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1.
Mol Cell ; 79(6): 1008-1023.e4, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32871104

RESUMO

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.


Assuntos
Proteínas de Ciclo Celular/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Proteínas Tirosina Quinases/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Tratamento Farmacológico , Proteína 7 com Repetições F-Box-WD/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
2.
Anticancer Res ; 40(9): 4961-4968, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878784

RESUMO

BACKGROUND/AIM: Despite advances in treatment modalities, the visual prognosis of retinoblastoma still remains unsatisfactory, underscoring the need to develop novel therapeutic approaches. MATERIALS AND METHODS: The effect on the growth of six human retinoblastoma cell lines and a normal human fibroblast cell line of CEP1347, a small-molecule kinase inhibitor originally developed for the treatment of Parkinson's disease and therefore with a known safety profile in humans, was examined. The role of the P53 pathway in CEP1347-induced growth inhibition was also investigated. RESULTS: CEP1347 selectively inhibited the growth of retinoblastoma cell lines expressing murine double minute 4 (MDM4), a P53 inhibitor. Furthermore, CEP1347 reduced the expression of MDM4 and activated the P53 pathway in MDM4-expressing retinoblastoma cells, which was required for the inhibition of their growth by CEP1347. CONCLUSION: We propose CEP1347 as a promising candidate for the treatment of retinoblastomas, where functional inactivation of P53 as a result of MDM4 activation is reportedly common.


Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Retinoblastoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Retinoblastoma/metabolismo , Retinoblastoma/patologia
3.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
4.
Nat Commun ; 11(1): 4666, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938943

RESUMO

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinß, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinß in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinß mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinß knock-out mice-indicating a functional and genetic interaction between Xinß and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinß modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cardiomiopatia Dilatada/genética , Comunicação Celular , Proteínas de Ciclo Celular/genética , Proliferação de Células , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/crescimento & desenvolvimento , Proteínas com Domínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Proteínas Nucleares/genética , Transdução de Sinais
5.
Nat Commun ; 11(1): 4766, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958778

RESUMO

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inflamação/patologia , Telômero/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antibacterianos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Criança , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Gastroenteropatias/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-18/genética , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transdução de Sinais , Telomerase/genética , Telomerase/metabolismo
6.
PLoS One ; 15(8): e0236881, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745107

RESUMO

PIERCE1, p53 induced expression 1 in Rb null cells, is a novel p53 target involved in the DNA damage response and cell cycle in mice. These facts prompted us to study the function of PIERCE1 with respect to p53-associated pathophysiology of cancer in humans. Unexpectedly, PIERCE1 did not respond to overexpression and activation of p53 in humans. In this study, we swapped p53 protein expression in human and mouse cells to find the clue of this difference between species. Human p53 expression in mouse cells upregulated PIERCE1 expression, suggesting that p53-responsive elements on the PIERCE1 promoter are crucial, but not the p53 protein itself. Indeed, in silico analyses of PIERCE1 promoters revealed that p53-responsive elements identified in mice are not conserved in humans. Consistently, chromatin immunoprecipitation-sequencing (ChIP-seq) analyses confirmed p53 enrichment against the PIERCE1 promoter region in mice, not in human cells. To complement the p53 study in mice, further promoter analyses suggested that the human PIERCE1 promoter is more similar to guinea pigs, lemurs, and dogs than to rodents. Taken together, our results confirm the differential responsiveness of PIERCE1 expression to p53 due to species differences in PIERCE1 promoters. The results also show partial dissimilarity after p53 induction between mice and humans.


Assuntos
Proteínas de Ciclo Celular , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/genética , Humanos , Camundongos , Regiões Promotoras Genéticas , Transcrição Genética/fisiologia
7.
PLoS One ; 15(8): e0235930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750054

RESUMO

Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNAi) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clock´s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels within ~two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rm´perRNAi or Rm´cry2RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´perRNAi and shorter periods for Rm´tim1RNAi and Rm´cry2RNAi.As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Baratas/fisiologia , Criptocromos/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Circadianas Period/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Relógios Circadianos , Ritmo Circadiano , Baratas/genética , Criptocromos/genética , Proteínas de Insetos/genética , Masculino , Proteínas Circadianas Period/genética , Fotoperíodo , Interferência de RNA
8.
Mol Cell ; 79(6): 917-933.e9, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32755595

RESUMO

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/ultraestrutura , Troca de Cromátide Irmã/genética , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Microscopia Crioeletrônica , DNA/genética , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
9.
Mol Cell ; 79(6): 902-916.e6, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32768407

RESUMO

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.


Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Adenosina Trifosfatases/genética , Anáfase/genética , Animais , Proteínas de Ciclo Celular/isolamento & purificação , Cromátides/genética , Proteínas Cromossômicas não Histona , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/isolamento & purificação , Imageamento Tridimensional , Mamíferos , Metáfase/genética , Prófase/genética
10.
Nat Commun ; 11(1): 4055, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792504

RESUMO

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.


