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1.
Elife ; 102021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477552

RESUMO

DNA double-strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to the expression of HR proteins, including BRCA1, BRCA2, PALB2, and RAD51, and promotes DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Rad51 Recombinase/metabolismo , Transativadores/metabolismo , Proteína BRCA1/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Fase G1 , Fase G2 , Recombinação Homóloga , Humanos , Fase S , Transativadores/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
2.
Cancer Sci ; 112(11): 4736-4747, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34536314

RESUMO

Glioblastomas (GBM) often acquire resistance against temozolomide (TMZ) after continuous treatment and recur as TMZ-resistant GBM (TMZ-R-GBM). Lomustine (CCNU) and nimustine (ACNU), which were previously used as standard therapeutic agents against GBM before TMZ, have occasionally been used for the salvage therapy of TMZ-R-GBM; however, their efficacy has not yet been thoroughly examined. Therefore, we investigated the antitumor effects of CCNU and ACNU against TMZ-R-GBM. As a model of TMZ-R-GBM, TMZ resistant clones of human GBM cell lines (U87, U251MG, and U343MG) were established (TMZ-R-cells) by the culture of each GBM cells under continuous TMZ treatment, and the antitumor effects of TMZ, CCNU, or ACNU against these cells were analyzed in vitro and in vivo. As a result, although growth arrest and apoptosis were triggered in all TMZ-R-cells after the administration of each drug, the antitumor effects of TMZ against TMZ-R-cells were significantly reduced compared to those of parental cells, whereas CCNU and ACNU demonstrated efficient antitumor effects on TMZ-R-cells as well as parental cells. It was also demonstrated that TMZ resistance of TMZ-R-cells was regulated at the initiation of DNA damage response. Furthermore, survival in mice was significantly prolonged by systemic treatment with CCNU or ACNU but not TMZ after implantation of TMZ-R-cells. These findings suggest that CCNU or ACNU may serve as a therapeutic agent in salvage treatment against TMZ-R-GBM.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Lomustina/uso terapêutico , Nimustina/uso terapêutico , Temozolomida/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Glioblastoma/metabolismo , Histonas/metabolismo , Humanos , Injeções Intraperitoneais , Lomustina/administração & dosagem , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Recidiva Local de Neoplasia/tratamento farmacológico , Nimustina/administração & dosagem , Terapia de Salvação/métodos , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nature ; 598(7880): 368-372, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34526721

RESUMO

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.


Assuntos
Microscopia Crioeletrônica , Reparo do DNA , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Transcrição Genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação
4.
BMC Cancer ; 21(1): 1025, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34525976

RESUMO

BACKGROUND: Mutations in driver genes such as IDH and BRAF have been identified in gliomas. Meanwhile, dysregulations in the p53, RB1, and MAPK and/or PI3K pathways are involved in the molecular pathogenesis of glioblastoma. RAS family genes activate MAPK through activation of RAF and PI3K to promote cell proliferation. RAS mutations are a well-known driver of mutation in many types of cancers, but knowledge of their significance for glioma is insufficient. The purpose of this study was to reveal the frequency and the clinical phenotype of RAS mutant in gliomas. METHODS: This study analysed RAS mutations and their clinical significance in 242 gliomas that were stored as unfixed or cryopreserved specimens removed at Kyoto University and Osaka National Hospital between May 2006 and October 2017. The hot spots mutation of IDH1/2, H3F3A, HIST1H3B, and TERT promoter and exon 2 and exon 3 of KRAS, HRAS, and NRAS were analysed with Sanger sequencing method, and 1p/19q codeletion was analysed with multiplex ligation-dependent probe amplification. DNA methylation array was performed in some RAS mutant tumours to improve accuracy of diagnosis. RESULTS: RAS mutations were identified in four gliomas with three KRAS mutations and one NRAS mutation in one anaplastic oligodendroglioma, two anaplastic astrocytomas (IDH wild-type in each), and one ganglioglioma. RAS-mutant gliomas were identified with various types of glioma histology. CONCLUSION: RAS mutation appears infrequent, and it is not associated with any specific histological phenotype of glioma.


