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AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here, we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A facilitates transcription silencing at DSBs in transcriptionally active chromatin by recruiting HDAC1 and RSF1 to control the distribution of activating histone marks, chromatin accessibility, and eviction of RNAPII. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.
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Cromatina , Reparación del ADN , Proteínas de Unión al ADN , Inmunidad , Factores de Transcripción , Humanos , Línea Celular Tumoral , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Recombinación Homóloga/genética , Inmunidad/genética , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/inmunología , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transactivadores , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.
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Trastornos por Deficiencias en la Reparación del ADN , Histonas , Niño , Humanos , Histonas/genética , Nucleotidiltransferasas/genética , Inmunidad , ADNRESUMEN
OBJECTIVE: Posterior left atrial wall isolation (PWI) plus traditional pulmonary vein isolation (PVI) has been proposed as a promising intervention to decrease atrial fibrillation (AF) recurrence. We aim to investigate the efficacy and safety of adding PWI to the traditional PVI in patients with AF. METHODS: A systematic review and meta-analysis was conducted using synthesizing randomized controlled trials (RCTs) retrieved by systematically searching PubMed, Web of Science, SCOPUS, EMBASE, and Cochrane through June 14, 2023. We used Stata version 17 to pool dichotomous data using risk ratio (RR) and continuous data using mean difference (MD), with a 95% confidence interval (CI) (PROSPERO ID: CRD42023446227). RESULTS: We included 11 RCTs with a total number of 1534 patients. Combined ablation with PWI + PVI was not associated with any significant difference over PVI only regarding the recurrence of clinical AF (RR: 0.86 with 95% CI [0.70-1.06]), all atrial arrhythmia (RR: 0.93 with 95% CI [0.82-1.07]), nonatrial fibrillation arrhythmia (RR: 1.22 with 95% CI [0.97-1.53]), early AF (RR: 0.89 with 95% CI [0.62-1.27]), and antiarrhythmic drugs at discharge (RR: 0.83 with 95% CI [0.67-1.04]). However, it was associated with increased total ablation duration (minutes) (MD: 12.58 with 95% CI [6.80-18.37]) and total procedure duration (minutes) (MD: 16.77 with 95% CI [9.63-23.91]), without any significant difference regarding adverse events (RR: 1.05 with 95% CI [0.63-1.74]). CONCLUSION: While the pooled data from PWI + PVI using point-by-point radiofrequency did not suggest a benefit in the recurrence of various atrial arrhythmias compared to PVI alone, PWI+PVI using direct posterior wall ablation, especially with cryoballoon, demonstrated a significant reduction in recurrence of AF/atrial arrhythmias. Also, PWI + PVI significantly increased the ablation and total procedure durations.
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Fibrilación Atrial , Ablación por Catéter , Venas Pulmonares , Fibrilación Atrial/cirugía , Humanos , Ablación por Catéter/métodos , Venas Pulmonares/cirugía , Atrios Cardíacos/cirugía , Recurrencia , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.
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Elementos de Facilitación Genéticos , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Proteína 1 de la Leucemia Linfocítica T Aguda , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Mutación , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Linfocitos T/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Drug-coated balloons (DCBs) are an established strategy for coronary artery disease. However, the new generation drug-eluting stent (DES) is recommended for patients with Acute myocardial infarction (AMI) for coronary artery revascularization. Our aim is to provide a comprehensive appraisal of the efficacy of DCBs in patients with AMI undergoing PCI. METHODS: We searched the WOS, PubMed, Scopus, and Cochrane CENTRAL till March 2023, for studies that compared DCBs versus DES in patients with AMI undergoing PCI. We used a random-effects model to compare major adverse cardiac events (MACE), cardiac death, all-cause death, myocardial infarction, target lesion revascularization (TLR), stent thrombosis, Late lumen Loss (LLL), and minimum lumen diameter (MLD) between the two groups. RESULTS: Thirteen studies comprising 2644 patients were included. The pooled OR showed non-inferiority of DCB over DES in terms of MACE (OR = 0.89, 95% CI [0.57 to 1.40], p = 0.63). When we defined MACE as a composite of cardiac death, MI, and TLR; the pooled OR favored DCB over DES (OR = 0.50, 95% CI [0.28 to 0.9], p = 0.02). Moreover, DCB was not inferior to DES in terms of all-cause mortality (OR = 0.88, 95% CI: 0.43 to 1.8, p = 0.73), cardiac mortality, (OR = 0.59, 95% CI: 0.22 to 1.56, p = 0.29), MI (OR = 0.88, 95% CI: 0.34 to 2.29, p = 0.79), stent thrombosis (OR = 1.21, 95% CI: 0.35 to 4.23, p = 0.76), TLR (OR = 0.9, 95% CI: 0.43 to 1.93, p = 0.8), LLL (MD = -0.6, 95% CI: -0.3 to 0.19, p = 0.64), or MLD (MD = -0.4, 95% CI: -0.33 to 0.25, p = 0.76). CONCLUSION: Our meta-analysis indicated that DCB intervention was not inferior to DES in the PCI setting in patients with AMI, and can be recommended as a feasible strategy in AMI. PROSPERO REGISTRATION: CRD42023412757.
