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1.
EMBO J ; 42(20): e110844, 2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37661798

RESUMO

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.


Assuntos
Neoplasias , Rad51 Recombinase , Animais , Camundongos , Envelhecimento/genética , Carcinogênese/genética , Transformação Celular Neoplásica , Dano ao DNA , Reparo do DNA , Recombinação Homóloga , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
2.
Nature ; 577(7792): E10, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911658

RESUMO

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

3.
Nature ; 569(7758): 672-678, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31092925

RESUMO

Autonomic nerve fibres in the tumour microenvironment regulate cancer initiation and dissemination, but how nerves emerge in tumours is currently unknown. Here we show that neural progenitors from the central nervous system that express doublecortin (DCX+) infiltrate prostate tumours and metastases, in which they initiate neurogenesis. In mouse models of prostate cancer, oscillations of DCX+ neural progenitors in the subventricular zone-a neurogenic area of the central nervous system-are associated with disruption of the blood-brain barrier, and with the egress of DCX+ cells into the circulation. These cells then infiltrate and reside in the tumour, and can generate new adrenergic neurons. Selective genetic depletion of DCX+ cells inhibits the early phases of tumour development in our mouse models of prostate cancer, whereas transplantation of DCX+ neural progenitors promotes tumour growth and metastasis. In humans, the density of DCX+ neural progenitors is strongly associated with the aggressiveness and recurrence of prostate adenocarcinoma. These results reveal a unique crosstalk between the central nervous system and prostate tumours, and indicate neural targets for the treatment of cancer.


Assuntos
Sistema Nervoso Central/patologia , Células-Tronco Neurais/patologia , Neurogênese , Neoplasias da Próstata/patologia , Adenocarcinoma/patologia , Neurônios Adrenérgicos/patologia , Animais , Carcinogênese , Diferenciação Celular , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Genes myc , Humanos , Ventrículos Laterais/patologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Neuropeptídeos/metabolismo , Bulbo Olfatório/patologia , Prognóstico
4.
PLoS Genet ; 16(11): e1009090, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33147208

RESUMO

Interferon ß (IFN-ß) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-ß binding motif, prevents binding of C/EBP-ß, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-ß expression in myeloid cells.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/imunologia , Interferon beta/genética , Células Mieloides/metabolismo , Alelos , Animais , Buffy Coat/citologia , Células Cultivadas , Humanos , Interferon beta/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Haematologica ; 105(5): 1216-1222, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31371412

RESUMO

Highly conserved among species and expressed in various types of cells, numerous roles have been attributed to the cellular prion protein (PrPC). In hematopoiesis, PrPC regulates hematopoietic stem cell self-renewal but the mechanisms involved in this regulation are unknown. Here we show that PrPC regulates hematopoietic stem cell number during aging and their determination towards myeloid progenitors. Furthermore, PrPC protects myeloid progenitors against the cytotoxic effects of total body irradiation. This radioprotective effect was associated with increased cellular prion mRNA level and with stimulation of the DNA repair activity of the Apurinic/pyrimidinic endonuclease 1, a key enzyme of the base excision repair pathway. Altogether, these results show a previously unappreciated role of PrPC in adult hematopoiesis, and indicate that PrPC-mediated stimulation of BER activity might protect hematopoietic progenitors from the cytotoxic effects of total body irradiation.