Assuntos
Neoplasias Encefálicas/genética , Genes ras/genética , Glioma/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrocitoma/genética , Neoplasias Encefálicas/patologia , Criança , Pré-Escolar , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Análise Mutacional de DNA/métodos , Enzimas Reparadoras do DNA/metabolismo , Éxons/genética , Feminino , Glioblastoma/genética , Glioma/patologia , Histonas/genética , Humanos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Oligodendroglioma/genética , Fenótipo , Regiões Promotoras Genéticas , Telomerase/genética , Proteínas Supressoras de Tumor/metabolismo , Adulto Jovem
5.
Biomolecules ; 11(7)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34356632

RESUMO

Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUbRL) and retracted conformations (pUbRT). Using NMR spectroscopy, we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUbRL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Relacionadas à Autofagia/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Proteínas Quinases/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/metabolismo , Termodinâmica , Ubiquitina/metabolismo
6.
PLoS Pathog ; 17(8): e1009812, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34343212

RESUMO

MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰ - 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip1; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors.


Assuntos
Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , DNA Viral/genética , Queratinócitos/metabolismo , Papiloma/genética , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Animais , Animais Recém-Nascidos , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Feminino , Genoma Viral , Recombinação Homóloga , Queratinócitos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Papiloma/virologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , RNA-Seq
7.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202589

RESUMO

BACKGROUND: Treatment resistance of glioblastoma multiforme to chemo- and radiotherapy remains a challenge yet to overcome. In particular, the O6-methylguanine-DNA-methyltransferase (MGMT) promoter unmethylated patients have only little benefit from chemotherapy treatment using temozolomide since MGMT counteracts its therapeutic efficacy. Therefore, new treatment options in radiotherapy need to be developed to inhibit MGMT and increase radiotherapy response. METHODS: Lomeguatrib, a highly specific MGMT inhibitor, was used to inactivate MGMT protein in vitro. Radiosensitivity of established human glioblastoma multiforme cell lines in combination with lomeguatrib was investigated using the clonogenic survival assay. Inhibition of MGMT was analyzed using Western Blot. Cell cycle distribution and apoptosis were investigated to determine the effects of lomeguatrib alone as well as in combination with ionizing radiation. RESULTS: Lomeguatrib significantly decreased MGMT protein and reduced radiation-induced G2/M arrest. A radiosensitizing effect of lomeguatrib was observed when administered at 1 µM and increased radioresistance at 20 µM. CONCLUSION: Low concentrations of lomeguatrib elicit radiosensitization, while high concentrations mediate a radioprotective effect.


Assuntos
Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/genética , Purinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Proteínas Supressoras de Tumor/metabolismo
8.
Cancer Treat Rev ; 99: 102261, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34332293

RESUMO

BACKGROUND: Temozolomide (TEM) is an active treatment in metastatic neuroendocrine tumors (NETs). Patients affected by glioblastoma multiforme or advanced melanoma treated with TEM who have deficiency of O6-methylguanine DNA methyltransferase (MGMT) have a better responses and survival. However, the predictive role of MGMT in patients with NETs treated with TEM is still debated. METHODS: We conducted a systematic review of the literature and meta-analysis, based on PRISMA methodology, searching in the main databases (PubMed, Embase, Scopus, Web of Science, Cochrane Library and clinical trial.gov) and the proceedings of the main international congresses, until April 26, 2021. RESULTS: Twelve out of 616 articles were selected for our analysis, regarding a total of 858 NET patients treated with TEM-based chemotherapy. The status of MGMT had been tested in 513 (60%) patients, using various methods. The pooled overall response rate (ORR) was higher in MGMT-deficient compared with MGMT-proficient NETs, with a risk difference of 0.31 (95% confidence interval, CI: 0.13-0.50; p < 0.001; I2: 73%) and risk ratio of 2.29 (95% CI: 1.34-3.91; p < 0.001; I2: 55%). The pooled progression free survival (PFS) (hazard ratio, HR = 0.56; 95% CI: 0.43-0.74; p < 0.001) and overall survival (OS) (HR = 0.41; 95% CI: 0.20-0.62; p = 0.011) were longer in MGMT-deficient versus MGMT-proficient NETs. CONCLUSIONS: Our meta-analysis suggested that MGMT status may be predictive of TEM efficacy. However, due to the high heterogeneity of the evaluated studies the risk of biases should be considered. On this hypothesis future homogeneous prospective studies are warranted.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Metilases de Modificação do DNA/deficiência , Enzimas Reparadoras do DNA/deficiência , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/enzimologia , Proteínas Supressoras de Tumor/deficiência , Ensaios Clínicos Fase II como Assunto , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Humanos , Intervalo Livre de Progressão , Ensaios Clínicos Controlados Aleatórios como Assunto , Temozolomida/administração & dosagem , Proteínas Supressoras de Tumor/metabolismo
9.
BMC Cancer ; 21(1): 851, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301221