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Enfermedad de la Arteria Coronaria , Stents Liberadores de Fármacos , Infarto del Miocardio , Intervención Coronaria Percutánea , Trombosis , Humanos , Intervención Coronaria Percutánea/efectos adversos , Stents Liberadores de Fármacos/efectos adversos , Resultado del Tratamiento , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/terapia , Infarto del Miocardio/etiología , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/terapia , Enfermedad de la Arteria Coronaria/complicaciones , Trombosis/etiología , MuerteRESUMEN
The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.
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Recombinación Homóloga , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Resistencia a Antineoplásicos , Factor de Transcripción E2F1/metabolismo , Células HEK293 , Humanos , Proteínas Inhibidoras de la Diferenciación/química , Masculino , Proteínas de Neoplasias/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/toxicidad , Poli(ADP-Ribosa) Polimerasas/metabolismo , Recombinasa Rad51/metabolismo , RecQ Helicasas/metabolismoRESUMEN
Objectives: To assess safety; efficacy and efficiency of mini-sternotomy in aortic valve replacement in comparison to conventional sternotomy on short term follow up. Method: This comparative study between 45 patients having aortic valve replacement via fullsternotomy versus 45 others planned for upper j-shaped mini-sternotomy, was conducted from May 2019 to February 2022 in Kafrelsheikh university hospital, Egypt. Data was collected and statistically analysed to assess outcomes. RESULTS: Mini-Sternotomy approach was compared to conventional approach on the aspects of cardio pulmonary bypass (CPB) (p=0.153) and cross clamp (CC) time (p=0.673),. There was significantly less postoperative bleeding (p<0.001), rate of blood transfusion (p<0.001), duration of ICU stay (p=0.013) and total hospitalstay (p=0.022) in ministernotomy approach in comparison to conventional sternotomy. CONCLUSIONS: For primary isolated AVR, lessinvasive techniques are a realistic, practical, and good alternative that offers better postoperative results than Full Sternotomy.
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Válvula Aórtica , Implantación de Prótesis de Válvulas Cardíacas , Humanos , Válvula Aórtica/cirugía , Esternotomía/métodos , Implantación de Prótesis de Válvulas Cardíacas/métodos , Resultado del Tratamiento , Estudios Retrospectivos , Procedimientos Quirúrgicos Mínimamente Invasivos/métodosRESUMEN
Radiotherapy can induce various adverse effects including fibrosis in cancer patients. Radiation-induced aberrant expression of profibrotic genes has been associated with dysregulated epigenetic mechanisms. Pan-BET (bromodomain and extraterminal domain) inhibitors, such as JQ1 and I-BET151, have been reported to attenuate the profibrotic response after irradiation. Despite their profound preclinical efficacy, the clinical utility of pan-inhibitors is limited due to observed cytotoxicicities. Recently, inhibitors were developed that selectively target the first (BD1) and second (BD2) bromodomain of the BET proteins (iBET-BD1 [GSK778] and iBET-BD2 [GSK046]). Here, their potential to attenuate radiation-induced fibroblast activation with low-toxicity was investigated. Our results indicated that cell proliferation and cell cycle progression in fibroblasts from BJ cells and six donors were reduced when treated with I-BET151 and iBET-BD1, but not with iBET-BD2. After irradiation, induction of DGKA and profibrotic markers, especially COL1A1 and ACTA2, was attenuated with all BET inhibitors. H3K27ac enrichment was similar at the DGKA enhancer region after I-BET151 treatment and irradiation, but was reduced at the COL1A1 transcription start site and the ACTA2 enhancer site. iBET-BD2 did not change H3K27ac levels in these regions. BRD4 occupancy at these regions was not altered by any of the compounds. Cell migration activity was measured as a characteristic independent of extracellular matrix production and was unchanged in fibroblasts after irradiation and BET inhibitor-treatment. In conclusion, iBET-BD2 efficiently suppressed radiation-induced expression of DGKA and profibrotic markers without showing cytotoxicity. Thus BD2-selective targeting is a promising new therapeutic avenue for further investigations to prevent or attenuate radiotherapy-induced fibrosis.
