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1.
Nat Commun ; 13(1): 2691, 2022 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-35577813

RESUMO

Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.

2.
Leukemia ; 36(2): 452-463, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34497325

RESUMO

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.


Assuntos
Diferenciação Celular , Janus Quinase 2/genética , Mutação , Complexo Repressor Polycomb 1/fisiologia , Mielofibrose Primária/patologia , Animais , Feminino , Lisina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mielofibrose Primária/etiologia , Mielofibrose Primária/metabolismo , Ubiquitinação
3.
Front Immunol ; 12: 777197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34868046

RESUMO

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple organ damage. Toll-like receptor 7 (TLR7), an innate immune RNA sensor expressed in monocytes/macrophages, dendritic cells (DCs), and B cells, promotes disease progression. However, little is known about the cellular mechanisms through which TLR7 drives lupus nephritis. Here, we show that the anti-mouse TLR7 mAb, but not anti-TLR9 mAb, protected lupus-prone NZBWF1 mice from nephritis. The anti-TLR7 mAb reduced IgG deposition in glomeruli by inhibiting the production of autoantibodies to the RNA-associated antigens. We found a disease-associated increase in Ly6Clow patrolling monocytes that expressed high levels of TLR7 and had upregulated expression of lupus-associated IL-10, CD115, CD31, and TNFSF15 in NZBWF1 mice. Anti-TLR7 mAb abolished this lupus-associated increase in patrolling monocytes in the circulation, spleen, and glomeruli. These results suggested that TLR7 drives autoantibody production and lupus-associated monocytosis in NZBWF1 mice and, that anti-TLR7 mAb is a promising therapeutic tool targeting B cells and monocytes/macrophages.


Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Nefrite Lúpica/etiologia , Nefrite Lúpica/metabolismo , Monócitos/imunologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Regulação da Expressão Gênica , Imunoglobulina G/imunologia , Imuno-Histoquímica , Imunofenotipagem , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/patologia , Camundongos , Monócitos/metabolismo
4.
PLoS One ; 16(9): e0257090, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34516556

RESUMO

Isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations and their key effector 2-hydroxyglutarate (2-HG) have been reported to promote oncogenesis in various human cancers. To elucidate molecular mechanism(s) associated with IDH1/2 mutations, we established mouse embryonic fibroblasts (MEF) cells and human colorectal cancer cells stably expressing cancer-associated IDH1R132C or IDH2R172S, and analyzed the change in metabolic characteristics of the these cells. We found that IDH1/2 mutants induced intracellular 2-HG accumulation and inhibited cell proliferation. Expression profile analysis by RNA-seq unveiled that glucose transporter 1 (Glut1) was induced by the IDH1/2 mutants or treatment with 2-HG in the MEF cells. Consistently, glucose uptake and lactate production were increased by the mutants, suggesting the deregulation of glucose metabolism. Furthermore, PI3K/Akt/mTOR pathway and Hif1α expression were involved in the up-regulation of Glut1. Together, these results suggest that Glut1 is a potential target regulated by cancer-associated IDH1/2 mutations.


Assuntos
Transtornos do Metabolismo de Glucose/genética , Transportador de Glucose Tipo 1/metabolismo , Isocitrato Desidrogenase/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mutação/genética , Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proliferação de Células , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Glutaratos/metabolismo , Glicólise , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Espaço Intracelular/metabolismo , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Transdução de Sinais
5.
Microbiol Spectr ; 9(1): e0070821, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34378948

