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1.
Nat Commun ; 12(1): 3686, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-34140498

RESUMEN

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.


Asunto(s)
Hipoxia de la Célula , Daño del ADN/genética , ADN Helicasas/metabolismo , Regulación de la Expresión Génica/genética , Enzimas Multifuncionales/metabolismo , Estructuras R-Loop/genética , ARN Helicasas/metabolismo , Respuesta de Proteína Desplegada/genética , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Inmunoprecipitación de Cromatina , ADN Helicasas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Enzimas Multifuncionales/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oxígeno/farmacología , Estructuras R-Loop/efectos de los fármacos , ARN Helicasas/genética , RNA-Seq , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba , Cinostatina/farmacología , eIF-2 Quinasa/metabolismo
2.
Mol Cancer Res ; 19(8): 1361-1374, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34050002

RESUMEN

Histone deacetylase inhibitors (HDACi) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action (MOA). Multiple activities of HDACis have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA-RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA (ssDNA) breaks detected by single-cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. IMPLICATIONS: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA-RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death.


Asunto(s)
ADN de Cadena Simple/efectos de los fármacos , Depsipéptidos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Estructuras R-Loop/efectos de los fármacos , Acetilación/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , ADN de Cadena Simple/genética , Humanos , Células PC-3 , Estructuras R-Loop/genética
3.
Nucleic Acids Res ; 48(21): 11942-11957, 2020 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-33137181

RESUMEN

Genomic 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.


Asunto(s)
ADN/química , G-Cuádruplex , Genoma Humano , Estructuras R-Loop , ARN/química , Aminoquinolinas/química , Aminoquinolinas/farmacología , Emparejamiento Base , ADN/genética , ADN/metabolismo , G-Cuádruplex/efectos de los fármacos , Inestabilidad Genómica , Guanina/química , Guanina/metabolismo , Humanos , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacología , Regiones Promotoras Genéticas , Estructuras R-Loop/efectos de los fármacos , ARN/genética , ARN/metabolismo , Telómero/efectos de los fármacos , Telómero/metabolismo , Telómero/ultraestructura , Homeostasis del Telómero
4.
Nat Commun ; 11(1): 4083, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32796829

RESUMEN

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.


Asunto(s)
Proteínas de Ciclo Celular/farmacología , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Estructuras R-Loop/efectos de los fármacos , Factores de Transcripción/farmacología , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Cromatina , Proteínas de Unión al ADN , Inestabilidad Genómica , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Fase S , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma
5.
Cell Prolif ; 53(9): e12875, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32761833

RESUMEN

OBJECTIVES: R-loop is a three-stranded nucleic acid structure of RNA/DNA hybrid, which occurs naturally during transcription, and more R-loop accumulation can trigger serious DNA damage. There has been increasing attention to the issue of R-loop accumulation acted as a target for cancer therapy. However, the regulation of R-loop-associated proteins is poorly explored. MATERIAL AND METHOD: Quantitative real-time PCR and Western blot were used to measure the expression of C1orf109 in cell lines. In addition, C1orf109L (C1orf109 longest isoform) protein binding partner was identified and validated using immunoprecipitation-mass spectrometric (IP-MS) and immunoprecipitation assays. DNA-RNA immunoprecipitation (DR-IP) and immunofluorescence determined the C1orf109L location on R-loop. R-loop accumulation was determined by immunofluorescence. Cell cycle was determined by flow cytometry. Finally, time-lapse assay and cell counting were conducted to determined cell survival in response to camptothecin (CPT). RESULTS: We found that C1orf109L could mediate cell cycle arrest in the G2/M phase and DNA damage depended on R-loop accumulation. Meanwhile, C1orf109L could bind with DHX9 to trigger R-loop accumulation. And C1orf109L was competitive with PARP1 binding to DHX9, which would block the function of DHX9-PARP1 to prevent the R-loop accumulation. Furthermore, C1orf109L could enhance the chemosensitivity of CPT, a chemotherapeutic drug capable of promoting R-loop formation. CONCLUSIONS: Our data demonstrate that C1orf109L triggers R-loop accumulation and DNA damage to arrest cell cycle.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/farmacología , ARN Helicasas DEAD-box/metabolismo , Daño del ADN/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Estructuras R-Loop/efectos de los fármacos
6.
J Med Chem ; 63(6): 3090-3103, 2020 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-32142285

RESUMEN

Targeting G-quadruplex structures is currently viewed as a promising anticancer strategy. Searching for potent and selective G-quadruplex binders, here we describe a small series of new monohydrazone derivatives designed as analogues of a lead which was proved to stabilize G-quadruplex structures and increase R loop levels in human cancer cells. To investigate the G-quadruplex binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G-quadruplex-forming sequences. The obtained results allowed the identification of a highly selective G-quadruplex ligand that, when studied in human cancer cells, proved to be able to stabilize both G-quadruplexes and R loops and showed a potent cell killing activity associated with the formation of micronuclei, a clear sign of genome instability.


Asunto(s)
Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , ADN/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Hidrazonas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Genoma/efectos de los fármacos , Humanos , Hidrazonas/síntesis química , Hidrazonas/metabolismo , Ligandos , Estructuras R-Loop/efectos de los fármacos
7.
Environ Health Perspect ; 128(1): 17002, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31939680

RESUMEN

BACKGROUND: Endocrine-disrupting chemicals have been shown to have broad effects on development, but their mutagenic actions that can lead to cancer have been less clearly demonstrated. Physiological levels of estrogen have been shown to stimulate DNA damage in breast epithelial cells through mechanisms mediated by estrogen-receptor alpha (ERα). Benzophenone-3 (BP-3) and propylparaben (PP) are xenoestrogens found in the urine of >96% of U.S. OBJECTIVES: We investigated the effect of BP-3 and PP on estrogen receptor-dependent transactivation and DNA damage at concentrations relevant to exposures in humans. METHODS: In human breast epithelial cells, DNA damage following treatment with 17ß-estradiol (E2), BP-3, and PP was determined by immunostaining with antibodies against γ-H2AX and 53BP1. Estrogenic responses were determined using luciferase reporter assays and gene expression. Formation of R-loops was determined with DNA: RNA hybrid-specific S9.6 antibody. Short-term exposure to the chemicals was also studied in ovariectomized mice. Immunostaining of mouse mammary epithelium was performed to quantify R-loops and DNA damage in vivo. RESULTS: Concentrations of 1µM and 5µM BP-3 or PP increased DNA damage similar to that of E2 treatment in a ERα-dependent manner. However, BP-3 and PP had limited transactivation of target genes at 1µM and 5µM concentrations. BP-3 and PP exposure caused R-loop formation in a normal human breast epithelial cell line when ERα was introduced. R-loops and DNA damage were also detected in mammary epithelial cells of mice treated with BP-3 and PP. CONCLUSIONS: Acute exposure to xenoestrogens (PP and BP-3) in mice induce DNA damage mediated by formation of ERα-dependent R-loops at concentrations 10-fold lower than those required for transactivation. Exposure to these xenoestrogens may cause deleterious estrogenic responses, such as DNA damage, in susceptible individuals. https://doi.org/10.1289/EHP5221.


Asunto(s)
Benzofenonas/toxicidad , Contaminantes Ambientales/toxicidad , Parabenos/toxicidad , Animales , Línea Celular Tumoral , Células Epiteliales , Humanos , Ratones , Estructuras R-Loop/efectos de los fármacos , Receptores de Estrógenos/efectos de los fármacos , Pruebas de Toxicidad
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