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1.
Gut ; 69(2): 329-342, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31439637

RESUMO

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Chaperonas de Histonas/fisiologia , Neoplasias Hepáticas/fisiopatologia , Estresse Oxidativo/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes/métodos , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Camundongos Endogâmicos BALB C , Camundongos Nus , Estresse Oxidativo/genética , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/biossíntese , Fatores de Elongação da Transcrição/genética , Regulação para Cima/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Cell Prolif ; 53(1): e12700, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31667976

RESUMO

OBJECTIVES: Long non-coding RNA (lncRNA) MATN1-AS1 is a newfound lncRNA that has been rarely explored in cancers. Herein, we would like to investigate its role in glioma. MATERIALS AND METHODS: qRT-PCR was conducted to examine gene expression in glioma. Then, MTT assay, colony formation assay and flow cytometry analysis were applied to evaluate the function of MATN1-AS1 on glioma cells. Western blot was performed to measure the protein levels of genes. Besides, the luciferase reporter assay, RNA pull-down assay, RIP assay and Spearman's correlation analysis were also performed as needed. RESULTS: Firstly, a data from TCGA showed that MATN1-AS1 might be largely implicated in glioma. Meanwhile, MATN1-AS1 upregulation confirmed in glioma predicted poor clinical outcomes. Functionally, MATN1-AS1 knockdown restrained cell proliferation but stimulated apoptosis in vitro and repressed tumour growth in vivo. Mechanistic investigations validated that MATN1-AS1 functioned as a ceRNA for miR-200b/c/429 to upregulate CHD1 which was also verified to exert a growth-promoting role in glioma cells here. Importantly, both CHD1 overexpression and miR-200b/c/429 inhibition could rescue the obstructive role of MATN1-AS1 silence in glioma cells. CONCLUSIONS: MATN1-AS1 promotes glioma progression through regulating miR-200b/c/429-CHD1 axis, suggesting MATN1-AS1 as a probable target for glioma treatment.


Assuntos
DNA Helicases/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Idoso , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Feminino , Glioma/genética , Glioma/patologia , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética
3.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 91-96, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31833535

RESUMO

Distamycin (DST) is a well-characterized DNA minor groove binder with antivirus activity and antitumor potency. Two separate gene clusters (a 28-kb cluster and a 7-kb cluster) have recently been identified to coordinately encode the biosynthetic machinery of DST in Streptomyces netropsis. Here we report a gene cassette, which is linked to the aforementioned smaller dst gene cluster and plays an important role in the self-resistance to DST in S. netropsis. This cassette consists of three uncharacterized genes that might be implicated in DNA replication/repair. Knockout of the cassette led to the decrease in the production of DST, while heterologous expression of part of the cassette in S. lividans made it become resistant to both DST and mitomycin C, another DNA-binding agent. More interestingly, homologs of these three genes were found in genomes of other actinomyces that produce DNA-binding antibiotics, suggesting that a novel common mechanism in addition to pumping may enable these strains to resist the cytotoxic metabolites they produced.


Assuntos
Antibacterianos/farmacologia , Reparo do DNA/genética , Replicação do DNA/genética , Distamicinas/farmacologia , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Streptomyces/genética , Antibacterianos/biossíntese , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/farmacologia , Distamicinas/biossíntese , Escherichia coli/genética , Técnicas de Inativação de Genes , Mitomicina/farmacologia , Família Multigênica/genética , Streptomyces/efeitos dos fármacos , Streptomyces lividans/efeitos dos fármacos
4.
Chemosphere ; 238: 124586, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31442775

RESUMO

Cyantraniliprole can effectively control lepidopteran pests and has been used all over the world. In general, the risk of cyantraniliprole seems low for fish, but the toxicity selectivity among different fish species was not clear. Here the acute toxicity and chronic effects of cyantraniliprole to juvenile tilapia (Oreochromis mossambicus) were assessed. The results showed that 96 h LC50 of cyantraniliprole to tilapia was 38.0 mg/L. After exposed for 28 days, specific growth rates of the blank control, solution control, and the treatments of 0.037, 0.37 and 3.7 mg/L of cyantraniliprole were 1.14, 0.95, 0.93, 0.82 and 0.70% per day, respectively. The results of micronucleus experiment and single cell gel electrophoresis showed that cyantraniliprole damaged DNA in liver cells of tilapia larvae. Quantitative PCR results showed that cyantraniliprole could induce the up-regulation of Rpa 3 that is responsible for the DNA repair. The significantly down-regulation of Chk 2 gene was related to p53 pathway. It is therefore proposed that cyantraniliprole causes DNA damage in liver cells of tilapia and activates DNA damage and repair pathways.