RESUMO

Chronic inflammation is a hallmark of human immunodeficiency virus (HIV) infection and a risk factor for the development and progression of age-related comorbidities. Although HIV-associated gut dysbiosis has been suggested to be involved in sustained chronic inflammation, there remains a limited understanding of the association between gut dysbiosis and chronic inflammation during HIV infection. Here, we investigated compositional changes in the gut microbiome and its role in chronic inflammation in patients infected with HIV. We observed that the gut microbiomes of patients with low CD4 counts had reduced alpha diversity compared to those in uninfected controls. Following CD4 recovery, alpha diversity was restored, but intergroup dissimilarity of bacterial composition remained unchanged between patients and uninfected controls. Patients with HIV had higher abundance of the classes Negativicutes, Bacilli, and Coriobacteriia, as well as depletion of the class Clostridia. These relative abundances positively correlated with inflammatory cytokines and negatively correlated with anti-inflammatory cytokines. We found that gut dysbiosis accompanying HIV infection was characterized by a depletion of obligate anaerobic Clostridia and enrichment of facultative anaerobic bacteria, reflecting increased intestinal oxygen levels and intestinal permeability. Furthermore, it is likely that HIV-associated dysbiosis shifts the immunological balance toward inflammatory Th1 responses and encourages proinflammatory cytokine production. Our results suggest that gut dysbiosis contributes to sustaining chronic inflammation in patients with HIV infection despite effective antiretroviral therapy and that correcting gut dysbiosis will be effective in improving long-term outcomes in patients. IMPORTANCE Chronic inflammation is a hallmark of HIV infection and is associated with the development and progression of age-related comorbidities. Although the gastrointestinal tract is a major site of HIV replication and CD4+ T-cell depletion, the role of HIV-associated imbalance of gut microbiome in chronic inflammation is unclear. Here, we aimed to understand the causal relationship between abnormalities in the gut microbiome and chronic inflammation in patients with HIV. Our results suggest HIV-associated gut dysbiosis presents a more aerobic environment than that of healthy individuals, despite prolonged viral suppression. This dysbiosis likely results from a sustained increase in intestinal permeability, which supports sustained bacterial translocation in HIV patients, despite effective therapy. Additionally, we observed that several bacterial taxa enriched in HIV patients were associated with increased expression of inflammatory cytokines. Collectively, these results suggest that gut dysbiosis plays an important role in chronic inflammation in HIV patients.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Microbioma Gastrointestinal , Infecções por HIV/tratamento farmacológico , Infecções por HIV/microbiologia , Adulto , Fármacos Anti-HIV/efeitos adversos , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Linfócitos T CD4-Positivos/imunologia , Doença Crônica/terapia , Disbiose/etiologia , Disbiose/microbiologia , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade
6.
J Hum Genet ; 66(11): 1053-1060, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33958709

RESUMO

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos/normas , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Programas de Rastreamento , Proteína 1 Homóloga a MutL/ultraestrutura , Proteína 2 Homóloga a MutS/ultraestrutura , Sequenciamento por Nanoporos , Polimorfismo de Nucleotídeo Único/genética , Conformação Proteica
7.
Transl Lung Cancer Res ; 10(3): 1292-1304, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33889510

RESUMO

BACKGROUND: Fetal adenocarcinoma of the lung is a rare variant of lung adenocarcinoma and is subcategorized into low-grade and high-grade (H-FLAC) fetal adenocarcinoma. We previously reported poor prognosis in pulmonary adenocarcinomas with an H-FLAC component; however, the genetic abnormalities involved in H-FLAC remain unclear. Therefore, this study aimed to elucidate molecular abnormalities as potential therapeutic targets for H-FLACs. METHODS: We performed immunohistochemical analysis and comprehensive genetic analyses using whole-exome sequencing in 16 lung cancer samples with an H-FLAC component. DNA was extracted from formalin-fixed paraffin-embedded tissues after macrodissection of the H-FLAC component. RESULTS: Cancer-related mutations were identified in TP53 (7/16 cases), KMT2C (6/16 cases), KRAS (4/16 cases), NF1 (3/16 cases), STK11 (3/16 cases), CTNNB1 (2/16 cases), and EGFR (1/16 cases). A high tumor mutation burden of ≥10 mutations per megabase was observed in 3/16 cases. A high microsatellite instability was not detected in any case. Based on the cosine similarity with the Catalogue of Somatic Mutations in Cancer mutational signatures, H-FLACs were hierarchically clustered into three types: common adenocarcinoma-like (five cases), surfactant-deficient (ten cases), and signatures 2 and 13-related (one case). All common adenocarcinoma-like cases presented thyroid transcription factor-1 (TTF-1) expression, whereas surfactant-deficient cases often presented loss of TTF-1 and surfactant protein expression and included cases with mutations in the surfactant system genes NKX2-1 and SFTPC. H-FLACs displayed low programmed death ligand-1 (PD-L1) expression (1-49% of tumor cells) in 5/16 cases, and no case displayed high PD-L1 expression (≥50% of tumor cells). CONCLUSIONS: This study indicates that lung cancers with an H-FLAC component rarely harbor currently targetable driver gene mutations for lung cancer but display a high frequency of KMT2C mutations. The microsatellite instability, tumor mutation burden, and PD-L1 expression status suggest a poor response to immune checkpoint therapy.

