Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 12.643
Filtrar
Mais filtros

Intervalo de ano de publicação
1.
Cell ; 185(4): 690-711.e45, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35108499

RESUMO

Single-cell (sc)RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these data, however, requires kinetic models capable of unveiling governing regulatory functions. Here, we introduce an analytical framework dynamo (https://github.com/aristoteleo/dynamo-release), which infers absolute RNA velocity, reconstructs continuous vector fields that predict cell fates, employs differential geometry to extract underlying regulations, and ultimately predicts optimal reprogramming paths and perturbation outcomes. We highlight dynamo's power to overcome fundamental limitations of conventional splicing-based RNA velocity analyses to enable accurate velocity estimations on a metabolically labeled human hematopoiesis scRNA-seq dataset. Furthermore, differential geometry analyses reveal mechanisms driving early megakaryocyte appearance and elucidate asymmetrical regulation within the PU.1-GATA1 circuit. Leveraging the least-action-path method, dynamo accurately predicts drivers of numerous hematopoietic transitions. Finally, in silico perturbations predict cell-fate diversions induced by gene perturbations. Dynamo, thus, represents an important step in advancing quantitative and predictive theories of cell-state transitions.


Assuntos
Análise de Célula Única , Transcriptoma/genética , Algoritmos , Feminino , Regulação da Expressão Gênica , Células HL-60 , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Cinética , Modelos Biológicos , RNA Mensageiro/metabolismo , Coloração e Rotulagem
2.
Cell ; 183(6): 1508-1519.e12, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33207184

RESUMO

The urgent need for an effective SARS-CoV-2 vaccine has forced development to progress in the absence of well-defined correlates of immunity. While neutralization has been linked to protection against other pathogens, whether neutralization alone will be sufficient to drive protection against SARS-CoV-2 in the broader population remains unclear. Therefore, to fully define protective humoral immunity, we dissected the early evolution of the humoral response in 193 hospitalized individuals ranging from moderate to severe. Although robust IgM and IgA responses evolved in both survivors and non-survivors with severe disease, non-survivors showed attenuated IgG responses, accompanied by compromised Fcɣ receptor binding and Fc effector activity, pointing to deficient humoral development rather than disease-enhancing humoral immunity. In contrast, individuals with moderate disease exhibited delayed responses that ultimately matured. These data highlight distinct humoral trajectories associated with resolution of SARS-CoV-2 infection and the need for early functional humoral immunity.


Assuntos
COVID-19 , Imunidade Humoral , Imunoglobulina A/imunologia , Imunoglobulina M/imunologia , Receptores de IgG/imunologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/mortalidade , Feminino , Células HL-60 , Humanos , Masculino
3.
Cell ; 173(3): 649-664.e20, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677511

RESUMO

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.


Assuntos
Sistemas CRISPR-Cas , Resistencia a Medicamentos Antineoplásicos , Genoma Humano , RNA Longo não Codificante/genética , Animais , Citarabina/farmacologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Farmacogenética , Proteínas/genética , RNA/análise , RNA Mensageiro/genética , Transdução de Sinais
4.
Genes Dev ; 34(21-22): 1452-1473, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33060135

RESUMO

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Modelos Biológicos , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/genética , Processamento Alternativo/genética , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ativação Enzimática/genética , Células HL-60 , Humanos , Quinase Ativadora de Quinase Dependente de Ciclina
5.
Mol Cell ; 76(4): 660-675.e9, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542297

RESUMO

Histone posttranslational modifications (PTMs) regulate chromatin structure and dynamics during various DNA-associated processes. Here, we report that lysine glutarylation (Kglu) occurs at 27 lysine residues on human core histones. Using semi-synthetic glutarylated histones, we show that an evolutionarily conserved Kglu at histone H4K91 destabilizes nucleosome in vitro. In Saccharomyces cerevisiae, the replacement of H4K91 by glutamate that mimics Kglu influences chromatin structure and thereby results in a global upregulation of transcription and defects in cell-cycle progression, DNA damage repair, and telomere silencing. In mammalian cells, H4K91glu is mainly enriched at promoter regions of highly expressed genes. A downregulation of H4K91glu is tightly associated with chromatin condensation during mitosis and in response to DNA damage. The cellular dynamics of H4K91glu is controlled by Sirt7 as a deglutarylase and KAT2A as a glutaryltransferase. This study designates a new histone mark (Kglu) as a new regulatory mechanism for chromatin dynamics.


Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , Glutaratos/metabolismo , Histonas/metabolismo , Mitose , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Lisina , Camundongos , Nucleossomos/genética , Células RAW 264.7 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Fatores de Tempo
6.
Nucleic Acids Res ; 52(15): 8800-8814, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-38943321

RESUMO

We characterized the regulatory mechanisms and role in human myeloid cell survival and differentiation of PRPF40A, a splicing factor lacking a canonical RNA Binding Domain. Upon PRPF40A knockdown, HL-60 cells displayed increased cell death, decreased proliferation and slight differentiation phenotype with upregulation of immune activation genes. Suggestive of both redundant and specific functions, cell death but not proliferation was rescued by overexpression of its paralog PRPF40B. Transcriptomic analysis revealed the predominant role of PRPF40A as an activator of cassette exon inclusion of functionally relevant splicing events. Mechanistically, the exons exclusively upregulated by PRPF40A are flanked by short and GC-rich introns which tend to localize to nuclear speckles in the nucleus center. These PRPF40A regulatory features are shared with other splicing regulators such as SRRM2, SON, PCBP1/2, and to a lesser extent TRA2B and SRSF2, as a part of a functional network that regulates splicing partly via co-localization in the nucleus.


Assuntos
Diferenciação Celular , Éxons , Células Mieloides , Humanos , Processamento Alternativo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proliferação de Células/genética , Éxons/genética , Células HL-60 , Íntrons/genética , Células Mieloides/metabolismo , Células Mieloides/citologia , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
7.
EMBO J ; 40(4): e105094, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33586225

RESUMO

The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here, we show that lagging-edge component PI(3,4)P2 also localizes to retracting leading-edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back-to-front gradient. These results uncover a surprising "reverse-fountain flow" of PI(3,4)P2 that regulates polarity.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Dictyostelium/fisiologia , Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Dictyostelium/citologia , Células HL-60 , Humanos
8.
PLoS Biol ; 20(2): e3001517, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35202387

RESUMO

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15's anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15's specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.


Assuntos
Camptotecina/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/genética , Obesidade/prevenção & controle , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Camptotecina/farmacocinética , Linhagem Celular , Linhagem Celular Tumoral , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Células HEK293 , Células HL-60 , Humanos , Células MCF-7 , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/etiologia , Obesidade/genética , Células PC-3
9.
Biochem J ; 481(10): 653-666, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38666590

RESUMO

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited ß-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT , Diferenciação Celular , Leucemia Mieloide Aguda , Ubiquitina-Proteína Ligases , Humanos , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/genética , Células HEK293 , Células HL-60 , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteólise , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Proc Natl Acad Sci U S A ; 119(31): e2201249119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35878025

RESUMO

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-ß/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca2+ mobilization from intracellular stores and Ca2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and ß-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.


Assuntos
Neutrófilos , Fagócitos , Receptores de Formil Peptídeo , Superóxidos , Cálcio/metabolismo , Fatores Quimiotáticos/metabolismo , Quimiotaxia , Células HL-60 , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Fagócitos/fisiologia , Receptores de Formil Peptídeo/metabolismo , Superóxidos/metabolismo
11.
J Biol Chem ; 299(3): 102971, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36736423

RESUMO

Acute myeloid leukemia (AML) is challenging to treat due to its heterogeneity, prompting a deep understanding of its pathogenesis mechanisms, diagnosis, and treatment. Here, we found reduced expression and acetylation levels of WISP2 in bone marrow mononuclear cells from AML patients and that AML patients with lower WISP2 expression tended to have reduced survival. At the functional level, overexpression of WISP2 in leukemia cells (HL-60 and Kasumi-1) suppressed cell proliferation, induced cell apoptosis, and exerted antileukemic effects in an in vivo model of AML. Our mechanistic investigation demonstrated that WISP2 deacetylation was regulated by the deacetylase histone deacetylase (HDAC)3. In addition, we determined that crosstalk between acetylation and ubiquitination was involved in the modulation of WISP2 expression in AML. Deacetylation of WISP2 decreased the stability of the WISP2 protein by boosting its ubiquitination mediated by NEDD4 and proteasomal degradation. Moreover, pan-HDAC inhibitors (valproic acid and trichostatin A) and an HDAC3-specific inhibitor (RGFP966) induced WISP2 acetylation at lysine K6 and prevented WISP2 degradation. This regulation led to inhibition of proliferation and induction of apoptosis in AML cells. In summary, our study revealed that WISP2 contributes to tumor suppression in AML, which provided an experimental framework for WISP2 as a candidate for gene therapy of AML.