RESUMO

BACKGROUND: This Phase 2a dose expansion study was performed to assess the safety, tolerability and preliminary efficacy of the maximum tolerated dose of the oral histone de-acetylase (HDAC) inhibitor CXD101 in patients with relapsed / refractory lymphoma or advanced solid organ cancers and to assess HR23B protein expression by immunohistochemistry as a biomarker of HDAC inhibitor sensitivity. METHODS: Patients with advanced solid-organ cancers with high HR23B expression or lymphomas received CXD101 at the recommended phase 2 dose (RP2D). Key exclusions: corrected QT > 450 ms, neutrophils < 1.5 × 109/L, platelets < 75 × 109/L, ECOG > 1. Baseline HR23B expression was assessed by immunohistochemistry. RESULTS: Fifty-one patients enrolled between March 2014 and September 2019, 47 received CXD101 (19 solid-organ cancer, 28 lymphoma). Thirty-four patients received ≥80% RP2D. Baseline characteristics: median age 57.4 years, median prior lines 3, male sex 57%. The most common grade 3-4 adverse events were neutropenia (32%), thrombocytopenia (17%), anaemia (13%), and fatigue (9%) with no deaths on CXD101. No responses were seen in solid-organ cancers, with disease stabilisation in 36% or patients; the overall response rate in lymphoma was 17% with disease stabilisation in 52% of patients. Median progression-free survival was 1.2 months (95% confidence interval (CI) 1.2-5.4) in solid-organ cancers and 2.6 months (95%CI 1.2-5.6) in lymphomas. HR23B status did not predict response. CONCLUSIONS: CXD101 showed acceptable tolerability with efficacy seen in Hodgkin lymphoma, T-cell lymphoma and follicular lymphoma. Further studies assessing combination approaches are warranted. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01977638 . Registered 07 November 2013.


Assuntos
Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Inibidores de Histona Desacetilases/uso terapêutico , Linfoma/tratamento farmacológico , Linfoma/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Adulto , Idoso , Biomarcadores Tumorais , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Imuno-Histoquímica/métodos , Linfoma/diagnóstico , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Resultado do Tratamento
10.
Nat Commun ; 12(1): 3520, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112784

RESUMO

The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Instabilidade Genômica , Histonas/metabolismo , Rad51 Recombinase/metabolismo , Animais , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , DNA Helicases/deficiência , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/genética , Epigênese Genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Histonas/deficiência , Histonas/genética , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Metilação , Camundongos , RNA Interferente Pequeno , Rad51 Recombinase/genética , Regulação para Cima
11.
Nat Commun ; 12(1): 3646, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131137

RESUMO

U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.


Assuntos
Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fatores de Alongamento de Peptídeos , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
12.
Nucleic Acids Res ; 49(11): 6331-6346, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096589

RESUMO

Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4) and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Adutos de DNA , Dano ao DNA , Células HEK293 , Células HeLa , Humanos , Mutagênicos/toxicidade , RNA/biossíntese , Fatores de Transcrição/química , Transcrição Genética
13.
J Biol Chem ; 297(1): 100882, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34144037

RESUMO

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53-p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.


Assuntos
Processamento Alternativo , Proteínas de Ciclo Celular/genética , Senescência Celular , Enzimas Reparadoras do DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Processamento de RNA/genética , Spliceossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo
14.
Nat Cell Biol ; 23(6): 595-607, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34108663

RESUMO

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.


Assuntos
Dano ao DNA , Reparo do DNA , Fator 1 de Elongação de Peptídeos/metabolismo , RNA Polimerase II/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Fator 1 de Elongação de Peptídeos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
15.
Biomolecules ; 11(6)2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067263