Assuntos
Dano ao DNA/efeitos dos fármacos , Inseticidas/toxicidade , Fígado/patologia , Pirazóis/toxicidade , Tilápia/embriologia , Tilápia/crescimento & desenvolvimento , ortoaminobenzoatos/toxicidade , Animais , Quinase do Ponto de Checagem 2/biossíntese , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/biossíntese , Brânquias/metabolismo , Hepatócitos/efeitos dos fármacos , Larva , Dose Letal Mediana , Alimentos Marinhos
5.
Elife ; 82019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31552827

RESUMO

Hsf1 is an ancient transcription factor that responds to protein folding stress by inducing the heat-shock response (HSR) that restore perturbed proteostasis. Hsp70 chaperones negatively regulate the activity of Hsf1 via stress-responsive mechanisms that are poorly understood. Here, we have reconstituted budding yeast Hsf1-Hsp70 activation complexes and find that surplus Hsp70 inhibits Hsf1 DNA-binding activity. Hsp70 binds Hsf1 via its canonical substrate binding domain and Hsp70 regulates Hsf1 DNA-binding activity. During heat shock, Hsp70 is out-titrated by misfolded proteins derived from ongoing translation in the cytosol. Pushing the boundaries of the regulatory system unveils a genetic hyperstress program that is triggered by proteostasis collapse and involves an enlarged Hsf1 regulon. The findings demonstrate how an apparently simple chaperone-titration mechanism produces diversified transcriptional output in response to distinct stress loads.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/biossíntese , DNA Fúngico/metabolismo , Temperatura Alta , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/efeitos da radiação
6.
Mol Med Rep ; 20(4): 3519-3526, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485614

RESUMO

Cisplatin has been widely used as a conventional treatment for patients with non­small cell lung cancer (NSCLC). However, primary and acquired cisplatin resistances are frequently developed during the treatment of patients with NSCLC, leading to an increased mortality rate. Accumulating evidence demonstrated that aberrantly expressed microRNAs (miRs) are involved in the development of chemoresistance. In the present study, sensitivity of NSCLC cells to cisplatin was identified to increase following overexpression of miR­608. Conversely, sensitivity to cisplatin was reduced following miR­608 knockdown. Reverse transcription­quantitative PCR and western blotting analyses identified that TEA domain transcription factor 2 (TEAD2), a key regulator of cell stemness, was negatively regulated by miR­608 in NSCLC cells. By repressing TEAD2, miR­608 decreased the expression level of several target genes of the Hippo­yes­associated protein signaling pathway. Furthermore, TEAD2 mRNA was confirmed to be targeted by miR­608 in NSCLC cells via a dual­luciferase reporter assay. Importantly, the increased cisplatin sensitivity induced by miR­608 overexpression was reversed by transfection of TEAD2 in NSCLC cells. The present data suggested that miR­608 may represent a novel candidate biomarker for the evaluation of cisplatin sensitivity in patients with NSCLC.


Assuntos
Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , Cisplatino/farmacologia , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares , MicroRNAs , Proteínas de Neoplasias , RNA Neoplásico , Fatores de Transcrição , Células A549 , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
7.
Elife ; 82019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502540

RESUMO

How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. We test the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, we manipulate expression of the classic temporal transcription factor Hunchback (Hb) specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then we monitor dendrite morphology and neuromuscular synaptic partnerships. We find that prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, our data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acts early in neurogenesis as a switch to control motor circuit wiring, and provide novel insight into the relationship between stem cell and circuit.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila/biossíntese , Expressão Gênica , Neurônios Motores/fisiologia , Vias Neurais/embriologia , Junção Neuromuscular/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/biossíntese , Animais , Drosophila , Neurônios Motores/citologia , Junção Neuromuscular/citologia , Células-Tronco/citologia
8.
Postgrad Med ; 131(8): 597-606, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31419922