Assuntos
Príons , Deficiência de Proteína , Células-Tronco Hematopoéticas , Humanos , Células Progenitoras Mieloides , Proteínas Priônicas/genética , Príons/genética
6.
Cell Mol Life Sci ; 72(18): 3559-73, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25894690

RESUMO

A general radioprotective effect by fibroblast growth factor (FGF) has been extensively described since the early 1990s; however, the molecular mechanisms involved remain largely unknown. Radiation-induced DNA double-strand breaks (DSBs) lead to a complex set of responses in eukaryotic cells. One of the earliest consequences is phosphorylation of histone H2AX to form nuclear foci of the phosphorylated form of H2AX (γH2AX) in the chromatin adjacent to sites of DSBs and to initiate the recruitment of DNA-repair molecules. Upon a DSB event, a rapid signaling network is activated to coordinate DNA repair with the induction of cell-cycle checkpoints. To date, three kinases (ATM, ATR, and DNA-PK) have been shown to phosphorylate histone H2AX in response to irradiation. Here, we report a kinome-targeted small interfering RNA (siRNA) screen to characterize human kinases involved in H2AX phosphorylation. By analyzing γH2AX foci at a single-nucleus level, we identified 46 kinases involved either directly or indirectly in H2AX phosphorylation in response to irradiation in human keratinocytes. Furthermore, we demonstrate that in response to irradiation, the FGFR4 signaling cascade promotes JNK1 activation and direct H2AX phosphorylation leading, in turn, to more efficient DNA repair. This can explain, at least partially, the radioprotective effect of FGF.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Histonas/metabolismo , Fosforilação/fisiologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Radiação , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo
7.
Genes Cells ; 19(3): 239-53, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24580727

RESUMO

Nrf2 is a major transcriptional activator of cytoprotective genes against oxidative/electrophilic stress, and Keap1 negatively regulates Nrf2. Emerging works have also suggested a role for Nrf2 as a regulator of differentiation in various cells, but the contribution of Nrf2 to the differentiation of hematopoietic stem cells (HSCs) remains elusive. Clarifying this point is important to understand Nrf2 functions in the development and/or resolution of inflammation. Here, we established two transgenic reporter mouse lines that allowed us to examine Nrf2 expression precisely in HSCs. Nrf2 was abundantly transcribed in HSCs, but its activity was maintained at low levels due to the Keap1-mediated degradation of Nrf2 protein. When we characterized Keap1-deficient mice, their bone marrow cells showed enhanced granulocyte-monocyte differentiation at the expense of erythroid and lymphoid differentiation. Importantly, Keap1-null HSCs showed lower expression of erythroid and lymphoid genes than did control HSCs, suggesting granulocyte-monocyte lineage priming in Keap1-null HSCs. This abnormal lineage commitment was restored by a concomitant deletion of Nrf2, demonstrating the Nrf2-dependency of the skewing. Analysis of Nrf2-deficient mice revealed that the physiological level of Nrf2 is sufficient to contribute to the lineage commitment. This study unequivocally shows that the Keap1-Nrf2 system regulates the cell fate determination of HSCs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas do Citoesqueleto/genética , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo , Fator 2 Relacionado a NF-E2/genética
8.
Hum Mol Genet ; 21(1): 121-35, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21968513

RESUMO

Fanconi anemia (FA) is a human rare genetic disorder characterized by congenital defects, bone marrow (BM) failure and predisposition to leukemia. The progressive aplastic anemia suggests a defect in the ability of hematopoietic stem cells (HSC) to sustain hematopoieis. We have examined the role of the nuclear FA core complex gene Fancg in the functionality of HSC. In Fancg-/- mice, we observed a decay of long-term HSC and multipotent progenitors that account for the reduction in the LSK compartment containing primitive hematopoietic cells. Fancg-/- lymphoid and myeloid progenitor cells were also affected, and myeloid progenitors show compromised in vitro functionality. HSC from Fancg-/- mice failed to engraft and to reconstitute at short and long term the hematopoiesis in a competitive transplantation assay. Fancg-/- LSK cells showed a loss of quiescence, an impaired migration in vitro in response to the chemokine CXCL12 and a defective homing to the BM after transplantation. Finally, the expression of several key genes involved in self-renewal, quiescence and migration of HSC was dysregulated in Fancg-deficient LSK subset. Collectively, our data reveal that Fancg should play a role in the regulation of physiological functions of HSC.