RESUMO

Although ubiquitination is widely assumed to be the only regulated step in the ubiquitin-proteasome pathway, recent studies have demonstrated several important mechanisms that regulate the activities of the 26S proteasome. Most proteasomes in cells are inactive but, upon binding a ubiquitinated substrate, become activated by a two-step mechanism requiring an association of the ubiquitin chain with Usp14 and then a loosely folded protein domain with the ATPases. The initial activation step is signaled by Usp14's UBL domain, and many UBL-domain-containing proteins (e.g., Rad23, Parkin) also activate the proteasome. ZFAND5 is a distinct type of activator that binds ubiquitin conjugates and the proteasome and stimulates proteolysis during muscle atrophy. The proteasome's activities are also regulated through subunit phosphorylation. Agents that raise cAMP and activate PKA stimulate within minutes Rpn6 phosphorylation and enhance the selective degradation of short-lived proteins. Likewise, hormones, fasting, and exercise, which raise cAMP, activate proteasomes and proteolysis in target tissues. Agents that raise cGMP and activate PKG also stimulate 26S activities but modify different subunit(s) and stimulate also the degradation of long-lived cell proteins. Both kinases enhance the selective degradation of aggregation-prone proteins that cause neurodegenerative diseases. These new mechanisms regulating proteolysis thus have clear physiological importance and therapeutic potential.


Assuntos
Atrofia Muscular/enzimologia , Doenças Neurodegenerativas/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Proteínas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
16.
Cancer Sci ; 112(9): 3699-3710, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34115910

RESUMO

Pyrosequencing (PSQ) represents the golden standard for MGMT promoter status determination. Binary interpretation of results based on the threshold from the average of several CpGs tested would neglect the existence of the "gray zone". How to define the gray zone and reclassify patients in this subgroup remains to be elucidated. A consecutive cohort of 312 primary glioblastoma patients were enrolled. CpGs 74-81 in the promoter region of MGMT were tested by PSQ and the protein expression was assessed by immunohistochemistry (IHC). Receiver operating characteristic curves were constructed to calculate the area under the curves (AUC). Kaplan-Meier plots were used to estimate the survival rate of patients compared by the log-rank test. The optimal threshold of each individual CpG differed from 5% to 11%. Patients could be separated into the hypomethylated subgroup (all CpGs tested below the corresponding optimal thresholds, n = 126, 40.4%), hypermethylated subgroup (all CpGs tested above the corresponding optimal thresholds, n = 108, 34.6%), and the gray zone subgroup (remaining patients, n = 78, 25.0%). Patients in the gray zone harbored an intermediate prognosis. The IHC score instead of the average methylation levels could successfully predict the prognosis for the gray zone (AUC for overall survival, 0.653 and 0.519, respectively). Combining PSQ and IHC significantly improved the efficiency of survival prediction (AUC: 0.662, 0.648, and 0.720 for PSQ, IHC, and combined, respectively). Immunohistochemistry is a robust method to predict prognosis for patients in the gray zone defined by PSQ. Combining PSQ and IHC could significantly improve the predictive ability for clinical outcomes.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Adolescente , Adulto , Idoso , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Ilhas de CpG/genética , Metilação de DNA , Feminino , Seguimentos , Glioblastoma/mortalidade , Glioblastoma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Prognóstico , Intervalo Livre de Progressão , Estudos Retrospectivos , Taxa de Sobrevida , Adulto Jovem
17.
Cancer Sci ; 112(7): 2835-2844, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33932065

RESUMO

This study aims to build a radiological model based on standard MR sequences for detecting methylguanine methyltransferase (MGMT) methylation in gliomas using texture analysis. A retrospective cross-sectional study was undertaken in a cohort of 53 glioma patients who underwent standard preoperative magnetic resonance (MR) imaging. Conventional visual radiographic features and clinical factors were compared between MGMT promoter methylated and unmethylated groups. Texture analysis extracted the top five most powerful texture features of MR images in each sequence quantitatively for detecting the MGMT promoter methylation status. The radiomic signature (Radscore) was generated by a linear combination of the five features and estimates in each sequence. The combined model based on each Radscore was established using multivariate logistic regression analysis. A receiver operating characteristic (ROC) curve, nomogram, calibration, and decision curve analysis (DCA) were used to evaluate the performance of the model. No significant differences were observed in any of the visual radiographic features or clinical factors between different MGMT methylated statuses. The top five most powerful features were selected from a total of 396 texture features of T1, contrast-enhanced T1, T2, and T2 FLAIR. Each sequence's Radscore can distinguish MGMT methylated status. A combined model based on Radscores showed differentiation between methylated MGMT and unmethylated MGMT both in the glioblastoma (GBM) dataset as well as the dataset for all other gliomas. The area under the ROC curve values for the combined model was 0.818, with 90.5% sensitivity and 72.7% specificity, in the GBM dataset, and 0.833, with 70.2% sensitivity and 90.6% specificity, in the overall gliomas dataset. Nomogram, calibration, and DCA also validated the performance of the combined model. The combined model based on texture features could be considered as a noninvasive imaging marker for detecting MGMT methylation status in glioma.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/enzimologia , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioma/diagnóstico por imagem , Glioma/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Neoplasias Encefálicas/patologia , Meios de Contraste , Estudos Transversais , Metilação de DNA , Reparo do DNA , Técnicas de Apoio para a Decisão , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/enzimologia , Glioblastoma/patologia , Glioma/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Nomogramas , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
18.
BMC Cancer ; 21(1): 493, 2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941107

