RESUMEN
XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end-joining (cNHEJ), a major DNA double-strand break (DSB) repair pathway. We report neurodevelopmental delays and significant behavioral alterations associated with microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is associated with a low level of apoptosis of neural cells and premature neurogenesis, which consists of an early shift of neural progenitors from proliferative to neurogenic divisions during brain development. We show that premature neurogenesis is related to an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.
Asunto(s)
Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , Humanos , Animales , Ratones , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Reparación del ADN , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Encéfalo/metabolismoRESUMEN
We developed an Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4-deficient condition, in particular its absence of immune deficiency.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , Linfocitos/inmunología , Mutación Missense , Inmunodeficiencia Combinada Grave/genética , Recombinación V(D)J , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/metabolismoRESUMEN
Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.
Asunto(s)
Lamina Tipo B/metabolismo , Complejo Shelterina/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Células Cultivadas , Humanos , Lamina Tipo B/química , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/químicaRESUMEN
The production of neurons from neural stem cells (NSCs) persists throughout life in the mouse ventricular-subventricular zone (V-SVZ). We have previously reported that NSCs from adult V-SVZ are contained in cell populations expressing the carbohydrate SSEA-1/LeX, which exhibit either characteristics of quiescent NSCs (qNSCs) or of actively dividing NSCs (aNSCs) based on the absence or the presence of EGF-receptor, respectively. Using the fluorescence ubiquitination cell cycle indicator-Cdt1 transgenic mice to mark cells in G0/G1 phase of the cell cycle, we uncovered a subpopulation of qNSCs which were primed to enter the cell cycle in vitro. Besides, we found that treatment with Syndecan-1, a heparan sulfate proteoglycan involved in NSC proliferation, hastened the division of qNSCs and increased proliferation of aNSCs shortening their G1 phase in vitro. Furthermore, administration of Syndecan-1 ameliorated the recovery of neurogenic populations in the V-SVZ after radiation-induced injury providing potential cure for neurogenesis decline during brain aging or after injury.
RESUMEN
Human glioblastoma (GBM) is the most common primary malignant brain tumor. A minor subpopulation of cancer cells, known as glioma stem-like cells (GSCs), are thought to play a major role in tumor relapse due to their stem cell-like properties, their high resistance to conventional treatments and their high invasion capacity. We show that ionizing radiation specifically enhances the motility and invasiveness of human GSCs through the stabilization and nuclear accumulation of the hypoxia-inducible factor 1α (HIF1α), which in turn transcriptionally activates the Junction-mediating and regulatory protein (JMY). Finally, JMY accumulates in the cytoplasm where it stimulates GSC migration via its actin nucleation-promoting activity. Targeting JMY could thus open the way to the development of new therapeutic strategies to improve the efficacy of radiotherapy and prevent glioma recurrence.
Asunto(s)
Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/metabolismo , Glioma/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/efectos de la radiación , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Nucleares/genética , Radiación Ionizante , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Transactivadores/genéticaRESUMEN
PURPOSE: Medulloblastoma is an important cause of mortality and morbidity in pediatric oncology. Here, we investigated whether the DNA repair inhibitor, AsiDNA, could help address a significant unmet clinical need in medulloblastoma care, by improving radiotherapy efficacy without increasing radiation-associated toxicity. EXPERIMENTAL DESIGN: To evaluate the brain permeability of AsiDNA upon systemic delivery, we intraperitoneally injected a fluorescence form of AsiDNA in models harboring brain tumors and in models still in development. Studies evaluated toxicity associated with combination of AsiDNA with radiation in the treatment of young developing animals at subacute levels, related to growth and development, and at chronic levels, related to brain organization and cognitive skills. Efficacy of the combination of AsiDNA with radiation was tested in two different preclinical xenografted models of high-risk medulloblastoma and in a panel of medulloblastoma cell lines from different molecular subgroups and TP53 status. Role of TP53 on the AsiDNA-mediated radiosensitization was analyzed by RNA-sequencing, DNA repair recruitment, and cell death assays. RESULTS: Capable of penetrating young brain tissues, AsiDNA showed no added toxicity to radiation. Combination of AsiDNA with radiotherapy improved the survival of animal models more efficiently than increasing radiation doses. Medulloblastoma radiosensitization by AsiDNA was not restricted to a specific molecular group or status of TP53. Molecular mechanisms of AsiDNA, previously observed in adult malignancies, were conserved in pediatric models and resembled dose increase when combined with irradiation. CONCLUSIONS: Our results suggest that AsiDNA is an attractive candidate to improve radiotherapy in medulloblastoma, with no indication of additional toxicity in developing brain tissues.