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Antineoplásicos , Proteínas Nucleares , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Fibrosis , Humanos , Proteínas Nucleares/metabolismo , Dominios Proteicos , Factores de Transcripción/metabolismoRESUMEN
Experiments demonstrate that magnetic nanoparticles, embedded in a tissue, very often form heterogeneous structures of various shapes and topologies. These structures (clusters) can significantly affect macroscopical properties of the composite system, in part its ability to generate heat under an alternating magnetic field (so-called magnetic hyperthermia). If the energy of magnetic interaction between the particles significantly exceeds the thermal energy of the system, the particles can form the closed ring-shaped clusters. In this work, we propose a relatively simple model of the heat production by the particles united in the 'ring' and immobilized in a host medium. Mathematically, this model is based on the phenomenological Debye equation of kinetics of the particles remagnetization. Magnetic interaction between all particles in the cluster is taken into account. Our results show that the appearance of the clusters can significantly decrease the thermal effect. This article is part of the theme issue 'Transport phenomena in complex systems (part 1)'.
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Hipertermia Inducida , Nanopartículas , Humanos , Hipertermia , Fenómenos Magnéticos , MagnetismoRESUMEN
Experiments show that clusters consisting of nano-sized ferromagnetic particles strongly affect the intensity of heat production during magnetic hyperthermia. In this paper, a theoretical study and mathematical modelling of the heat production by clusters of single-domain ferromagnetic particles, immobilized in a host medium, are presented. Two situations of strong and weak magnetic anisotropy of the particles are considered. Our results show that, in the case of strong anisotropy, the clusterization weakens the thermal effect, whereas in the case of weak anisotropy it enhances it. This article is part of the theme issue 'Patterns in soft and biological matters'.
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The paper deals with the theoretical study of the effect of magnetic interparticle interaction on magnetic hyperthermia, produced by the particles under the action of a linearly polarized oscillating field. The particles are homogeneously distributed and immobilized in a rigid medium. The supposed size of the magnetite particles is about 20-30 nm. For these particles, the characteristic time of the Neel remagnetization is much longer than the time of observation. This is why we concluded that the dissipation occurs as a result of the particle magnetic moment oscillation in the pit of energy of magnetic anisotropy. This article is part of the theme issue 'Heterogeneous materials: metastable and non-ergodic internal structures'.
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BACKGROUND: Cancer chemotherapy is associated with various tissue toxicities that limit its use. Cyclophosphamide (CYC) is one of the most commonly used antineoplastic and immunosuppressive agent. Thyroid dysfunction is a critical side effect of anticancer drugs. Atorvastatin (ATV) is antihyperlipedemic drug with different tissue protective activities. The aim of this study was to determine the potential protective effect of ATV against CYC-induced thyroid injury in rats. METHODS: ATV was administered in the presence and absence of CYC. Thirty-two adult Wistar rats were randomly divided into four groups: control group, ATV group (20 mg/kg/day, p.o. for 14 day), CYC group (200 mg/kg, i.p. on day 9) and ATV/CYC group. Triiodothyronine (T3), thyroxine (T4), reduced glutathione (GSH), malondialdehyde (MDA), total nitrite/nitrate (NOx), p38 mitogen-activated protein kinase (P38MAPK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK) were measured. In addition, thyroid histopathology and caspase 3 immunohistochemistry were performed. RESULTS: CYC significantly increased thyroid MDA, NOx, P38MAPK, ERK and JNK with decrease in GSH, T3 and T4 levels. Histopathological features of thyroid lesions and increased caspase 3 immune expression were appeared. ATV significantly normalized distributed oxidative, inflammatory and apoptotic indicators, resulting in an improvement of histopathological features and reduction of caspase 3 immunoexpression. CONCLUSION: These findings suggest that ATV protects against CYC-induced thyroid injury by regulating the JNK/ERK/p38-MAPK signaling pathway.
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Quinasas MAP Reguladas por Señal Extracelular , Transducción de Señal , Ratas , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Atorvastatina/uso terapéutico , Atorvastatina/farmacología , Caspasa 3/metabolismo , Glándula Tiroides/metabolismo , Ratas Wistar , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ciclofosfamida/toxicidadRESUMEN
The use of percutaneous coronary intervention (PCI) in patients with chronic total occlusion (CTO) is still a subject of debate, with conflicting outcomes reported in different studies when compared to non-CTO lesions. This meta-analysis aims to clarify the clinical outcomes of PCI in CTO cases compared to non-CTO lesions, both in the short and long-term. PubMed, Scopus, Web of Science, Ovid, and Cochrane Central were searched until March 2023 for relevant studies addressing short- and long-term outcomes of PCI in CTO vs non-CTO lesions. Dichotomous data were pooled as odds ratio (OR) with its 95% confidence interval (CI) in a random Der-Simonian lair effect model using STATA 17 MP. Eight studies with a total of 690,123 patients were included. In terms of short-term outcomes, CTO PCI was associated with higher rates of vessel perforation (ORâ¯=â¯2.16, 95% CI: 1.31-3.57) and cardiac tamponade (ORâ¯=â¯5.19, 95% CI: 4.29-6.28). Additionally, CTO PCI showed lower rates of procedural success (ORâ¯=â¯0.84, 95% CI: 0.73-0.96). Moreover, in the long-term, CTO PCI had higher rates of MACE (ORâ¯=â¯1.02, 95% CI: 1.01-1.04), however, it showed lower rates of cardiac death (ORâ¯=â¯0.61, 95% CI: 0.38-0.98), with no significant difference in other reported outcomes. Our findings underscore the challenges and adverse outcomes associated with using PCI to treat CTO lesions in the short term. This suggests that interventional cardiologists should carefully evaluate the risks and benefits before proceeding with PCI in CTO lesions.
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Oclusión Coronaria , Intervención Coronaria Percutánea , Humanos , Oclusión Coronaria/cirugía , Intervención Coronaria Percutánea/efectos adversos , Enfermedad Crónica , Oportunidad Relativa , Resultado del Tratamiento , Factores de RiesgoRESUMEN
Monitoring bubble cavitations and bubble dynamics are essential in enhancing non-invasive ultrasonic ablation methods like histotripsy that mechanically fractionates tissue into acellular debris using microcavitation. Histotripsy can totally fractionate tissue into a liquid-appearing homogenate with no cellular features with enough pulses. In this paper, we present the analysis of the dynamics of cavitation bubbles in a viscoelastic medium subjected to a histotripsy pulse using different fidelities in depicting compressibility and viscoelasticity effects. The mathematical formulation is described based on the Keller-Miksis equation in two models for cavitation bubbles in viscoelastic tissue through histotripsy process; the first model is in neo-Hookean, and the second is in quadratic law Kelvin-Voigt model. The governing model is solved analytically based on the modified Plesset-Zwick method. Analysis of the results reveals that the parameters of Young modulus, viscosity effects and stiffening parameter reduce the growth of cavitation microbubbles through the histotripsy process. The cavitation bubble growth increases when the gel concentration decreases during the histotripsy process.
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Microburbujas , Ultrasonido , Módulo de Elasticidad , ViscosidadRESUMEN
Deregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1-G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Elementos de Facilitación Genéticos , Factor de Transcripción GATA2/metabolismo , Regulación Leucémica de la Expresión Génica , Reordenamiento Génico , Leucemia Mieloide Aguda/patología , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Carcinogénesis , Aberraciones Cromosómicas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Factor de Transcripción GATA2/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína del Locus del Complejo MDS1 y EV11/genética , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Translocación Genética , Células Tumorales CultivadasRESUMEN
Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.
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Felodipine (FLD), a calcium channel antagonist, is commonly prescribed for the treatment of hypertension either with Metoprolol (MET) or Ramipril (RAM) in two different drug combinations. FLD has high plasma protein binding ability affecting its extraction recoveries from plasma samples. Hence, a specific ultrasound assisted dispersive liquid-liquid microextraction (UA-DLLME) method coupled with HPLC using photodiode array detector was developed and validated for the simultaneous determination of FLD, MET and RAM in rat plasma after oral administration of these combinations. The factors affecting UA-DLLME were carefully optimized. In this study, UA-DLLME method could provide simple and efficient plasma extraction procedures with superior recovery results. Under optimum condition, all target drugs were separated within 13min. The validation procedures was carried out in agreement with US-FDA guidelines and shown to be suitable for anticipated purposes. Linear calibration ranges were obtained in the range 0.05-2.0µgmL-1 for FLD and MET and 0.1-2.0µgmL-1 for RAM with detection limits of 0.013-0.031µgmL-1 for all the studied drug combinations. The%RSD for inter-day and intra-day precisions was in range of 0.63-3.85% and the accuracy results were in the range of 92.13-100.5%. The validated UA-DLLME-HPLC method was successfully applied for the bioavailability studies of FLD, MET and RAM. The pharmacokinetic parameters were calculated for all the investigated drugs in rats after single-dose administrations of two different drug combinations. Although FLD was bioequivalent in the two formulations, a small increase in plasma levels of MET and RAM was found in the presence of FLD.
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Cromatografía Líquida de Alta Presión/métodos , Felodipino/sangre , Microextracción en Fase Líquida/métodos , Sonicación/métodos , Animales , Disponibilidad Biológica , Monitoreo de Drogas , Estabilidad de Medicamentos , Felodipino/química , Felodipino/farmacocinética , Límite de Detección , Modelos Lineales , Masculino , Ratas , Ratas Wistar , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors. MATERIALS AND METHODS: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis. RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection. CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.
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Proteína BRCA1/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína 1 de Unión al Supresor Tumoral P53/fisiología , Ciclo Celular , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Células HeLa , Recombinación Homóloga , HumanosRESUMEN
End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.