Assuntos
Cromatina/metabolismo , Glucose/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunoprecipitação da Cromatina , Epigênese Genética/genética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
11.
PLoS Genet ; 16(8): e1008981, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745133

RESUMO

Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.


Assuntos
Proteínas de Ciclo Celular/genética , Heterogeneidade Genética , Genética Populacional , Repetições Minissatélites/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Estônia/epidemiologia , Feminino , Regulação da Expressão Gênica/genética , Genótipo , Humanos , Masculino , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , RNA-Seq , Sequenciamento Completo do Genoma
12.
PLoS Genet ; 16(8): e1008962, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750047

RESUMO

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.


Assuntos
Cromatina/genética , Proteínas de Drosophila/genética , Mitose/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Proteínas de Ciclo Celular/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/genética , Histonas/genética , Interfase/genética , Fosforilação , Proteínas do Grupo Polycomb/genética , Troca de Cromátide Irmã/genética , Treonina/genética
13.
J Environ Pathol Toxicol Oncol ; 39(2): 101-111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32749120

RESUMO

Long noncoding RNAs (lncRNAs) have been reported to be involved in cancer initiation and evolution, including colorectal cancer (CRC). Nuclear-enriched abundant transcript 1 (NEAT1) exerts important functions in multiple cancers; however, the specific modulatory mechanism in CRC demands in-depth exploration. The expression levels of NEAT1, microRNA-195-5p (miR-195-5p), and centrosomal protein 55 (CEP55) were examined using quantitative real-time polymerase chain reaction (qRT-PCR), and protein expression of CEP55 was detected by Western blot assay. Cell proliferation and apoptosis were measured by 3-(4,5-dimethylthiazole-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry. Transwell migration and invasion assays were applied to evaluate cell metastasis ability. Dual-luciferase reporter assay was used to analyze the correlation among NEAT1, miR-195-5p and CEP55. The expression of NEAT1 was up-regulated in CRC tissues and cells, and overall survival was lower with high expression of NEAT1. Knockdown of NEAT1 repressed cell proliferation, migration, and invasion, while inducing apoptosis in CRC cells. NEAT1 targeted miR-195-5p and inhibited the expression of miR-195-5p. Silence of NEAT1 inhibited CRC cell proliferation, migration, and invasion, and promoted apoptosis by up-regulating miR-195-5p. MiR-195-5p targeted and suppressed CEP55 expression, and CEP55 reverted the effects induced by miR-195-5p. NEAT1 regulated the expression of CEP55 through miR-195-5p. NEAT1 promotes colorectal cancer cellular processes by regulating CEP55 expression via the sponging of miR-195-5p. Therefore, NEAT1 might play a crucial role in CRC treatments.


Assuntos
Neoplasias Colorretais/genética , RNA Longo não Codificante/genética , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Mucosa Gástrica/citologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Estimativa de Kaplan-Meier , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo
14.
Nat Commun ; 11(1): 3796, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732900

RESUMO

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Divisão Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo
15.
Nat Commun ; 11(1): 4053, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792481

RESUMO

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequenciamento Completo do Exoma/métodos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina D1/genética , Variações do Número de Cópias de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Nus , Piperazinas/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pteridinas/uso terapêutico , Piridinas/uso terapêutico
16.
Nat Commun ; 11(1): 4102, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796823

RESUMO

Emerging evidence suggests that intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. However, the extent of heterogeneity within the villi stromal compartment and how IntSCs regulate the structure and function of specialized intestinal lymphatic capillary called lacteal remain elusive. Here we show that selective hyperactivation or depletion of YAP/TAZ in PDGFRß+ IntSCs leads to lacteal sprouting or regression with junctional disintegration and impaired dietary fat uptake. Indeed, mechanical or osmotic stress regulates IntSC secretion of VEGF-C mediated by YAP/TAZ. Single-cell RNA sequencing delineated novel subtypes of villi fibroblasts that upregulate Vegfc upon YAP/TAZ activation. These populations of fibroblasts were distributed in proximity to lacteal, suggesting that they constitute a peri-lacteal microenvironment. Our findings demonstrate the heterogeneity of IntSCs and reveal that distinct subsets of villi fibroblasts regulate lacteal integrity through YAP/TAZ-induced VEGF-C secretion, providing new insights into the dynamic regulatory mechanisms behind lymphangiogenesis and lymphatic remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Transcrição/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Fibroblastos/ultraestrutura , Citometria de Fluxo , Imunofluorescência , Hibridização in Situ Fluorescente , Mucosa Intestinal/ultraestrutura , Linfangiogênese/genética , Linfangiogênese/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fator C de Crescimento do Endotélio Vascular/genética
17.
PLoS Biol ; 18(8): e3000762, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760088

RESUMO

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older "mother" and a younger "daughter" centriole. Stem cells either inherit the mother or daughter-centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-structured illumination microscopy (3D-SIM) and live-cell imaging, we show in fly neural stem cells (neuroblasts) that the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) accumulate on the daughter centriole during mitosis, thereby generating molecularly distinct mother and daughter centrioles before interphase. Cnb's asymmetric localization, potentially involving a direct relocalization mechanism, is regulated by Polo-mediated phosphorylation, whereas Polo's daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments, we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.