RESUMO

The association between cancer and dysglycemia has been well documented. It is underappreciated, however, that sustained dysglycemia could potentially be a catalyst toward a pro-cancer physiologic milieu and/or increase the burden of cancer. Hyperglycemia, hyperinsulinemia and energy metabolism at large impact a cascade of growth pathways, epi/genetic modifications, and mitochondrial changes that could feasibly link to tumor processes. Oxidative stress is a recurring motif in cell dysfunction: in diabetes, oxidative stress and reactive oxygen species (ROS) feature prominently in the damage and demise of pancreatic beta cells, as well as cell damage contributing to diabetes-related complications. Oxidative stress may be one intersection at which metabolic and oncogenic processes cross paths with deleterious results in the development of precancer, cancer, and cancer progression. This would augur for tight glucose control. Regrettably, some medical societies have recently relaxed hemoglobin A1c targets. A framework for the hyperglycemic state is presented that helps account and translate the full scope of effects of dysglycemia to ultimately improve clinical best practices.


Assuntos
Hiperglicemia/epidemiologia , Hiperglicemia/fisiopatologia , Neoplasias/epidemiologia , Neoplasias/fisiopatologia , Proteínas de Ligação a DNA/biossíntese , Progressão da Doença , Metabolismo Energético/fisiologia , Hemoglobina A Glicada , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/genética , Hiperinsulinismo/fisiopatologia , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Neoplasias/genética , Obesidade/epidemiologia , Estresse Oxidativo/fisiologia , Prognóstico , Proteínas Proto-Oncogênicas/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
9.
Pathologica ; 111(2): 58-61, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31388196

RESUMO

Introduction: The 2011 WHO Classification for lung adenocarcinoma enlightened the need for a wise use of immunohistochemistry to preserve tissue for both diagnosis and molecular studies. The current recommendation is to use a panel comprising TTF1 and p40 to classify tumors with no clear squamous or glandular differentiation as many studies have showed the higher specificity of p40 over p63 as marker of squamous differentiation. However, the co-expression of both markers opens a new scenario with subsequent classification and potentially treatment issues. Materials and methods: We report a case of a non-small lung cell carcinoma (NSCLC) with coexistent expression of TTF1 and p40 in the same tumour cells. To our knowledge, this peculiar immunohistochemical profile is very rare, and thus a review of the clinical and molecular features including molecular variances of the tumour was performed. Review of the pertinent literature was also carried out. Results: Two additional articles describing unusual cases of NSCLC with coexistent expression of TTF1 and p40 were found and compared to our case. Interestingly, they all carried out aberrant mutation in TP53 oncogene and were of advance stage. Conclusion: The positivity for both "squamous" and "adenocarcinomatous" markers and mutations of TP53 could be the expression of a not fully recognized variant of NSCLC with possible implications for classification, diagnosis and therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Epitopos Imunodominantes/genética , Neoplasias Pulmonares/genética , Mutação/genética , Fragmentos de Peptídeos/genética , Fatores de Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Ligação a DNA/biossíntese , Humanos , Epitopos Imunodominantes/biossíntese , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/biossíntese , Fatores de Transcrição/biossíntese
10.
Inorg Chem ; 58(17): 11351-11363, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31433627

RESUMO

Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from Listeria innocua to structurally characterize these steps for Co2+, Zn2+, and La3+ ions. The structures reveal that differences in their ion coordination chemistry determine alternative metal ion-binding sites on the areas of the surface surrounding the translocation pore that captures nine La3+, three Co2+, or three Zn2+ ions as aquo clusters and passes them on for translocation. Inside these pores, ion-selective conformational changes at key residues occur before a gating residue to actively move ions through the constriction zone. Ions upstream of the Asp130 gate residue are typically hydrated, while ions downstream directly interact with the protein matrix. Inside the cavity, ions move along negatively charged residues to the ferroxidase center, where seven main residues adapt to the three different ions by dynamically changing their conformations. In total, we observed more than 20 metal-binding sites per Dps monomer, which clearly highlights the metal-binding capacity of this protein family. Collectively, our results provide a detailed structural description of the preparative steps for amino acid-assisted biomineralization in Dps proteins, demonstrating unexpected protein matrix plasticity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Listeria/química , Metais Pesados/química , Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/biossíntese , Modelos Moleculares , Eletricidade Estática
11.
Mol Cell ; 75(6): 1178-1187.e4, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31402096