Assuntos
Proteínas de Sinalização Intercelular CCN , Leucemia Mieloide Aguda , Proteínas Repressoras , Humanos , Acetilação , Apoptose , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/genética , Ácido Valproico/farmacologia , Proteínas de Sinalização Intercelular CCN/genética , Proteínas Repressoras/genética , Células HL-60
12.
J Cell Physiol ; 239(6): e31288, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38685860

RESUMO

Galectin-12 is a tissue-specific galectin that has been largely defined by its role in the regulation of adipocyte differentiation and lipogenesis. This study aimed to evaluate the role of galectin-12 in the differentiation and polarization of neutrophils within a model of acute myeloid leukemia HL-60 cells. All-trans retinoic acid and dimethyl sulfoxide were used to induce differentiation of HL-60 cells which led to the generation of two phenotypes of neutrophil-like cells with opposite changes in galectin-12 gene (LGALS12) expression and different functional responses to N-formyl- l-methionyl- l-leucyl- l-phenylalanine. These phenotypes showed significant differences of differentially expressed genes on a global scale based on bioinformatics analysis of available Gene Expression Omnibus (GEO) data sets. We also demonstrated that HL-60 cells could secrete and accumulate galectin-12 in cell culture medium under normal growth conditions. This secretion was found to be entirely inhibited upon neutrophilic differentiation and was accompanied by an increase in intracellular lipid droplet content and significant enrichment of 22 lipid gene ontology terms related to lipid metabolism in differentiated cells. These findings suggest that galectin-12 could serve as a marker of neutrophilic plasticity or polarization into different phenotypes and that galectin-12 secretion may be influenced by lipid droplet biogenesis.


Assuntos
Galectinas , Leucemia Promielocítica Aguda , Neutrófilos , Humanos , Diferenciação Celular , Galectinas/metabolismo , Galectinas/genética , Células HL-60 , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Metabolismo dos Lipídeos/genética , Neutrófilos/metabolismo , Fenótipo , Tretinoína/farmacologia
13.
Biochem Cell Biol ; 102(3): 275-284, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38484367

RESUMO

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.


Assuntos
Furina , Peroxidase , Humanos , Células HL-60 , Furina/metabolismo , Furina/genética , Peroxidase/metabolismo , Granulócitos/metabolismo , Granulócitos/citologia , Diferenciação Celular , Subtilisinas/metabolismo , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/genética , Clorometilcetonas de Aminoácidos/farmacologia
14.
Biochem Biophys Res Commun ; 710: 149541, 2024 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-38608490

RESUMO

For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ, CLEC6A, CLEC7A, CASP1, IL-1ß, and EGR3, as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation.


Assuntos
Leucemia Promielocítica Aguda , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Glicosilação , Tretinoína/farmacologia , Tretinoína/metabolismo , Diferenciação Celular , Células HL-60 , Linhagem Celular Tumoral
15.
Blood Cells Mol Dis ; 108: 102871, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39013336

RESUMO

A graft source for allogeneic hematopoietic stem cell transplantation is umbilical cord blood, which contains umbilical cord blood mononuclear cells (MNCs and mesenchymal stem cells, both an excellent source of extracellular microparticles (MPs). MPs act as cell communication mediators, which are implicated in reactive oxygen species formation or detoxification depending on their origin. Oxidative stress plays a crucial role in both the development of cancer and its treatment by triggering apoptotic mechanisms, in which CD34+ cells are implicated. The aim of this work is to investigate the oxidative stress status and the apoptosis of HL-60 and mononuclear cells isolated from umbilical cord blood (UCB) following a 24- and 48-hour exposure to CD34 + microparticles (CD34 + MPs). The activity of superoxide dismutase, glutathione reductase, and glutathione S-transferase, as well as lipid peroxidation in the cells, were employed as oxidative stress markers. A 24- and 48-hour exposure of leukemic and mononuclear cells to CD34 + -MPs resulted in a statistically significant increase in the antioxidant activity and lipid peroxidation in both cells types. Moreover, CD34 + MPs affect the expression of BCL2 and FAS and related proteins and downregulate the hematopoietic differentiation program in both HL-60 and mononuclear cells. Our results indicate that MPs through activation of antioxidant enzymes in both homozygous and nonhomozygous cells might serve as a means for graft optimization and enhancement.


