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1.
Oncogene ; 43(11): 804-820, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38279062

RESUMO

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.


Assuntos
Cromatina , Glioma , Humanos , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glioma/genética
2.
Cancer Gene Ther ; 27(5): 319-329, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31138900

RESUMO

The Holliday Junction-Recognition Protein (HJURP) was reported as overexpressed in several cancers and also strongly correlated with poor prognosis of patients, especially in glioblastoma (GBM), the most common and deadly type of primary brain tumor. HJURP is responsible for loading the histone H3 variant-the Centromeric Protein A (CENP-A)-at the centromeres in a cell cycle-regulated manner, being required for proper chromosome segregation. Here we investigated HJURP association with survival and radioresistance of different GBM cell lines. HJURP knockdown compromised the clonogenic capacity and severely impaired survival of five distinct GBM cells, while nontumor astrocytes were not affected. U251MG cells showed a robust cell cycle arrest in G2/M phases followed by a drastic increment in cell death after HJURP silencing, while U138MG and U343MG cell lines presented augmented senescence with a comparable increase in cell death. Importantly, we verified that the impact on cell cycle dynamics and clonogenic survival were associated with loss CENP-A at the centromeres. Moreover, radiation resistance was also impacted by HJURP modulation in several GBM cell lines. U87MG, T98G, U138MG, and U343MG cells were all sensitized to ionizing radiation after HJURP reduction. These data reinforce the requirement of HJURP for proliferative capacity and radioresistance of tumor cells, underlining its potential as a promising therapeutic target for GBM.


Assuntos
Neoplasias Encefálicas/radioterapia , Proteína Centromérica A/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glioblastoma/radioterapia , Células-Tronco Neoplásicas/efeitos da radiação , Neoplasias Encefálicas/patologia , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Centrômero/metabolismo , Centrômero/efeitos da radiação , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Humanos , Células-Tronco Neoplásicas/patologia , Tolerância a Radiação/genética , Ensaio Tumoral de Célula-Tronco
3.
Pharmacogenomics J ; 19(1): 72-82, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30131588

RESUMO

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.


Assuntos
Galanina/genética , Esclerose Múltipla/genética , Receptor Tipo 2 de Galanina/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular , Feminino , Células HEK293 , Humanos , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética , Adulto Jovem
4.
Pigment Cell Melanoma Res ; 32(2): 303-314, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30457212

RESUMO

RMEL3 is a recently identified lncRNA associated with BRAFV600E mutation and melanoma cell survival. Here, we demonstrate strong and moderate RMEL3 upregulation in BRAF and NRAS mutant melanoma cells, respectively, compared to melanocytes. High expression is also more frequent in cutaneous than in acral/mucosal melanomas, and analysis of an ICGC melanoma dataset showed that mutations in RMEL3 locus are preponderantly C > T substitutions at dipyrimidine sites including CC > TT, typical of UV signature. RMEL3 mutation does not correlate with RMEL3 levels, but does with poor patient survival, in TCGA melanoma dataset. Accordingly, RMEL3 lncRNA levels were significantly reduced in BRAFV600E melanoma cells upon treatment with BRAF or MEK inhibitors, supporting the notion that BRAF-MEK-ERK pathway plays a role to activate RMEL3 gene transcription. RMEL3 overexpression, in immortalized fibroblasts and melanoma cells, increased proliferation and survival under serum starvation, clonogenic ability, and xenografted melanoma tumor growth. Although future studies will be needed to elucidate the mechanistic activities of RMEL3, our data demonstrate that its overexpression bypasses the need of mitogen activation to sustain proliferation/survival of non-transformed cells and suggest an oncogenic role for RMEL3.


Assuntos
Citoproteção , Melanoma/genética , Melanoma/patologia , RNA Longo não Codificante/metabolismo , Soro/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Clonais , Citoproteção/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células NIH 3T3 , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , RNA Longo não Codificante/genética
5.
J Mol Model ; 24(9): 225, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30088101

RESUMO

Receptor-interacting protein kinase 2 (RIPK2) plays an essential role in autoimmune response and is suggested as a target for inflammatory diseases. A pharmacophore model was built from a dataset with ponatinib (template) and 18 RIPK2 inhibitors selected from BindingDB database. The pharmacophore model validation was performed by multiple linear regression (MLR). The statistical quality of the model was evaluated by the correlation coefficient (R), squared correlation coefficient (R2), explanatory variance (adjusted R2), standard error of estimate (SEE), and variance ratio (F). The best pharmacophore model has one aromatic group (LEU24 residue interaction) and two hydrogen bonding acceptor groups (MET98 and TYR97 residues interaction), having a score of 24.739 with 14 aligned inhibitors, which were used in virtual screening via ZincPharmer server and the ZINC database (selected in function of the RMSD value). We determined theoretical values of biological activity (logRA) by MLR, pharmacokinetic and toxicology properties, and made molecular docking studies comparing binding affinity (kcal/mol) results with the most active compound of the study (ponatinib) and WEHI-345. Nine compounds from the ZINC database show satisfactory results, yielding among those selected, the compound ZINC01540228, as the most promising RIPK2 inhibitor. After binding free energy calculations, the following molecular dynamics simulations showed that the receptor protein's backbone remained stable after the introduction of ligands.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo
6.
Nucleic Acids Res ; 46(8): 3891-3905, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29529298

RESUMO

CHD1 is a conserved chromatin remodeling enzyme required for development and linked to prostate cancer in adults, yet its role in human cells is poorly understood. Here, we show that targeted disruption of the CHD1 gene in human cells leads to a defect in early double-strand break (DSB) repair via homologous recombination (HR), resulting in hypersensitivity to ionizing radiation as well as PARP and PTEN inhibition. CHD1 knockout cells show reduced H2AX phosphorylation (γH2AX) and foci formation as well as impairments in CtIP recruitment to the damaged sites. Chromatin immunoprecipitation following a single DSB shows that the reduced levels of γH2AX accumulation at DSBs in CHD1-KO cells are due to both a global reduction in H2AX incorporation and poor retention of H2AX at the DSBs. We also identified a unique N-terminal region of CHD1 that inhibits the DNA binding, ATPase, and chromatin assembly and remodeling activities of CHD1. CHD1 lacking the N terminus was more active in rescuing the defects in γH2AX formation and CtIP recruitment in CHD1-KO cells than full-length CHD1, suggesting the N terminus is a negative regulator in cells. Our data point to a role for CHD1 in the DSB repair process and identify a novel regulatory region of the protein.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular , Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Técnicas de Inativação de Genes , Histonas/metabolismo , Recombinação Homóloga , Humanos , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais
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