8.
Autophagy ; 17(11): 3776-3793, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33706682

RESUMO

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, fbxo22-/- mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy.Abbreviations: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas F-Box/metabolismo , Hormese , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Células Cultivadas , Proteínas F-Box/fisiologia , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Ubiquitinação
9.
Nat Commun ; 12(1): 1826, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758188

RESUMO

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.


Assuntos
Envelhecimento/genética , Hematopoiese Clonal/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/genética , Serina-Treonina Quinases TOR/metabolismo , Idoso , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Apoptose/genética , Ciclo Celular/genética , Proliferação de Células/genética , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Técnicas de Introdução de Genes , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
10.
Gastroenterology ; 160(6): 2089-2102.e12, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33577875

RESUMO

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.


Assuntos
Enterocolite Pseudomembranosa/terapia , Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Viroma , Adulto , Idoso , Bacteriófagos , Clostridioides difficile , Enterocolite Pseudomembranosa/microbiologia , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/virologia , Humanos , Masculino , Metagenômica , Microviridae , Pessoa de Meia-Idade , Proteobactérias , Viroma/genética
11.
Stem Cells ; 39(4): 429-442, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400835

RESUMO

Tissues and cells derived from pluripotent stem cells (PSC) are likely to become widely used in disease modeling, drug screening, and regenerative medicine. For these applications, the in vitro PSC differentiation process must be elaborately investigated and controlled to reliably obtain the desired end products. However, because traditional experimental methods, such as one factor at a time or brute-force approaches, are impractical for detailed screening of complex PSC cultivation conditions, more strategic and effective screening based on statistical design of experiments (DOE) ought to be indispensable. Among various DOE approaches, we regard robust parameter design (RPD) as particularly suited for differentiation protocol optimization due to its suitability for multifactorial screening. We confirmed the adaptability of RPD for investigating human induced PSC lineage specification toward anterior-posterior gut tube endodermal cells and clarified both the contribution of each cell signaling pathway and the effect of cell signaling condition alteration on marker RNA expression levels, while increasing the efficiency of the screening in 243-fold (18 vs 4374) compared with that of a brute-force approach. Specific induction of anterior foregut, hepatic, pancreatic, or mid-hindgut cells was achieved using seven iPSC strains with the optimal culture protocols established on the basis of RPD analysis. RPD has the potential to enable efficient construction and optimization of PSC differentiation protocols, and its use is recommended from fundamental research to mass production of PSC-derived products.


Assuntos
Técnicas de Cultura de Células , Endoderma/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Intestinos/citologia , Fígado/citologia , Pâncreas/citologia , Projetos de Pesquisa , Biomarcadores/metabolismo , Ácido Butírico/farmacologia , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Análise Fatorial , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Tretinoína/farmacologia , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
12.
Science ; 371(6526): 265-270, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446552

RESUMO

Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, we identified glutaminase 1 (GLS1) as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.


Assuntos
Envelhecimento/metabolismo , Senescência Celular/fisiologia , Glutaminase/metabolismo , Tecido Adiposo/enzimologia , Envelhecimento/genética , Amônia/metabolismo , Animais , Sobrevivência Celular , Senescência Celular/genética , Genes Essenciais , Glutaminase/genética , Humanos , Concentração de Íons de Hidrogênio , Pulmão/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pele/enzimologia
13.
Clin Cancer Res ; 27(1): 267-275, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32958699