RESUMO

BACKGROUND: Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/ß-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/ß-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. METHODS: Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of ß-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. RESULTS: Wnt/ß-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. CONCLUSIONS: Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.


Assuntos
Neoplasias Encefálicas/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Glioblastoma/metabolismo , Proteínas de Neoplasias/efeitos dos fármacos , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Idoso , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Celecoxib/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Metilases de Modificação do DNA/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Etoricoxib/farmacologia , Feminino , Glioblastoma/tratamento farmacológico , Humanos , Isoxazóis/farmacologia , Lactonas/farmacologia , Masculino , Metilação , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Receptores de Prostaglandina E Subtipo EP4/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Sulfonas/farmacologia , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/efeitos dos fármacos , beta Catenina/metabolismo
19.
DNA Repair (Amst) ; 103: 103128, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33991872

RESUMO

The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of multiple DNA repair pathways, including nucleotide excision repair (NER), which eliminates a broad variety of helix-distorting DNA lesions that can otherwise cause deleterious mutations and genomic instability. In mammalian NER, DNA damage sensors, DDB and XPC acting in global genomic NER (GG-NER), and, CSB and RNAPII acting in transcription-coupled NER (TC-NER) sub-pathways, undergo an array of post-translational ubiquitination at the DNA lesion sites. Accumulating evidence indicates that ubiquitination orchestrates the productive assembly of NER preincision complex by driving well-timed compositional changes in DNA damage-assembled sensor complexes. Conversely, the deubiquitination is also intimately involved in regulating the damage sensing aftermath, via removal of degradative ubiquitin modification on XPC and CSB to prevent their proteolysis for the factor recycling. This review summaries the relevant research efforts and latest findings in our understanding of ubiquitin-mediated regulation of NER and active participation by new regulators of NER, e.g., Cullin-Ring ubiquitin ligases (CRLs), ubiquitin-specific proteases (USPs) and ubiquitin-dependent segregase, valosin-containing protein (VCP)/p97. We project hypothetical step-by-step models in which VCP/p97-mediated timely extraction of damage sensors is integral to overall productive NER. The USPs and proteasome subtly counteract in fine-tuning the vital stability and function of NER damage sensors.


Assuntos
Reparo do DNA , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , Animais , DNA/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/metabolismo , Raios Ultravioleta
20.
Acta Biochim Biophys Sin (Shanghai) ; 53(7): 893-902, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-33954334

RESUMO

Pre-mRNA processing factor 19 (PRPF19) is a multifaceted protein and participates in DNA damage response and pre-mRNA processing. The role of PRPF19 in cancer is unclear. Here, we report that the expression of PRPF19 in human tongue cancer is associated with unfavorable prognosis. Overexpression of PRPF19 promotes while knockdown of PRPF19 inhibits tongue cancer cell migration, proliferation, and tumor growth. Overexpression of PRPF19 increases the resistance of tongue cancer cells to radiation and cisplatin treatment. Furthermore, PRPF19 regulates the expression of solute carrier family 40 member 1 (SLC40A1) and mono-ADP ribosylhydrolase 2 (MACROD2), knockdown of SLC40A1 or MACROD2 decreases the sensitivity of tongue cancer cells to radiation and cisplatin treatment. Thus, our results establish a key role of PRPF19 in tongue cancer growth and chemoradiotherapy resistance, targeting PRPF19 would be an effective therapeutic strategy for tongue cancer, especially for those resistant to chemoradiotherapy.


Assuntos
Movimento Celular , Proliferação de Células , Quimiorradioterapia , Cisplatino/farmacologia , Enzimas Reparadoras do DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA/metabolismo , Tolerância a Radiação , Neoplasias da Língua , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Fatores de Processamento de RNA/genética , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Neoplasias da Língua/genética , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologia , Neoplasias da Língua/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
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