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1.
EMBO Mol Med ; 11(7): e9982, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31273933

RESUMO

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.


Assuntos
Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Clorambucila/farmacologia , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cricetinae , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos SCID , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS One ; 12(9): e0184415, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28910317

RESUMO

The first aim of this research was to assess the effectiveness, in terms of emotional and behavioral reactions, of moderately vs. highly TVWs (Threatening Visual Warnings) displayed on tobacco packs. Given the key role that emotional reactions play in explaining the effect of TVWs on behaviors, psychophysiological and self-report methods were used-for the first time in this context-to measure the emotions provoked by TVWs. The second aim of this research was to determine whether increasing the size of warnings, and their display on plain packaging (compared with branded packaging) would improve their effectiveness. A within-subjects experiment was conducted. Three variables were manipulated: health warning threat level (high vs. moderate), image size (40% vs. 75%) and pack type (plain vs. branded). A convenience sample of 48 French daily smokers participated. They were exposed to eight different packs of cigarettes in a research lab at the University of Rennes. Smokers' emotions and behavioral intentions were recorded through self-reports. Emotions were also evaluated using psychophysiological measurements: electrodermal activity and facial electromyography. The results revealed that TVWs with a high threat level are the most effective in increasing negative emotions (fear, disgust, valence, arousal) and behavioral intentions conducive to public health (desire to quit, etc.). They also highlight the appeal of increasing the size of the warnings and displaying them on plain packs, because this influences emotions, which is the first step toward behavioral change. Increasing the threat level of TVWs from moderate to high seems beneficial for public health. Our results also confirm the relevance of recent governmental decisions to adopt plain packaging and larger TVWs (in the UK, France, Ireland, Canada, New Zealand, Hungary, etc.).


Assuntos
Embalagem de Medicamentos/métodos , Emoções/fisiologia , Rotulagem de Produtos/métodos , Embalagem de Produtos/métodos , Fumar/psicologia , Adulto , Atenção/fisiologia , Atitude Frente a Saúde , Feminino , França , Humanos , Masculino , Pessoa de Meia-Idade , Autorrelato , Prevenção do Hábito de Fumar , Tabaco , Adulto Jovem
4.
EMBO Mol Med ; 9(10): 1398-1414, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28729482

RESUMO

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.


Assuntos
Acetaldeído/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Rad51 Recombinase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Rad51 Recombinase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 8: 15983, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28714477

RESUMO

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.


Assuntos
Proteína BRCA2/genética , Segregação de Cromossomos/genética , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Endonucleases/genética , Anáfase , Linhagem Celular Tumoral , Células HeLa , Humanos , Mitose
6.
Mol Cell ; 61(3): 449-460, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26748828

RESUMO

G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.


Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Biomarcadores Tumorais/deficiência , Quadruplex G/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Ácidos Picolínicos/farmacologia , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Masculino , Camundongos Nus , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Interferência de RNA , Telômero/efeitos dos fármacos , Telômero/genética , Telômero/metabolismo , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ensaios Antitumorais Modelo de Xenoenxerto
8.
EMBO J ; 34(3): 410-24, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25582120

RESUMO

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.


Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , Telômero/metabolismo , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Autoantígeno Ku , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Telômero/genética , Proteínas de Xenopus
9.
Nat Commun ; 4: 2697, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24162189

RESUMO

ARF is a tumour suppressor activated by oncogenic stress, which stabilizes p53. Although p53 is a key component of the response to DNA damage, a similar function for ARF has not been ascribed. Here we show that primary mouse and human cells lacking the tumour suppressor BRCA2 accumulate DNA damage, which triggers checkpoint signalling and ARF activation. Furthermore, senescence induced by Brca2 deletion in primary mouse and human cells is reversed by the loss of ARF, a phenotype recapitulated in cells lacking RAD51. Surprisingly, ARF is not necessary for p53 accumulation per se but for altering the spectrum of genes activated by this transcription factor. Specifically, ARF enables p53 transcription of Dusp4 and Dusp7, which encode a pair of phosphatases known to inactivate the MAP kinases ERK1/2. Our results ascribe a previously unanticipated function to the ARF tumour suppressor in genome integrity, controlled by replicative stress and ATM/ATR-dependent checkpoint responses.


Assuntos
Proteína BRCA2/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Cricetinae , Dano ao DNA , Epigênese Genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Células HeLa , Humanos , Camundongos , Fenótipo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Transdução de Sinais , Fatores de Transcrição
10.
Nat Struct Mol Biol ; 17(12): 1461-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21076401

RESUMO

The tumor suppressor protein BRCA2 is a key component of the homologous recombination pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here we show that BRCA2 associates with telomeres during the S and G2 phases of the cell cycle and facilitates the loading of RAD51 onto telomeres. Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals--a hallmark of telomere fragility that is associated with replication defects. These findings suggest that BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication and imply that BRCA2 has an essential role in maintaining telomere integrity during unchallenged cell proliferation. Mouse mammary tumors that lacked Brca2 accumulated telomere dysfunction-induced foci. Human breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction.


Assuntos
Proteína BRCA2/fisiologia , Rad51 Recombinase/metabolismo , Telômero/metabolismo , Animais , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Reparo do DNA , Fase G2 , Deleção de Genes , Instabilidade Genômica , Camundongos , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Fase S , Telômero/química
11.
Curr Biol ; 20(6): 521-6, 2010 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-20226664

RESUMO

Telomeres are protected by capping structures consisting of core protein complexes that bind with sequence specificity to telomeric DNA. In their absence, telomeres trigger a DNA damage response, materialized in accumulation at the telomere of damage response proteins, e.g., phosphorylated histone H2AX (gammaH2AX), into telomere-dysfunction-induced foci. Telomere uncapping occurs transiently in every cell cycle in G2, following DNA replication, but little is known about how protective structures are reassembled or whether this process is controlled by the cell-cycle surveillance machinery. Here, we report that telomere capping is monitored at the G2/M transition by the p53/p21 damage response pathway. Unlike their wild-type counterparts, human and mouse cells lacking p53 or p21 progress into mitosis prematurely with persisting uncapped telomeres. Furthermore, artificially uncapped telomeres delay mitotic entry in a p53- and p21-dependent manner. Uncapped telomeres that persist in mitotic p53-deficient cells are shorter than average and religate to generate end-to-end fusions. These results suggest that a p53-dependent pathway monitors telomere capping after DNA replication and delays G2/M progression in the presence of unprotected telomeres. This mechanism maintains a cell-cycle stage conducive for capping reactions and prevents progression into stages during which uncapped telomeres are prone to deleterious end fusions.


Assuntos
Mitose/fisiologia , Telômero/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitose/genética , Proteínas Serina-Treonina Quinases/fisiologia , RNA Interferente Pequeno/genética , Telômero/genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/fisiologia
12.
J Cell Biol ; 185(4): 587-600, 2009 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-19451272

RESUMO

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular , Quinase do Ponto de Checagem 2 , Dano ao DNA , Humanos , Proteínas Nucleares , Fosforilação , Rad51 Recombinase , Proteína de Replicação A , Transdução de Sinais
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