RESUMO

PURPOSE: Irinotecan/5-fluorouracil (5-FU; FOLFIRI) or oxaliplatin/5-FU (FOLFOX), combined with bevacizumab or cetuximab, are approved, first-line treatments for metastatic colorectal cancer (mCRC). We aimed at identifying germline variants associated with survival in patients with mCRC treated with these regimens in Cancer and Leukemia Group B/SWOG 80405. EXPERIMENTAL DESIGN: Patients with mCRC receiving either FOLFOX or FOLFIRI were randomized to either cetuximab or bevacizumab. DNA from peripheral blood was genotyped for approximately 700,000 SNPs. The association between SNPs and overall survival (OS) was tested in 613 patients of genetically estimated European ancestry using Cox proportional hazards models. RESULTS: The four most significant SNPs associated with OS were three haplotypic SNPs between microsomal glutathione S-transferase 1 (MGST1) and LIM domain only 3 (LMO3, representative HR, 1.56; P = 1.30 × 10-6), and rs11644916 in AXIN1 (HR, 1.39, P = 4.26 × 10-6). AXIN1 is a well-established tumor suppressor gene in colorectal cancer, and rs11644916 (G>A) conferred shorter OS. Median OS for patients with the AA, AG, or GG genotypes was 18.4, 25.6, or 36.4 months, respectively. In 90 patients with stage IV colorectal cancer from The Cancer Genome Atlas (TCGA), rs11649255 in AXIN1 [in almost complete linkage disequilibrium (LD) with rs11644916], was associated with shorter OS (HR, 2.24, P = 0.0096). Using rs11648673 in AXIN1 (in very high LD with rs11644916 and with functional evidence), luciferase activity in three colorectal cancer cell lines was reduced. CONCLUSIONS: This is the first large genome-wide association study ever conducted in patients with mCRC treated with first-line standard treatment in a randomized phase III trial. A common SNP in AXIN1 conferred worse OS and the effect was replicated in TCGA. Further studies in colorectal cancer experimental models are required.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Produtos Biológicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias Colorretais/mortalidade , Resistencia a Medicamentos Antineoplásicos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Produtos Biológicos/farmacologia , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Estudo de Associação Genômica Ampla , Humanos , Leucovorina/farmacologia , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Polimorfismo de Nucleotídeo Único , Medição de Risco/métodos , Adulto Jovem
14.
Bioinformatics ; 37(9): 1211-1217, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-33165508

RESUMO

MOTIVATION: In recent years, nanopore sequencing technology has enabled inexpensive long-read sequencing, which promises reads longer than a few thousand bases. Such long-read sequences contribute to the precise detection of structural variations and accurate haplotype phasing. However, deciphering precise DNA sequences from noisy and complicated nanopore raw signals remains a crucial demand for downstream analyses based on higher-quality nanopore sequencing, although various basecallers have been introduced to date. RESULTS: To address this need, we developed a novel basecaller, Halcyon, that incorporates neural-network techniques frequently used in the field of machine translation. Our model employs monotonic-attention mechanisms to learn semantic correspondences between nucleotides and signal levels without any pre-segmentation against input signals. We evaluated performance with a human whole-genome sequencing dataset and demonstrated that Halcyon outperformed existing third-party basecallers and achieved competitive performance against the latest Oxford Nanopore Technologies' basecallers. AVAILABILITYAND IMPLEMENTATION: The source code (halcyon) can be found at https://github.com/relastle/halcyon.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA , Software
15.
Cancer Sci ; 111(12): 4336-4347, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33037737

RESUMO

Monomer tubulin polymerize into microtubules, which are highly dynamic and play a critical role in mitosis. Therefore, microtubule dynamics are an important target for anticancer drugs. The inhibition of tubulin polymerization or depolymerization was previously targeted and exhibited efficacy against solid tumors. The novel small molecule PTC596 directly binds tubulin, inhibits microtubule polymerization, downregulates MCL-1, and induces p53-independent apoptosis in acute myeloid leukemia cells. We herein investigated the efficacy of PTC-028, a structural analog of PTC596, for myelodysplastic syndrome (MDS). PTC-028 suppressed growth and induced apoptosis in MDS cell lines. The efficacy of PTC028 in primary MDS samples was confirmed using cell proliferation assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit growth and induce apoptosis in MDS cells. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We also assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using the MDS cell line, MDS-L, and the AkaBLI bioluminescence imaging system, which is composed of AkaLumine-HCl and Akaluc. PTC-028 prolonged the survival of mice in xenograft models. The present results suggest a chemotherapeutic strategy for MDS through the disruption of microtubule dynamics in combination with DNA hypomethylating agents.