Assuntos
Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interfase , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Optogenética/métodos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
18.
PLoS Biol ; 18(8): e3000817, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813728

RESUMO

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence/fluorescence in situ hybridization (immuno-FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.


Assuntos
Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Cromossomos/ultraestrutura , Meiose , Complexo Sinaptonêmico/ultraestrutura , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Análise Citogenética , Hibridização in Situ Fluorescente , Fase S/genética , Complexo Sinaptonêmico/metabolismo
19.
Pediatrics ; 146(Suppl 1): S60-S65, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32737234

RESUMO

Charlie Gard (August 4, 2016, to July 28, 2017) was an infant in the United Kingdom who was diagnosed with an encephalopathic form of mitochondrial DNA depletion syndrome caused by a mutation in the RRM2B gene. Charlie's parents raised £1.3 million (∼$1.6 million US) on a crowdfunding platform to travel to New York to pursue experimental nucleoside bypass treatment, which was being used to treat a myopathic form of mitochondrial DNA depletion syndrome caused by mutations in a different gene (TK2). The case made international headlines about what was in Charlie's best interest. In the medical ethics community, it raised the question of whether best interest serves as a guidance principle (a principle that provides substantive directions as to how decisions are to be made), an intervention principle (a principle specifying the conditions under which third parties are to intervene), both guidance and intervention, or neither. I show that the United Kingdom uses best interest as both guidance and intervention, and the United States uses best interest for neither. This explains why the decision to withdraw the ventilator without attempting nucleoside bypass treatment was the correct decision in the United Kingdom and why the opposite conclusion would have been reached in the United States.


Assuntos
Proteínas de Ciclo Celular/genética , Encefalomiopatias Mitocondriais/terapia , Defesa do Paciente/ética , Respiração Artificial/ética , Ribonucleotídeo Redutases/genética , Suspensão de Tratamento/ética , Tomada de Decisão Clínica/ética , Crowdsourcing/economia , História do Século XXI , Humanos , Lactente , Masculino , Futilidade Médica/ética , Encefalomiopatias Mitocondriais/genética , Cidade de Nova Iorque , Poder Familiar , Defesa do Paciente/legislação & jurisprudência , Transferência de Pacientes/ética , Transferência de Pacientes/legislação & jurisprudência , Guias de Prática Clínica como Assunto , Timidina Quinase/genética , Reino Unido , Estados Unidos , Suspensão de Tratamento/legislação & jurisprudência
20.
Gene ; 763: 145030, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32755658

RESUMO

OBJECTIVE: To investigate the impact and the mechanism of Gadd45α mediating p38MAPK pathway on the retinal ganglion cells (RGCs) injury in chronic ocular hypertension (COH) rats. METHODS: COH model in rats were established and intraocular pressure (IOP) was tested. Retrograde labeling was used for counting RGCs and TUNEL staining was performed for RGCs apoptosis. Western Blotting was conducted to examine the expression of Gadd45α and p38MAPK pathway. Besides, RGC-5 cells cultured in vitro were treated with H2O2. Cell viability was detected by CCK-8, ROS level tested by DCFH-DA assay, and cell apoptosis examined by flow cytometry. RESULTS: COH rats had increased expression of Gadd45α and p-p38/p38 protein 1-4 weeks after surgery. Rats in COH group enhanced obviously in IOP, RGC apoptosis rate and the protein expression of Gadd45α, p-p38/p38, Bax/Bcl-2 and cleaved caspase-3, but declined appreciably in RGC counting. However, the above indicators of COH rats were effectively improved by Gadd45α shRNA treatment. Additionally, RGC-5 cells in H2O2 group reduced in cell viability and went up in ROS level and apoptosis rate. The H2O2-induced RGC-5 cells treated with Gadd45α shRNA were improved apparently in those indicators, and cells treated with pcDNA Gadd45α showed an opposite trend. Moreover, p38 MAPK inhibitor SB203580 can effectively reverse the damage of pcDNA Gadd45α from H2O2-induced RGC-5 cells. CONCLUSION: Silencing Gadd45α can reduce the RGC damage in COH rats by inhibiting p38MAPK pathway and such a protective role may be associated with the suppression of RGC apoptosis and the mitigation of oxidative stress.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Sistema de Sinalização das MAP Quinases , Hipertensão Ocular/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Proteínas de Ciclo Celular/genética , Células Cultivadas , Humanos , Masculino , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley
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