Assuntos
Proteína do Grupo de Complementação G da Anemia de Fanconi/deficiência , Anemia de Fanconi/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Medula Óssea/metabolismo , Movimento Celular , Quimiocina CXCL12/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Feminino , Hematopoese , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
9.
Development ; 138(2): 203-13, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21148188

RESUMO

During embryonic development, Igf2 gene transcription is highly regulated through the use of several promoters whose specific roles are not defined. Here, we show that loss-of-function of one of these promoters, Igf2-P2, results in growth defects that are temporally and quantitatively different from those seen in Igf2-null mutants. In particular, Igf2-P2 mutants exhibit skeletal abnormalities characterized by thin and short bones with reduced mineralization and medullar cavity and with altered bone remodeling. These abnormalities are associated with decreased numbers of embryonic mesenchymal chondroprogenitors, adult mesenchymal stem cells and osteoprogenitors. Differentiation of osteoprogenitors into osteoblasts is impaired in the Igf2-P2 mutant mice in a cell-autonomous manner, and osteopontin is a target of the IGF2 signaling pathway during this differentiation. Igf2-P2 mutant mice also display impaired formation of giant osteoclasts owing to a defective micro-environment. These results support a model wherein transcriptional activity of the Igf2-P2 promoter regulates the fate of mesenchymal progenitors during bone development and remodeling in the adult, and regulates osteogenesis in a cell-autonomous and non-autonomous manner.


Assuntos
Fator de Crescimento Insulin-Like II/deficiência , Fator de Crescimento Insulin-Like II/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Nanismo/embriologia , Nanismo/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Mutantes , Mutação , Osteogênese/genética , Osteogênese/fisiologia , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/metabolismo
10.
Sci Rep ; 14(1): 21005, 2024 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-39251615

RESUMO

Using a new red blood cell (RBC) metabolite extraction protocol, we performed a metabolomic analysis on RBCs in rheumatoid arthritis (RA) patients treated or not with methotrexate (MTX), with the two following objectives: to compare the RBC metabolic profiles of MTX-naïve RA patients and healthy controls (HC), and to investigate whether RBC profiles before and after MTX treatment in RA differed between responders and non-responders. Plasma analysis was performed in parallel. Metabolites were extracted and identified in RBCs and plasma by liquid chromatography-mass spectrometry. We compared the metabolomic fingerprints of 31 DMARD-naïve RA patients and 39 HCs. We also compared the RBC and plasma metabolomes of 25 RA patients who responded or not to MTX therapy before (M0) and after a 3-month treatment period (M3). Significance was determined by Storey's false discovery rate (FDR) q-values to correct for multiple testing. RA patients and HCs differed in the metabolomic signature of RBCs. The signature mainly contained amino acids (AA). Eleven metabolites, including 4 metabolites belonging to the carbohydrate subclass and 2 amino acids (creatine and valine) showed accumulation in RBCs from RA patients. Conversely, citrulline (fold change = 0.83; q = 0.025), histidine (fold change = 0.86; q = 0.014) and ergothioneine (EGT) (fold change = 0.66; q = 0.024), were lower in RBC of RA patients. Five plasma metabolites, including succinic acid and hydroxyproline, were higher in RA patients, and 7 metabolites, including DHEA sulfate, alanine, threonine and ornithine, were lower. Among RA patients undergoing MTX treatment pre-treatment (M0), EGT values were significantly lower in non-responders. In conclusion, low RBC levels of EGT, a food-derived AA barely detectable in plasma, characterize DMARD naïve RA patients and lack of response to MTX treatment.