RESUMO

In complex genetic loci, individual enhancers interact most often with specific basal promoters. Here we investigate the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. We show that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, we propose that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Motivos de Nucleotídeos , Elementos de Resposta , Transativadores/metabolismo , Fatores de Transcrição/biossíntese , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transativadores/genética , Fatores de Transcrição/genética
12.
Genes Cells ; 24(10): 650-666, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31442350

RESUMO

Neural progenitor cells (NPCs, also known as radial glial progenitors) produce neurons and then glial cells such as astrocytes during development of the mouse neocortex. Given that this sequential generation of neural cells is critical for proper brain formation, the neurogenic potential of NPCs must be precisely controlled. Here, we show that the transcription factor Plag1 plays an important role in the regulation of neurogenic potential in mouse neocortical NPCs. We found that Hmga2, a key neurogenic factor in neocortical NPCs, induces expression of the Plag1 gene. Analysis of the effects of over-expression or knockdown of Plag1 indicated that Plag1 promotes the production of neurons at the expense of astrocyte production in embryonic neocortical cultures. Furthermore, over-expression of Plag1 promoted and knockdown of Plag1 suppressed neuronal differentiation of neocortical NPCs in vivo. Transcriptomic analysis showed that Plag1 increases the expression of a set of neuronal genes in NPCs. Our results thus identify Plag1 as a regulator of neuronal gene expression and neuronal differentiation in NPCs of the developing mouse neocortex.


Assuntos
Proteínas de Ligação a DNA/genética , Neocórtex/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Camundongos , Neocórtex/citologia , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia
13.
Respir Res ; 20(1): 163, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31331325

RESUMO

BACKGROUND: Pulmonary fibrosis is a progressive and irreversible disease for which therapeutic options are currently limited. A recent in vivo study showed that tenofovir, a nucleotide analogue reverse transcriptase inhibitor, had direct antifibrotic effects on skin and liver fibrosis. Another study in vitro revealed that NS5ATP9 inhibited the activation of human hepatic stellate cells. Because of the similarity of fibrotic diseases, we hypothesized that tenofovir alafenamide fumarate (TAF), the prodrug of tenofovir, and NS5ATP9, is related to and plays a role in the suppression of pulmonary fibrosis. METHODS: We investigated the influence of NS5ATP9 on fibrosis in vitro. Human lung fibroblasts (HFL1) were transfected with short interfering RNAs or overexpression plasmids of NS5ATP9 before stimulation by human recombinant transforming growth factor-ß1. The effect of TAF was evaluated in a bleomycin-induced fibrosis murine model. Male C57BL/6 mice were treated with bleomycin on day 0 by intratracheal injection and intragastrically administered TAF or vehicle. Left lung sections were fixed for histological analysis, while homogenates of the right lung sections and HFL1 cells were analyzed by western blotting and quantitative reverse transcription polymerase chain reaction. RESULTS: NS5ATP9 suppressed the activation of lung fibroblasts. Upregulation of collagen type 3 (α 1 chain) and α-smooth muscle actin was observed in HFL1 cells when NS5ATP9 was silenced, and vice-versa. TAF also showed anti-fibrotic effects in mice, as demonstrated by histological analysis of fibrosis and expression of extracellular matrix components in the lung sections. Additionally, TAF inhibited transforming growth factor-ß1 and phosphorylated-Smad3 synthesis in HFL1 cells and the murine model, which was accompanied by upregulation of NS5ATP9. CONCLUSIONS: Our results suggest that NS5ATP9 forms a negative feedback pathway in pulmonary fibrosis and TAF has anti-fibrotic properties as it upregulates the expression level of NS5ATP9. As TAF has been shown to be safe and well-tolerated in humans, TAF and NS5ATP9 may be useful for developing novel therapeutics for pulmonary fibrosis.