Asunto(s)
ADN/farmacología , Meduloblastoma/tratamiento farmacológico , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína p53 Supresora de Tumor/genética , Adulto , Animales , Línea Celular Tumoral , Niño , ADN/efectos adversos , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Xenoinjertos , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/radioterapia , Pediatría , RNA-Seq , Fármacos Sensibilizantes a Radiaciones/efectos adversosRESUMEN
High fidelity of genetic transmission in neural stem and progenitor cells (NSPCs) has been long time considered to be crucial for brain development and homeostasis. However, recent studies have identified recurrent DSB clusters in dividing NSPCs, which may underlie the diversity of neuronal cell types. This raised the interest in understanding how NSPCs sense and repair DSBs and how this mechanism could be altered by environmental genotoxic stress caused by pollutants or ionizing radiation. Here, we show that embryonic mouse neural stem and progenitor cells (NSPCs) have significantly higher capacity than mouse embryonic fibroblasts (MEFs) to maintain their chromosome stability in response to acute (γ-radiation) and chronic (tritiated thymidine -3H-T- incorporation into DNA) genotoxic stress. Cells deficient for XLF/Cernunnos, which is involved in non-homologous end joining DNA (NHEJ) repair, highlighted important variations in fidelity of DNA repair pathways between the two cell types. Strikingly, a progressive and generalized chromosome instability was observed in MEFs cultured with 3H-T at long-term, whereas NSPCs cultured in the same conditions, preserved their chromosome stability thanks to higher DNA repair activity further enhanced by an adaptive response and also to the elimination of damaged cells by apoptosis. This specific DNA damage response of NSPCs may rely on the necessity for preservation of their genome stability together with their possible function in creating neuronal genetic diversity.
Asunto(s)
Inestabilidad Cromosómica/genética , Daño del ADN , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Células-Madre Neurales/metabolismo , Animales , Reparación del ADN/genética , Ratones , Factores de TiempoRESUMEN
Some cancer cells elongate their telomeres through the ALT (alternative lengthening of telomeres) pathway, which is based on homologous recombination for the addition of telomere repeats without telomerase activity. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF), two homologous lysine acetyltransferases, exert opposite effects on the ALT pathway, inhibiting or favoring it respectively. Here we show that ALT cells are particularly sensitive to the inhibition of acetyltransferases activities using Anacardic Acid (AA). AA treatment recapitulates the effect of PCAF knockdown on several ALT features, suggesting that AA decreased the ALT mechanism through the inhibition of lysine transferase activity of PCAF, but not that of GCN5. Furthermore, AA specifically sensitizes human ALT cells to radiation as compared to telomerase-positive cells suggesting that the inhibition of lysine acetyltransferases activity may be used to increase the radiotherapy efficiency against ALT cancers.
RESUMEN
Deciphering the mechanisms that regulate the quiescence of adult neural stem cells (NSCs) is crucial for the development of therapeutic strategies based on the stimulation of their endogenous regenerative potential in the damaged brain. We show that LeXbright cells sorted from the adult mouse subventricular zone exhibit all the characteristic features of quiescent NSCs. Indeed, they constitute a subpopulation of slowly dividing cells that is able to enter the cell cycle to regenerate the irradiated niche. Comparative transcriptomic analyses showed that they express hallmarks of NSCs but display a distinct molecular signature from activated NSCs (LeX+EGFR+ cells). Particularly, numerous membrane receptors are expressed on quiescent NSCs. We further revealed a different expression pattern of Syndecan-1 between quiescent and activated NSCs and demonstrated its role in the proliferation of activated NSCs. Our data highlight the central role of the stem cell microenvironment in the regulation of quiescence in adult neurogenic niches.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Ciclo Celular , Diferenciación Celular , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Nicho de Células Madre , Células Madre Adultas/efectos de la radiación , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Diferenciación Celular/genética , Diferenciación Celular/efectos de la radiación , Metabolismo Energético , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células-Madre Neurales/efectos de la radiación , Neurogénesis , Estrés Oxidativo , Transducción de Señal , Nicho de Células Madre/genética , Nicho de Células Madre/efectos de la radiaciónRESUMEN
The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf-/-PAXX-/- E18.5 embryos is severely affected with the block of B- and T-cell maturation at the stage of IgH and TCRß gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development.
Asunto(s)
Sistema Nervioso Central/crecimiento & desarrollo , Proteínas de Unión al ADN/metabolismo , Síndromes de Inmunodeficiencia/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Cementos de Resina/metabolismoRESUMEN
Neural stem cells (NSCs) enter quiescence in early embryonic stages to create a reservoir of dormant NSCs able to enter proliferation and produce neuronal precursors in the adult mammalian brain. Various approaches of fluorescent-activated cell sorting (FACS) have emerged to allow the distinction between quiescent NSCs (qNSCs), their activated counterpart (aNSCs), and the resulting progeny. In this article, we review two FACS techniques that can be used alternatively. We also show that their association with transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice allows an unprecedented overlook on the cell cycle dynamics of adult NSCs.