Assuntos
Antirreumáticos , Artrite Reumatoide , Ergotioneína , Eritrócitos , Metabolômica , Metotrexato , Humanos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/sangue , Artrite Reumatoide/metabolismo , Metotrexato/uso terapêutico , Ergotioneína/sangue , Eritrócitos/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Metabolômica/métodos , Antirreumáticos/uso terapêutico , Adulto , Idoso , Metaboloma/efeitos dos fármacos
11.
Antioxidants (Basel) ; 13(9)2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39334742

RESUMO

Few therapeutic options are available to treat COVID-19. The KEAP1/NRF2 pathway, the major redox-responsive pathway, has emerged as a potential therapeutic target for COVID-19 as it regulates redox homeostasis and inflammation that are altered during SARS-CoV-2 infection. Here, we characterized the effects of NRF2-agonist Sulfodyne®, a stabilized natural Sulforaphane, in cellular and animal models of SARS-CoV-2 infection. In pulmonary or colonic epithelial cell lines, Sulfodyne® elicited a more efficient inhibition of SARS-CoV-2 replication than NRF2-agonists DMF and CDDO. This antiviral activity was not dependent on NRF2 but was associated with the regulation of several metabolic pathways, including the inhibition of ER stress and mTOR signaling, which are activated during SARS-CoV-2 infection. Sulfodyne® also decreased SARS-CoV-2 mediated inflammatory responses by inhibiting the delayed induction of IFNB1 and type I IFN-stimulated genes in infected epithelial cell lines and by reducing the activation of human by-stander monocytes recruited after SARS-CoV-2 infection. In K18-hACE2 mice infected with SARS-CoV-2, Sulfodyne® treatment reduced both early lung viral load and disease severity by fine-tuning IFN-beta levels. Altogether, these results provide evidence for multiple mechanisms that underlie the antiviral and anti-inflammatory activities of Sulfodyne® and pinpoint Sulfodyne® as a potent therapeutic agent against pathogenic effects of SARS-CoV-2 infection.

12.
Blood ; 117(6): e57-66, 2011 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-21135259

RESUMO

Emerging metabolomic tools can now be used to establish metabolic signatures of specialized circulating hematopoietic cells in physiologic or pathologic conditions and in human hematologic diseases. To determine metabolomes of normal and sickle cell erythrocytes, we used an extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry-based metabolite profiling method. Comparison of these 2 metabolomes identified major changes in metabolites produced by (1) endogenous glycolysis characterized by accumulation of many glycolytic intermediates; (2) endogenous glutathione and ascorbate metabolisms characterized by accumulation of ascorbate metabolism intermediates, such as diketogulonic acid and decreased levels of both glutathione and glutathione disulfide; (3) membrane turnover, such as carnitine, or membrane transport characteristics, such as amino acids; and (4) exogenous arginine and NO metabolisms, such as spermine, spermidine, or citrulline. Finally, metabolomic analysis of young and old normal red blood cells indicates metabolites whose levels are directly related to sickle cell disease. These results show the relevance of metabolic profiling for the follow-up of sickle cell patients or other red blood cell diseases and pinpoint the importance of metabolomics to further depict the pathophysiology of human hematologic diseases.


Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/fisiopatologia , Eritrócitos/metabolismo , Metaboloma , Adulto , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Modelos Biológicos , Estresse Oxidativo , Espectrometria de Massas por Ionização por Electrospray , Adulto Jovem
13.
J Invest Dermatol ; 143(1): 105-114.e12, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36007550

RESUMO

Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor ex vivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for ex vivo expansion of human KC precursors.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Epidérmicas , Queratinócitos , Humanos , Diferenciação Celular , Epiderme/metabolismo , Queratinócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo
14.
Stem Cell Res Ther ; 14(1): 201, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37568164