Assuntos
Benzimidazóis/farmacologia , Síndromes Mielodisplásicas/tratamento farmacológico , Pirazinas/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Decitabina/farmacologia , Fase G2/efeitos dos fármacos , Células HL-60 , Xenoenxertos , Humanos , Camundongos , Síndromes Mielodisplásicas/genética , Paclitaxel/farmacologia , Pirazinas/uso terapêutico , Análise de Sequência de RNA/métodos , Tubulina (Proteína)/efeitos dos fármacos , Moduladores de Tubulina/uso terapêutico , Vincristina/farmacologia
16.
Cell Metab ; 32(5): 814-828.e6, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-32949498

RESUMO

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-CreERT2-tdTomato mouse model to analyze the in vivo characteristics of p16high cells at a single-cell level. We found tdTomato-positive p16high cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16high cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16high cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Animais , Linhagem Celular , Senescência Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Análise de Célula Única
17.
Biomolecules ; 10(8)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32824461

RESUMO

Although gastric cancer is one of the most common causes of cancer death in the world, mechanisms underlying this type of tumor have not been fully understood. In this study, we found that IQGAP3, a member of the IQGAP gene family, was significantly up-regulated in human gastric cancer starting from the early stages of tumor progression. Overexpression of IQGAP3 in 293T and NIH3T3 cells, which have no endogenous IQGAP3 expression, resulted in morphological change with multiple dendritic-like protrusions and enhanced migration. Overexpression of IQGAP3 also led to reduced cell-cell adhesion in 293T cells, likely as a result of its interactions with e-cadherin or ß-catenin proteins. Additionally, IQGAP3 accumulated along the leading edge of migrating cells and at the cleavage furrow of dividing cells. In contrast, suppression of IQGAP3 by short-interfering RNA (siRNA) markedly reduced invasion and anchorage-independent growth of MKN1 and TMK-1 gastric cancer cells. We further confirmed that IQGAP3 interacted with Rho family GTPases, and had an important role in cytokinesis. Taken together, we demonstrated that IQGAP3 plays critical roles in migration and invasion of human gastric cancer cells, and regulates cytoskeletal remodeling, cell migration and adhesion. These findings may open a new avenue for the diagnosis and treatment of gastric cancer.


Assuntos
Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima , Animais , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Gradação de Tumores , Invasividade Neoplásica , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
18.
Cell Host Microbe ; 28(3): 380-389.e9, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32652061

RESUMO

The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.


Assuntos
Bacteriófagos/genética , Clostridioides difficile/virologia , Endopeptidases/genética , Microbioma Gastrointestinal/genética , Terapia por Fagos/métodos , Prófagos/genética , Animais , Antibacterianos/farmacologia , Bacteriófagos/isolamento & purificação , Infecções por Clostridium/terapia , Modelos Animais de Doenças , Endopeptidases/farmacologia , Fezes/microbiologia , Feminino , Genoma Bacteriano , Genoma Viral , Humanos , Metagenoma , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de DNA , Organismos Livres de Patógenos Específicos , Proteínas Virais/genética , Proteínas Virais/farmacologia
19.
Sci Rep ; 10(1): 9275, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518284

RESUMO

Cancer cells adapt to various stress conditions by optimizing gene expression profiles via transcriptional and translational regulation. However, whether and how EXOSC9, a component of the RNA exosome complex, regulates adaptation to stress conditions and tumorigenicity in cancer cells remain unclear. Here, we examined the effects of EXOSC9 depletion on cancer cell growth under various stress conditions. EXOSC9 depletion attenuated growth and survival under various stress conditions in cancer cells. Interestingly, this also decreased the number of P-bodies, which are messenger ribonucleoprotein particles (mRNPs) required for stress adaptation. Meanwhile, EXOSC2/EXOSC4 depletion also attenuated P-body formation and stress resistance with decreased EXOSC9 protein. EXOSC9-mediated stress resistance and P-body formation were found to depend on the intact RNA-binding motif of this protein. Further, RNA-seq analyses identified 343 EXOSC9-target genes, among which, APOBEC3G contributed to defects in stress resistance and P-body formation in MDA-MB-231 cells. Finally, EXOSC9 also promoted xenografted tumor growth of MDA-MB-231 cells in an intact RNA-binding motif-dependent manner. Database analyses further showed that higher EXOSC9 activity, estimated based on the expression of 343 target genes, was correlated with poorer prognosis in some cancer patients. Thus, drugs targeting activity of the RNA exosome complex or EXOSC9 might be useful for cancer treatment.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico/fisiologia , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Estruturas Citoplasmáticas/metabolismo , Dano ao DNA , Estresse do Retículo Endoplasmático , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/genética , Exossomos/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Proteínas de Ligação a RNA/genética , Ensaios Antitumorais Modelo de Xenoenxerto
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