Asunto(s)
Encéfalo/citología , Ciclo Celular , Separación Celular/métodos , Citometría de Flujo/métodos , Microscopía Fluorescente/métodos , Células-Madre Neurales/citología , Animales , Encéfalo/fisiología , Proliferación Celular , Células Cultivadas , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Células-Madre Neurales/fisiologíaRESUMEN
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.
Asunto(s)
Complejos de Coordinación/toxicidad , G-Cuádruplex/efectos de los fármacos , Platino (Metal)/química , Telómero/efectos de los fármacos , Acridinas/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Cisplatino/toxicidad , Daño del ADN/efectos de los fármacos , Humanos , Ligandos , Microscopía Fluorescente , Compuestos Organoplatinos/toxicidad , Telómero/metabolismo , Acortamiento del Telómero/efectos de los fármacos , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF) are two homologous acetyltransferases that are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. Our results suggest that GCN5 is present at telomeres and opposes telomere recombination, in contrast to PCAF that may indirectly favour them in ALT cells.
Asunto(s)
Estudios de Asociación Genética , Homeostasis del Telómero/genética , Telómero/genética , Factores de Transcripción p300-CBP/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Unión Proteica , Intercambio de Cromátides Hermanas , Translocación GenéticaRESUMEN
Identifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.
Asunto(s)
Proteínas Hedgehog/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Fase de Descanso del Ciclo Celular , Transducción de Señal , Animales , Ciclo Celular , Eliminación de Gen , Ratones , Ratones Noqueados , Ratones Transgénicos , Neurogénesis , Neuronas , Receptores Patched/genética , Nicho de Células MadreRESUMEN
Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals, they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However, little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ), we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months, the proliferative status of activated NSCs was already altered by 6 months, with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures, as well as a modulation of the TGFß signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications.
Asunto(s)
Ciclo Celular , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Factores de Edad , Envejecimiento , Animales , Biomarcadores , Recuento de Células , Análisis por Conglomerados , Biología Computacional/métodos , Perfilación de la Expresión Génica , Ratones , Ratones TransgénicosRESUMEN
Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.
Asunto(s)
Citometría de Flujo/métodos , Ventrículos Laterales/citología , Células-Madre Neurales/citología , Animales , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Ratones , Ratones Transgénicos , Neurogénesis/fisiología , Neuronas/citología , Bulbo OlfatorioRESUMEN
c-Fos is a proto-oncogene involved in diverse cellular functions. Its deregulation has been associated to abnormal development and oncogenic progression. c-fos-/- mice are viable but present a reduction in their body weight and brain size. We examined the importance of c-Fos during neocortex development at 13.5, 14.5 and 16.5 days of gestation. At E14.5, neocortex thickness, apoptosis, mitosis and expression of markers along the different stages of Neural Stem Progenitor Cells (NSPCs) differentiation in c-fos-/- and wild-type mice were analyzed. A ~15% reduction in the neocortex thickness of c-fos-/- embryos was observed which correlates with a decrease in the number of differentiated cells and an increase in apoptosis at the ventricular zone. No difference in mitosis rate was observed, although the mitotic angle was predominantly vertical in c-fos-/- embryos, suggesting a reduced trend of NSPCs to differentiate. At E13.5, changes in differentiation markers start to be apparent and are still clearly observed at E16.5. A tendency of more AP-1/DNA complexes present in nuclear extracts of cerebral cortex from c-fos-/- embryos with no differences in the lipid synthesis activity was found. These results suggest that c-Fos is involved in the normal development of NSPCs by means of its AP-1 activity.
Asunto(s)
Diferenciación Celular/genética , Genes fos/genética , Neocórtex/embriología , Células-Madre Neurales/citología , Neurogénesis/genética , Animales , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.
Asunto(s)
Antineoplásicos/farmacología , Pirazinas/farmacología , Sulfonas/farmacología , Homeostasis del Telómero , Telómero/efectos de los fármacos , Telómero/metabolismo , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular , Línea Celular Tumoral , ADN Helicasas/genética , ADN Helicasas/metabolismo , Técnicas de Silenciamiento del Gen , Glioma/tratamiento farmacológico , Glioma/genética , Células HeLa , Recombinación Homóloga , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Proteína de la Leucemia Promielocítica , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteína de Replicación A/metabolismo , Telomerasa/metabolismo , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
The prion protein (PrP) is highly conserved and ubiquitously expressed, suggesting that it plays an important physiological function. However, despite decades of investigation, this role remains elusive. Here, by using animal and cellular models, we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1, the major mammalian endonuclease essential for base excision repair, and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus, PrP is required to maintain genomic stability in response to genotoxic stresses.