RESUMO

BACKGROUND: Human multilineage-differentiating stress enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be easily obtained from various adult or fetal tissues. Regenerative effects of Muse cells have been shown in some disease models. Muse cells specifically home in damaged tissues where they exert pleiotropic effects. Exposition of the small intestine to high doses of irradiation (IR) delivered after radiotherapy or nuclear accident results in a lethal gastrointestinal syndrome (GIS) characterized by acute loss of intestinal stem cells, impaired epithelial regeneration and subsequent loss of the mucosal barrier resulting in sepsis and death. To date, there is no effective medical treatment for GIS. Here, we investigate whether Muse cells can prevent lethal GIS and study how they act on intestinal stem cell microenvironment to promote intestinal regeneration. METHODS: Human Muse cells from Wharton's jelly matrix of umbilical cord (WJ-Muse) were sorted by flow cytometry using the SSEA-3 marker, characterized and compared to bone-marrow derived Muse cells (BM-Muse). Under gas anesthesia, GIS mice were treated or not through an intravenous retro-orbital injection of 50,000 WJ-Muse, freshly isolated or cryopreserved, shortly after an 18 Gy-abdominal IR. No immunosuppressant was delivered to the mice. Mice were euthanized either 24 h post-IR to assess early small intestine tissue response, or 7 days post-IR to assess any regenerative response. Mouse survival, histological stainings, apoptosis and cell proliferation were studied and measurement of cytokines, recruitment of immune cells and barrier functional assay were performed. RESULTS: Injection of WJ-Muse shortly after abdominal IR highly improved mouse survival as a result of a rapid regeneration of intestinal epithelium with the rescue of the impaired epithelial barrier. In small intestine of Muse-treated mice, an early enhanced secretion of IL-6 and MCP-1 cytokines was observed associated with (1) recruitment of monocytes/M2-like macrophages and (2) proliferation of Paneth cells through activation of the IL-6/Stat3 pathway. CONCLUSION: Our findings indicate that a single injection of a small quantity of WJ-Muse may be a new and easy therapeutic strategy for treating lethal GIS.


Assuntos
Alprostadil , Células-Tronco Mesenquimais , Adulto , Camundongos , Humanos , Animais , Diferenciação Celular/fisiologia , Alprostadil/metabolismo , Células-Tronco Mesenquimais/metabolismo , Interleucina-6/metabolismo , Intestinos
15.
J Biol Chem ; 286(23): 20870-9, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21478550

RESUMO

The inhibitor of DNA binding 2, dominant negative helix-loop-helix protein, ID2, acts as an oncogene and elevated levels of ID2 have been reported in several malignancies. Whereas some inducers of the ID2 gene have been characterized, little is known regarding the proteins capable to repress its expression. We developed siRNA microarrays to perform a large scale loss-of-function screen in human adult keratinocytes engineered to express GFP under the control of the upstream region of ID2 gene. We screened the effect of siRNA-dependent inhibition of 220 cancer-associated genes on the expression of the ID2::GFP reporter construct. Three genes NBN, RAD21, and p63 lead to a repression of ID2 promoter activity. Strikingly NBN and RAD21 are playing on major role in cell cycle progression and mitosis arrest. These results underline the pregnant need to silence ID2 expression at transcript level to promote cell cycle exit. Central to this inhibitory mechanism we find p63, a key transcription factor in epithelial development and differentiation, which binds specific cis-acting sequence within the ID2 gene promoter both in vitro and in vivo. P63 would not suppress ID2 expression, but would rather prevent excessive expression of that protein to enable the onset of keratinocyte differentiation.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteína 2 Inibidora de Diferenciação/biossíntese , Queratinócitos/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Queratinócitos/citologia , Mitose/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Interferente Pequeno/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
16.
Blood ; 115(3): 443-52, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-19797522

RESUMO

Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin(-)Sca-1(+)c-Kit(+)CD34(-) (LSKCD34(-)) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34(-) hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34(-) hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34(-) hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34(-) hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.


Assuntos
Hematopoese/efeitos dos fármacos , Imagem Molecular/métodos , Espécies Reativas de Oxigênio/farmacologia , Animais , Antígenos CD34/metabolismo , Antígenos Ly/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Fêmur/citologia , Fêmur/metabolismo , Fêmur/fisiologia , Tecnologia de Fibra Óptica/métodos , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estudos de Validação como Assunto , Irradiação Corporal Total
17.
Haematologica ; 97(4): 491-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22180429

RESUMO

BACKGROUND: Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important. DESIGN AND METHODS: Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation. RESULTS: Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice. CONCLUSIONS: This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.


Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Compostos Heterocíclicos/farmacologia , Animais , Benzilaminas , Ciclamos , Sinergismo Farmacológico , Hematopoese/efeitos dos fármacos , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Heparitina Sulfato/síntese química , Cinética , Camundongos , Camundongos Endogâmicos C57BL
18.
PLoS Biol ; 7(6): e1000123, 2009 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-19513100

RESUMO

How cell proliferation subsides as cells terminally differentiate remains largely enigmatic, although this phenomenon is central to the existence of multicellular organisms. Here, we show that GATA-1, the master transcription factor of erythropoiesis, forms a tricomplex with the retinoblastoma protein (pRb) and E2F-2. This interaction requires a LXCXE motif that is evolutionary conserved among GATA-1 orthologs yet absent from the other GATA family members. GATA-1/pRb/E2F-2 complex formation stalls cell proliferation and steers erythroid precursors towards terminal differentiation. This process can be disrupted in vitro by FOG-1, which displaces pRb/E2F-2 from GATA-1. A GATA-1 mutant unable to bind pRb fails to inhibit cell proliferation and results in mouse embryonic lethality by anemia. These findings clarify the previously suspected cell-autonomous role of pRb during erythropoiesis and may provide a unifying molecular mechanism for several mouse phenotypes and human diseases associated with GATA-1 mutations.


Assuntos
Fator de Transcrição E2F2/metabolismo , Eritropoese , Fator de Transcrição GATA1/metabolismo , Proteína do Retinoblastoma/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Divisão Celular , Proliferação de Células , Células Eritroides/citologia , Células Eritroides/metabolismo , Fator de Transcrição GATA1/química , Fator de Transcrição GATA1/deficiência , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteína do Retinoblastoma/deficiência , Fatores de Transcrição/metabolismo
19.
Blood Adv ; 6(6): 1766-1779, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35100346

RESUMO

Bone marrow (BM) resident macrophages interact with a population of long-term hematopoietic stem cells (LT-HSCs) but their role on LT-HSC properties after stress is not well defined. Here, we show that a 2 Gy-total body irradiation (TBI)-mediated death of LT-HSCs is associated with increased percentages of LT-HSCs with reactive oxygen species (ROS) and of BM resident macrophages producing nitric oxide (NO), resulting in an increased percentage of LT-HSCs with endogenous cytotoxic peroxynitrites. Pharmacological or genetic depletion of BM resident macrophages impairs the radio-induced increases in the percentage of both ROS+ LT-HSCs and peroxynitrite+ LT-HSCs and results in a complete recovery of a functional pool of LT-HSCs. Finally, we show that after a 2 Gy-TBI, a specific decrease of NO production by BM resident macrophages improves the LT-HSC recovery, whereas an exogenous NO delivery decreases the LT-HSC compartment. Altogether, these results show that BM resident macrophages are involved in the response of LT-HSCs to a 2 Gy-TBI and suggest that regulation of NO production can be used to modulate some deleterious effects of a TBI on LT-HSCs.


Assuntos
Medula Óssea , Irradiação Corporal Total , Células-Tronco Hematopoéticas , Macrófagos , Espécies Reativas de Oxigênio , Irradiação Corporal Total/efeitos adversos
20.
Blood Cancer Discov ; 3(4): 285-297, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35290450

RESUMO

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients' erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. SIGNIFICANCE: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265.


Assuntos
Anidrases Carbônicas , Neoplasias Hematológicas , Policitemia Vera , Animais , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas , Janus Quinase 2/genética , Camundongos , Policitemia Vera/tratamento farmacológico
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