Assuntos
Adenina/análogos & derivados , Bleomicina/toxicidade , Proteínas de Ligação a DNA/biossíntese , Fibrose Pulmonar/metabolismo , Proteína Smad3/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
14.
Med Oncol ; 36(8): 66, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183633

RESUMO

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Neuroblastoma/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína GAP-43/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para Cima
15.
Virchows Arch ; 475(4): 407-414, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201505

RESUMO

Nuclear membrane proteins reportedly play important roles in maintaining nuclear structures and coordinating cell activities. Studying profiles of nuclear membrane proteins may help us evaluate the biological and/or clinical nature of malignant tumors. Using immunohistochemistry with antibodies for emerin, lamin A/C, lamin B, and LAP2, we examined 105 lung cancer tissues from 33 small cell lung carcinomas (SCLCs) and 72 non-SCLCs (34 adenocarcinomas, 30 squamous cell carcinomas, and 8 large cell carcinomas). Emerin had negative or local/weak positivity in 79% of SCLCs and 1% of non-SCLCs, and lamin A/C had similar positivity in 91% of SCLCs and 3% of non-SCLCs. LAP2's expression was similar between SCLCs and non-SCLCs. RT-PCR analyses for these four nuclear membrane proteins over 7 cell lines showed that mRNA of emerin and lamin A/C were distinctly downregulated in the SCLC cell lines, supporting the immunohistochemical results. In conclusion, we suggest that downregulation of the nuclear membrane proteins emerin and lamin A/C is characteristic of SCLC cells, and this constitutional abnormality of the nuclear membrane may be related to the biological and/or clinical nature of SCLC. In addition, knowing the nuclear protein profile in SCLC cells may contribute to our understanding of nuclear fragility known as the crush artifact in pulmonary biopsy specimens.


Assuntos
Lamina Tipo A/biossíntese , Neoplasias Pulmonares/patologia , Proteínas de Membrana/biossíntese , Proteínas Nucleares/biossíntese , Carcinoma de Pequenas Células do Pulmão/patologia , Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Humanos , Lamina Tipo A/análise , Lamina Tipo B/análise , Lamina Tipo B/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/análise , Proteínas Nucleares/análise , Carcinoma de Pequenas Células do Pulmão/metabolismo
16.
Exp Hematol ; 73: 50-63.e2, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30986496

RESUMO

The most frequently occurring genetic abnormality in pediatric B-lymphocyte-lineage acute lymphoblastic leukemia is the t(12;21) chromosomal translocation that results in a ETV6-RUNX1 (also known as TEL-AML1) fusion gene. Expression of ETV6-RUNX1 induces a preleukemic condition leading to acquisition of secondary driver mutations, but the mechanism is poorly understood. SPI-B (encoded by SPIB) is an important transcriptional activator of B-cell development and differentiation. We hypothesized that SPIB is directly transcriptionally repressed by ETV6-RUNX1. Using chromatin immunoprecipitation, we identified a regulatory region in the first intron of SPIB that interacts with ETV6-RUNX1. Mutation of the RUNX1 binding site in SPIB intron 1 prevented transcriptional repression in transient transfection assays. Next, we sought to determine to what extent gene expression in REH cells can be altered by ectopic SPI-B expression. SPI-B expression was forced using CRISPR-mediated gene activation and also using a retroviral vector. Forced expression of SPI-B resulted in altered gene expression and, at high levels, impaired cell proliferation and induced apoptosis. Finally, we identified CARD11 and CDKN1A (encoding p21) as transcriptional targets of SPI-B involved in regulation of proliferation and apoptosis. Taken together, this study identifies SPIB as an important target of ETV6-RUNX1 in regulation of B-cell gene expression in t(12;21) leukemia.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/biossíntese , Regulação Leucêmica da Expressão Gênica , Íntrons , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Elementos de Resposta , Fatores de Transcrição/biossíntese , Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD/biossíntese , Proteínas Adaptadoras de Sinalização CARD/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Guanilato Ciclase/biossíntese , Guanilato Ciclase/genética , Humanos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Fatores de Transcrição/genética , Translocação Genética
17.
Oncol Res ; 27(7): 809-818, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30982491

RESUMO

RAS-responsive element-binding protein 1 (RREB1) is a transcription factor that is implicated in RAS signaling and multiple tumors. However, the role of RREB1 in acute myeloid leukemia has not been studied. We found that RREB1 is overexpressed in AML patients and myeloid leukemia cell lines (NB4 and HL-60), and RREB1 expression was significantly decreased during granulocytic differentiation of myeloid leukemia cells induced by all-trans retinoic acid (ATRA). Then we performed a RREB1 knockdown assay in NB4 and HL-60 cells; the results showed that knockdown of RREB1 upregulated expression of CD11b, CEBPß, and microRNA-145 (miR-145), which hinted that knockdown of RREB1 enhanced granulocytic differentiation of myeloid leukemia cells. In addition, inhibitor of miR-145 can offset the enhanced effect on granulocytic differentiation mediated by downregulation of RREB1. These collective findings demonstrated that RREB1 blocks granulocytic differentiation of myeloid leukemia cells by inhibiting the expression of miR-145 and downstream targets of the RAS signal pathway. These may provide a promising therapeutic target for AML patients.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Granulócitos/patologia , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Granulócitos/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , MicroRNAs/genética , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Tretinoína/farmacologia , Proteínas ras/metabolismo
18.
Virchows Arch ; 475(4): 457-466, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31016435

RESUMO

Overexpression of ETS genes is involved in prostate cancer (PrCa), but there is little information on the non-ERG components of this family. We have investigated ETV1, ETV4, and ETV5 overexpression, with or without PTEN loss, and their association with grade group (GG), pathological stage, focality, and PSA recurrence in PrCa. ETS gene expression was analyzed by qPCR in 104 cases. ETV1 and PTEN immunohistochemistry was assessed in TMA sections from 194 additional cases (PSMAR-Biobank, Barcelona, Spain). ETS mRNA overexpression was found in 23.1%, being ETV1 the most frequently overexpressed (18.3%). ETV1 protein overexpression was detected in 30.4% cases (moderate in 19.6%, strong in 10.8%). PTEN protein expression loss was detected in 36.1% cases and was not associated with ETV1. Strong-moderate ETV1 protein overexpression reaches its highest values in GG3-4, whereas its negativity was significantly more common in GG1 tumors (p = 0.034). ETV1-overexpressing tumors were more often unifocal (p = 0.0007) and high stage (p = 0.032). PTEN loss was less common in GG1 (p = 0.012) and showed a trend to be less frequent in pT2 (p = 0.062) tumors. Strong ETV1 immunostaining (histoscore > 177) was associated with shorter time to PSA recurrence in the univariate (p = 0.002) and in the multivariate analysis (p = 0.018). Moreover, when strong ETV1 overexpression was not combined with PTEN loss, its association with PSA recurrence was even stronger (p = 0.0004). In conclusion, non-ERG ETS overexpression, particularly ETV1 overexpression, has a non-negligible role in PrCa. Strong ETV1 protein expression has a negative impact on prostate cancer outcome that seems to be independent of PTEN status.


Assuntos
Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/biossíntese , Neoplasias da Próstata/patologia , Fatores de Transcrição/biossíntese , Idoso , Citoplasma/metabolismo , Proteínas de Ligação a DNA/análise , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Fatores de Transcrição/análise
19.
Nat Cell Biol ; 21(4): 442-451, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886345

RESUMO

The cytosolic accumulation of mitochondrial precursors is hazardous to cellular fitness and is associated with a number of diseases. However, it is not observed under physiological conditions. Individual mechanisms that allow cells to avoid cytosolic accumulation of mitochondrial precursors have recently been discovered, but their interplay and regulation remain elusive. Here, we show that cells rapidly launch a global transcriptional programme to restore cellular proteostasis after induction of a 'clogger' protein that reduces the number of available mitochondrial import sites. Cells upregulate the protein folding and proteolytic systems in the cytosol and downregulate both the cytosolic translation machinery and many mitochondrial metabolic enzymes, presumably to relieve the workload of the overstrained mitochondrial import system. We show that this transcriptional remodelling is a combination of a 'wideband' core response regulated by the transcription factors Hsf1 and Rpn4 and a unique mitoprotein-induced downregulation of the oxidative phosphorylation components, mediated by an inactivation of the HAP complex.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico/genética , Transcrição Genética , Citosol/enzimologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Fosforilação Oxidativa , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo
20.
Gynecol Oncol ; 153(2): 416-424, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30797591

RESUMO

PURPOSE: PARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers. METHODS: ERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality. RESULTS: Low ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis. CONCLUSION: The data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.


Assuntos
Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/metabolismo , Proteínas de Ligação a DNA/deficiência , Endonucleases/deficiência , Compostos Organoplatínicos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Endonucleases/biossíntese , Endonucleases/genética , Feminino , Humanos , Imuno-Histoquímica , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/biossíntese , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Análise Serial de Tecidos , Transfecção
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