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1.
Nature ; 628(8007): 433-441, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38509368

RESUMEN

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1-6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription-replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription-replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.


Asunto(s)
Replicación del ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas , Transcripción Genética , Humanos , Roturas del ADN de Doble Cadena , Replicación del ADN/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reparación del ADN por Recombinación , Fase S , Transcripción Genética/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo
2.
J Pathol ; 259(1): 10-20, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36210634

RESUMEN

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.


Asunto(s)
Neoplasias Colorrectales , Replicación del ADN , Proteínas de Unión al ADN , Animales , Humanos , Ratones , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/genética , Daño del ADN , Proteínas de Unión al ADN/metabolismo
3.
Mol Cell ; 82(18): 3382-3397.e7, 2022 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-36002001

RESUMEN

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.


Asunto(s)
Replicación del ADN , Mitosis , Afidicolina/farmacología , Proteína BRCA2/genética , Sitios Frágiles del Cromosoma/genética , ADN/genética , Daño del ADN , Inestabilidad Genómica , Humanos , Mitosis/genética
4.
Front Pharmacol ; 13: 860682, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35548337

RESUMEN

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.

6.
Cancer Genomics Proteomics ; 16(6): 593-601, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31659113

RESUMEN

BACKGROUND/AIM: Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. MATERIALS AND METHODS: In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. RESULTS AND CONCLUSION: A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.


Asunto(s)
Bases de Datos Genéticas , Geminina/metabolismo , Células Madre Pluripotentes/metabolismo , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Proteínas de Ciclo Celular/metabolismo , Humanos , MicroARNs/metabolismo
7.
Trends Biochem Sci ; 44(9): 752-764, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31054805

RESUMEN

Strict regulation of DNA replication is of fundamental significance for the maintenance of genome stability. Licensing of origins of DNA replication is a critical event for timely genome duplication. Errors in replication licensing control lead to genomic instability across evolution. Here, we present accumulating evidence that aberrant replication licensing is linked to oncogene-induced replication stress and poses a major threat to genome stability, promoting tumorigenesis. Oncogene activation can lead to defects in where along the genome and when during the cell cycle licensing takes place, resulting in replication stress. We also discuss the potential of replication licensing as a specific target for novel anticancer therapies.


Asunto(s)
Replicación del ADN , ADN/genética , Inestabilidad Genómica/genética , Estrés Fisiológico/genética , Humanos
8.
J Pathol ; 246(2): 134-140, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29952003

RESUMEN

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Asunto(s)
Adenoma/genética , Carcinoma/genética , Neoplasias del Colon/genética , Geminina/genética , Genes Supresores de Tumor , Inestabilidad Genómica , Neoplasias Pulmonares/genética , Adenoma/inducido químicamente , Adenoma/metabolismo , Adenoma/patología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Azoximetano , Carcinoma/inducido químicamente , Carcinoma/metabolismo , Carcinoma/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Geminina/deficiencia , Geminina/metabolismo , Predisposición Genética a la Enfermedad , Histonas/metabolismo , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Fosforilación , Uretano
9.
Dig Dis Sci ; 63(10): 2582-2592, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29876779

RESUMEN

AIM: The present study investigates the role of innate and adaptive immune system of intestinal mucosal barrier function in cirrhosis. METHODS: Forty patients with decompensated (n = 40, group A), 27 with compensated cirrhosis (n = 27, group B), and 27 controls (n = 27, group C) were subjected to duodenal biopsy. Expression of α-defensins 5 and 6 at the intestinal crypts was evaluated by immunohistochemistry and immunofluorescence. Serum endotoxin, intestinal T-intraepithelial, and lamina propria B-lymphocytes were quantified. RESULTS: Cirrhotic patients presented higher endotoxin concentrations (p < 0.0001) and diminished HD5 and HD6 expression compared to healthy controls (p = 0.000287, p = 0.000314, respectively). The diminished HD5 and HD6 expressions were also apparent among the decompensated patients compared to compensated group (p = 0.025, p = 0.041, respectively). HD5 and HD6 expressions were correlated with endotoxin levels (r = -0.790, p < 0.0001, r = - 0.777, p < 0.0001, respectively). Although intraepithelial T-lymphocytes were decreased in group A compared to group C (p = 0.002), no notable alterations between groups B and C were observed. The B-lymphocytic infiltrate did not differ among the investigated groups. CONCLUSIONS: These data demonstrate that decreased expression of antimicrobial peptides may be considered as a potential pathophysiological mechanism of intestinal barrier dysfunction in liver cirrhosis, while remodeling of gut-associated lymphoid tissue as an acquired immune response to bio-pathogens remains an open field to illuminate.


Asunto(s)
Inmunidad Mucosa , Cirrosis Hepática/inmunología , Células de Paneth/metabolismo , alfa-Defensinas/metabolismo , Endotoxinas/sangre , Femenino , Humanos , Cirrosis Hepática/metabolismo , Linfocitos , Tejido Linfoide/citología , Masculino , Persona de Mediana Edad , Estudios Prospectivos
10.
Redox Biol ; 6: 100-105, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26204503

RESUMEN

Hydrogen peroxide (Η2Ο2) is produced during a variety of cellular procedures. In this paper, the regulatory role of Η2Ο2, in Escherichia coli phagocytosis by the human polymorphonuclears, was investigated. White blood cells were incubated with dihydrorhodamine (DHR) in order to study H2O2 synthesis and E. coli-FITC to study phagocytosis. Flow cytometry revealed increased synthesis of H2O2 in polymorphonuclears which incorporated E. coli-FITC. The blocking of H2O2 synthesis by specific inhibitors, N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for superoxide dismutase (SOD), decreased E. coli phagocytosis, as well. Immunoblot analysis of white blood cell protein extracts revealed that the blocking of NADPH oxidase and SOD decreased ERK-1/2 phosphorylation, while it had no effect on JNK and p38. Confocal microscopy showed that phosphorylation of MAPKs and phagocytosis solely occur in the polymorphonuclear and not in mononuclear cells. The use of specific MAPKs inhibitors showed that all of them are necessary for phagocytosis, but only phospho-p38 affects H2O2 synthesis. The blocking of JNK phosphorylation, in the presence of E. coli, evoked a further decrease of cytoplasmic p47 thus increasing its translocation onto the plasma membrane for the assembly of NADPH oxidase. It appears that newly synthesised H2O2 invigorates the phosphorylation and action of ERK-1/2 in E. coli phagocytosis, while phospho-JNK and phospho-p38 appear to regulate H2O2 production.


Asunto(s)
Escherichia coli/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Neutrófilos/metabolismo , Transducción de Señal/inmunología , Ditiocarba/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/química , Etilmaleimida/farmacología , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Regulación de la Expresión Génica/inmunología , Humanos , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/inmunología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Coloración y Etiquetado , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología
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