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1.
Cell Rep ; 14(6): 1435-1447, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26854234

RESUMO

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase checkpoint pathway maintains genome integrity; however, the role of the sirtuin 2 (SIRT2) acetylome in regulating this pathway is not clear. We found that deacetylation of ATR-interacting protein (ATRIP), a regulatory partner of ATR, by SIRT2 potentiates the ATR checkpoint. SIRT2 interacts with and deacetylates ATRIP at lysine 32 (K32) in response to replication stress. SIRT2 deacetylation of ATRIP at K32 drives ATR autophosphorylation and signaling and facilitates DNA replication fork progression and recovery of stalled replication forks. K32 deacetylation by SIRT2 further promotes ATRIP accumulation to DNA damage sites and binding to replication protein A-coated single-stranded DNA (RPA-ssDNA). Collectively, these results support a model in which ATRIP deacetylation by SIRT2 promotes ATR-ATRIP binding to RPA-ssDNA to drive ATR activation and thus facilitate recovery from replication stress, outlining a mechanism by which the ATR checkpoint is regulated by SIRT2 through deacetylation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Pontos de Checagem do Ciclo Celular/genética , Replicação do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteína de Replicação A/genética , Sirtuína 2/genética , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Proteína de Replicação A/metabolismo , Transdução de Sinais , Sirtuína 2/metabolismo
2.
Nucleic Acids Res ; 42(18): 11517-27, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25217585

RESUMO

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Replicação do DNA , Desoxicitidina/análogos & derivados , Proteínas Serina-Treonina Quinases/fisiologia , Estresse Fisiológico/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desoxicitidina/farmacologia , Feminino , Humanos , Quinases Relacionadas a NIMA , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/análise , Neoplasias de Mama Triplo Negativas/genética , Gencitabina
3.
Cancer Res ; 74(10): 2677-87, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24626090

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to use a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes, including 55 genes linked to DNA damage responses (DDR), that demonstrated gemcitabine sensitization when silenced, including CHD7, which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 patients with resected PDAC receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , DNA Helicases/biossíntese , Proteínas de Ligação a DNA/biossíntese , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/cirurgia , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirurgia , Modelos de Riscos Proporcionais , Distribuição Aleatória , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
4.
Proc Natl Acad Sci U S A ; 110(33): 13546-51, 2013 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-23898190

RESUMO

Sirtuin 2 (SIRT2) is a sirtuin family deacetylase that directs acetylome signaling, protects genome integrity, and is a murine tumor suppressor. We show that SIRT2 directs replication stress responses by regulating the activity of cyclin-dependent kinase 9 (CDK9), a protein required for recovery from replication arrest. SIRT2 deficiency results in replication stress sensitivity, impairment in recovery from replication arrest, spontaneous accumulation of replication protein A to foci and chromatin, and a G2/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 at lysine 48 in response to replication stress in a manner that is partially dependent on ataxia telangiectasia and Rad3 related (ATR) but not cyclin T or K, thereby stimulating CDK9 kinase activity and promoting recovery from replication arrest. Moreover, wild-type, but not acetylated CDK9, alleviates the replication stress response impairment of SIRT2 deficiency. Collectively, our results define a function for SIRT2 in regulating checkpoint pathways that respond to replication stress through deacetylation of CDK9, providing insight into how SIRT2 maintains genome integrity and a unique mechanism by which SIRT2 may function, at least in part, as a tumor suppressor protein.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Quinase 9 Dependente de Ciclina/metabolismo , Replicação do DNA/fisiologia , Sirtuína 2/metabolismo , Acetilação , Animais , Western Blotting , Linhagem Celular , Cromatografia Líquida , Ensaio de Unidades Formadoras de Colônias , Imunofluorescência , Humanos , Camundongos , Proteína de Replicação A/metabolismo , Espectrometria de Massas em Tandem
5.
Cancer ; 119(17): 3148-55, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23720157

RESUMO

BACKGROUND: Mixed lineage kinase domain-like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC). METHODS: Tissue from 80 patients was collected from a prospectively maintained database of patients with PAC who underwent pancreaticoduodenectomy between January 2000 and October 2008. Immunohistochemistry analysis was performed and scored using an established scoring system. Kaplan-Meier survival curves were generated for recurrence-free survival (RFS) and overall survival (OS) for all patients and for patients receiving adjuvant chemotherapy. MLKL scores were correlated with RFS and OS using univariate and multivariate Cox regression analyses incorporating clinically relevant covariates. RESULTS: The median age of the patients was 63 years and 53% were men. Low MLKL expression was associated with decreased OS (6 months vs 17 months; P = .006). In the subset of 59 patients who received adjuvant chemotherapy, low MLKL expression was associated with decreased RFS (5 months vs 15 months; P = .006) and decreased OS (6 months vs 19 months; P < .0001). On multivariate analysis, low MLKL expression was associated with poor OS in all patients (hazards ratio, 4.6 [95% confidence interval, 1.6-13.8]; P = .006) and in patients receiving adjuvant chemotherapy (hazards ratio, 8.1 [95% confidence interval, 2.2-29.2]; P = .002). CONCLUSIONS: Low expression of MLKL is associated with decreased OS in patients with resected PAC and decreased RFS and OS in the subset of patients with resected PAC who receive adjuvant chemotherapy. The use of this biomarker in patients with PAC may provide important prognostic information.


Assuntos
Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Biomarcadores Tumorais/análise , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Proteínas Quinases/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Razão de Chances , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais
6.
Br J Nutr ; 102(4): 571-5, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19203424

RESUMO

The objectives of the present cross-sectional study were to assess the screening, prevalence and management of malnutrition and identify any co-existence with obesity in adult hospital in-patients. The Malnutrition Universal Screening Tool (MUST) was applied to all medical, surgical, orthopaedic and critical care in-patients in an acute hospital in North-East England on a single day in 2007. An audit was also performed of malnutrition screening using a locally developed tool. Patients were excluded from study if they had been an in-patient less than 24 h or if discharged on the day of study. Of 328 patients meeting inclusion criteria, 100% had full data collection (143 males, 185 females, median length of stay 8 d (range 1-90 d), median age 76 years (range 17-101 years)). Only 226 patients (68.9%) had been screened for malnutrition and thirty-one (13.7%) were at highest malnutrition risk, of which only 45.2% were appropriately referred to nutrition and dietetic services. The prevalence of malnutrition (MUST > or = 1) was 44%. The prevalence of highest risk (MUST > or = 2) increased with age (20.6% < 60 years, 29.7% 60-79 years and 39.4% > or = 80 years). In total 37.8% (n 70) of female patients had a MUST score of > or = 2 compared with 24.5% (n 35) of males. Obesity (BMI > 30 kg/m2) was identified in 9.5% of those with a MUST score > or = 2. We have shown that malnutrition is a common problem affecting over 40% of patients in this hospital-wide study. Currently malnutrition is often unrecognised and undertreated in clinical practice. Hospitals must develop comprehensive strategies to both identify and treat in-patients with this common condition.


Assuntos
Desnutrição/diagnóstico , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Erros de Diagnóstico , Feminino , Hospitais Gerais , Humanos , Masculino , Desnutrição/epidemiologia , Desnutrição/terapia , Pessoa de Meia-Idade , Obesidade/diagnóstico , Obesidade/epidemiologia , Obesidade/terapia , Prevalência , Risco , Reino Unido , Adulto Jovem
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