor suppressive effects for miR-591 in orthotopic neuroblastoma xenografts. However, miR-558 markedly increased colony formation, proliferation, and tumor growth in vivo. Our data reveal host-gene independent functions of MYCN-target microRNAs and demonstrate that MYCN represses both tumor suppressive and proproliferative microRNAs.