Assuntos
Antígenos CD34 , Apoptose , Micropartículas Derivadas de Células , Sangue Fetal , Células-Tronco Hematopoéticas , Estresse Oxidativo , Humanos , Sangue Fetal/citologia , Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Micropartículas Derivadas de Células/metabolismo , Células HL-60 , Peroxidação de Lipídeos , Leucócitos Mononucleares/metabolismo , Superóxido Dismutase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
16.
Nat Immunol ; 13(5): 457-64, 2012 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-22447027

RESUMO

To kill invading bacteria, neutrophils must interpret spatial cues, migrate and reach target sites. Although the initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we found that two mitogen-activated protein kinases (MAPKs) had opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase Erk potentiated activity of the G protein-coupled receptor kinase GRK2 and inhibited neutrophil migration, whereas the MAPK p38 acted as a noncanonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 controlled neutrophil 'stop' and 'go' activity, which ensured that neutrophils reached their final destination as the first line of host defense.


Assuntos
Quimiotaxia de Leucócito , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Neutrófilos/imunologia , Receptores de Formil Peptídeo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Células HEK293 , Células HL-60 , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Knockout , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
17.
J Biol Inorg Chem ; 29(3): 331-338, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38717473

RESUMO

Two new lanthanide-complexes based on the 5-nitropicolinate ligand (5-npic) were obtained and fully characterized. Single-crystal X-ray diffraction revealed that these compounds are isostructural to a Dy-complex, previously published by us, based on dinuclear monomers link together with an extended hydrogen bond network, providing a final chemical formula of [Ln2(5-npic)6(H2O)4]·(H2O)2, where Ln = Dy (1), Gd (2), and Tb (3). Preliminary photoluminescent studies exhibited a ligand-centered emission for all complexes. The potential antitumoral activity of these materials was assayed in a prostatic cancer cell line (PC-3; the 2nd most common male cancerous disease), showing a significant anticancer activity (50-60% at 500 µg·mL-1). In turn, a high biocompatibility by both, the complexes and their precursors in human immunological HL-60 cells, was evidenced. In view of the strongest toxic effect in the tumoral cell line provided by the free 5-npic ligand (~ 40-50%), the overall anticancer complex performance seems to be triggered by the presence of this molecule.


Assuntos
Antineoplásicos , Elementos da Série dos Lantanídeos , Ácidos Picolínicos , Humanos , Elementos da Série dos Lantanídeos/química , Elementos da Série dos Lantanídeos/farmacologia , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Masculino , Ensaios de Seleção de Medicamentos Antitumorais , Modelos Moleculares , Células HL-60 , Cristalografia por Raios X , Estrutura Molecular , Linhagem Celular Tumoral , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos
18.
BMC Cancer ; 24(1): 125, 2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38267906

RESUMO

BACKGROUND: T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1. METHODS: Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively. RESULTS: The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001). CONCLUSION: Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.


Assuntos
Glutamina , Leucemia Mieloide Aguda , Humanos , Ácido Glutâmico , Receptor Celular 2 do Vírus da Hepatite A , Células HL-60
19.
Chem Res Toxicol ; 37(10): 1738-1746, 2024 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-39361003

RESUMO

6-PPD (N-[1,3-dimethylbutyl]-N'-phenyl-p-phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/H2O2. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.


Assuntos
Glutationa , Peroxidase , Humanos , Peroxidase/metabolismo , Células HL-60 , Glutationa/metabolismo , Fenilenodiaminas/metabolismo , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Oxirredução
20.
FASEB J ; 37(1): e22676, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36468834

RESUMO

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.


Assuntos
Ácidos Graxos Voláteis , Leucócitos , Receptores de Superfície Celular , Humanos , Ácidos Graxos Voláteis/metabolismo , Leucócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Diferenciação Celular , Células HL-60
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA