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1.
Genes Dev ; 29(24): 2603-16, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26680303

RESUMO

Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.


Assuntos
Diferenciação Celular/genética , Células Eritroides/citologia , Eritropoese/fisiologia , Eritropoetina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Transdução de Sinais , Anemia Hemolítica/genética , Animais , Proliferação de Células/genética , Ativação Enzimática/genética , Eritropoese/genética , Eritropoetina/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Técnicas de Introdução de Genes , Camundongos , Mutação , Proteínas Nucleares/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Ligação Proteica/genética , Fatores de Transcrição/metabolismo
2.
N Engl J Med ; 378(16): 1479-1493, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29669226

RESUMO

BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ßA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-thalassemia. METHODS: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent ß-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. RESULTS: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-ß0/ß0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a ß0/ß0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe ß-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).


Assuntos
Terapia Genética , Globinas beta/genética , Talassemia beta/terapia , Adolescente , Adulto , Antígenos CD34 , Criança , Transfusão de Eritrócitos/estatística & dados numéricos , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Hemoglobinas/análise , Hemoglobinas/genética , Humanos , Lentivirus/genética , Masculino , Mutação , Transplante Autólogo , Adulto Jovem , Talassemia beta/genética
3.
Nature ; 525(7569): 380-3, 2015 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-26331539

RESUMO

Whether cancer is maintained by a small number of stem cells or is composed of proliferating cells with approximate phenotypic equivalency is a central question in cancer biology. In the stem cell hypothesis, relapse after treatment may occur by failure to eradicate cancer stem cells. Chronic myeloid leukaemia (CML) is quintessential to this hypothesis. CML is a myeloproliferative disorder that results from dysregulated tyrosine kinase activity of the fusion oncoprotein BCR-ABL. During the chronic phase, this sole genetic abnormality (chromosomal translocation Ph(+): t(9;22)(q34;q11)) at the stem cell level causes increased proliferation of myeloid cells without loss of their capacity to differentiate. Without treatment, most patients progress to the blast phase when additional oncogenic mutations result in a fatal acute leukaemia made of proliferating immature cells. Imatinib mesylate and other tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL have improved patient survival markedly. However, fewer than 10% of patients reach the stage of complete molecular response (CMR), defined as the point when BCR-ABL transcripts become undetectable in blood cells. Failure to reach CMR results from the inability of TKIs to eradicate quiescent CML leukaemia stem cells (LSCs). Here we show that the residual CML LSC pool can be gradually purged by the glitazones, antidiabetic drugs that are agonists of peroxisome proliferator-activated receptor-γ (PPARγ). We found that activation of PPARγ by the glitazones decreases expression of STAT5 and its downstream targets HIF2α and CITED2, which are key guardians of the quiescence and stemness of CML LSCs. When pioglitazone was given temporarily to three CML patients in chronic residual disease in spite of continuous treatment with imatinib, all of them achieved sustained CMR, up to 4.7 years after withdrawal of pioglitazone. This suggests that clinically relevant cancer eradication may become a generally attainable goal by combination therapy that erodes the cancer stem cell pool.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , PPAR gama/agonistas , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , Tiazolidinedionas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , PPAR gama/metabolismo , Pioglitazona , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT5/metabolismo , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Transativadores/metabolismo
4.
N Engl J Med ; 376(9): 848-855, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28249145

RESUMO

Sickle cell disease results from a homozygous missense mutation in the ß-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling ß-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling ß-globin remained high (approximately 50% of ß-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).


Assuntos
Anemia Falciforme/terapia , Terapia Genética , Globinas beta/genética , Adolescente , Anemia Falciforme/sangue , Ensaios Clínicos como Assunto , Expressão Gênica , Terapia Genética/efeitos adversos , Vetores Genéticos , Hemoglobina A/metabolismo , Humanos , Lentivirus , Masculino
5.
Nature ; 514(7521): 242-6, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25156257

RESUMO

ß-Thalassaemia major (ß-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing ß-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human ß-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of ß-TM erythroblasts, which may provide a rationale for new targeted therapies of ß-TM.


Assuntos
Eritroblastos/metabolismo , Eritropoese , Proteínas de Choque Térmico HSP70/metabolismo , alfa-Globinas/metabolismo , Talassemia beta/sangue , Talassemia beta/metabolismo , Apoptose , Medula Óssea/metabolismo , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Citoplasma/metabolismo , Ativação Enzimática , Eritroblastos/citologia , Eritroblastos/patologia , Eritropoese/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Humanos , Cinética , Terapia de Alvo Molecular , Ligação Proteica , Redobramento de Proteína , Talassemia beta/patologia
6.
Exp Physiol ; 104(7): 1074-1089, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31012180

RESUMO

NEW FINDINGS: What is the central question of this study? Do Fog2Rb-/Rb- mice present a defect of small intestine homeostasis? What is the main finding and its importance? The importance of interactions between FOG-2 and pRb in adipose tissue physiology has previously been demonstrated. Here it is shown that this interaction is also intrinsic to small intestine homeostasis and exerts extrinsic control over mouse metabolism. Thus, this association is involved in maintaining small intestine morphology, and regulating crypt proliferation and lineage differentiation. It therefore affects mouse growth and adaptation to a high-fat diet. ABSTRACT: GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. We have shown that GATA-1 and FOG-2 contain an LXCXE pRb-binding motif. Interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation, whereas the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Fog2-knock-in mice have defective pRb binding and are resistant to obesity, due to efficient white-into-brown fat conversion. Our aim was to investigate the pathophysiological impact of FOG-2-pRb interaction on the small intestine and mouse growth. Histological analysis of the small intestine revealed architectural changes in Fog2Rb-/Rb- mice, including villus shortening, with crypt expansion and a change in muscularis propria thickness. These differences were more marked in the proximo-distal part of the small intestine and were associated with an increase in crypt cell proliferation and disruption of the goblet and Paneth cell lineage. The small intestine of the mutants was unable to adapt to a high-fat diet, and had significantly lower plasma lipid levels on such a diet. Fog2Rb-/Rb- mice displayed higher levels of glucose-dependent insulinotropic peptide release, and lower levels of insulin-like growth factor I release on a regular diet. Their intestinal lipid absorption was impaired, resulting in restricted weight gain. In addition to the intrinsic effects of the mutation on adipose tissue, we show here an extrinsic relationship between the intestine and the effect of FOG-2 mutation on mouse metabolism. In conclusion, the interaction of FOG-2 with pRb coordinates the crypt-villus axis and controls small intestine homeostasis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Dieta Hiperlipídica/efeitos adversos , Homeostase/fisiologia , Intestino Delgado/metabolismo , Domínios Proteicos Ricos em Prolina/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Intestino Delgado/citologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Ligação Proteica/fisiologia , Distribuição Aleatória , Fatores de Transcrição/genética
7.
Biochem Biophys Res Commun ; 472(4): 624-30, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26972250

RESUMO

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.


Assuntos
Adipogenia/efeitos dos fármacos , Azepinas/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Lisina/metabolismo , Triazóis/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Regulação para Baixo/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Fatores de Transcrição
8.
Nature ; 467(7313): 318-22, 2010 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-20844535

RESUMO

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.


Assuntos
Transfusão de Sangue , Terapia Genética , Proteína HMGA2/metabolismo , Globinas beta/genética , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/terapia , Adolescente , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Pré-Escolar , Células Clonais/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Proteína HMGA2/genética , Homeostase , Humanos , Lentivirus/genética , Masculino , MicroRNAs/genética , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Ativação Transcricional , Adulto Jovem , Talassemia beta/metabolismo
9.
Stem Cells ; 31(10): 2162-71, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23554255

RESUMO

Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by nonarrayed DNA barcodes subsequently analyzed by high-throughput DNA sequencing. However, key limitations include statistical biases compromising quantification and the lack of applicability to deconvolute individual cell fate in vivo after pooling single cells differentially exposed to different conditions ex vivo. Here, we have derived an arrayed lentiviral library of DNA barcodes and obtained a proof-of-concept of its resolving capacity by quantifying hematopoietic regeneration after engraftment of mice with genetically modified autologous cells. This method has helped clarify and bridge the seemingly opposed clonal-succession and continuous-recruitment models of hematopoietic stem cell behavior and revealed that myeloid-lymphoid biases are common occurrences in steady-state hematopoiesis. Arrayed lentiviral barcoding should prove a versatile and powerful approach to deconvolute cell dynamics in vivo with applications in hematology, embryology, and cancer biology.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Lentivirus/genética , Animais , Rastreamento de Células/métodos , Código de Barras de DNA Taxonômico , Vetores Genéticos , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo
10.
Stem Cells ; 31(9): 1785-94, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23712774

RESUMO

A patient with ß(E)/ß(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, ß-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the ß-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with ß-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.


Assuntos
Globinas/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/metabolismo , Transdução Genética , Talassemia beta/patologia , Adulto , Animais , Diferenciação Celular/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/metabolismo , Globinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Mutagênicos/toxicidade , Regeneração/efeitos dos fármacos , Integração Viral/efeitos dos fármacos
11.
Biochem Biophys Res Commun ; 429(1-2): 1-5, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23137537

RESUMO

Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.


Assuntos
Azepinas/farmacologia , Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Leucemia Eritroblástica Aguda/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Triazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismo
12.
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
14.
Viruses ; 12(12)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33353185

RESUMO

Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlation between infectivity and S/G2/M cell cycle stage. Two of the clones tested, B12 and E2, reached sensitivity levels higher than those of UT7/Epo-S1 and CD36+ erythroid progenitor cells. These findings highlight the importance of cell cycle status for sensitivity to B19V, and we propose a promising new straightforward cell-based method for quantifying B19V infectious units.


Assuntos
Ciclo Celular , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/fisiologia , Biomarcadores , Linhagem Celular , Células Eritroides/metabolismo , Células Eritroides/virologia , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/virologia , Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Técnicas de Diagnóstico Molecular , Infecções por Parvoviridae/metabolismo , Sensibilidade e Especificidade , Tropismo Viral
15.
Mol Cell Biol ; 25(17): 7412-22, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16107690

RESUMO

The contribution of erythropoietin to the differentiation of the red blood cell lineage remains elusive, and the demonstration of a molecular link between erythropoietin and the transcription of genes associated with erythroid differentiation is lacking. In erythroid cells, expression of the tissue inhibitor of matrix metalloproteinase (TIMP-1) is strictly dependent on erythropoietin. We report here that erythropoietin regulates the transcription of the TIMP-1 gene upon binding to its receptor in erythroid cells by triggering the activation of phosphatidylinositol 3-kinase (PI3K)/Akt. We found that Akt directly phosphorylates the transcription factor GATA-1 at serine 310 and that this site-specific phosphorylation is required for the transcriptional activation of the TIMP-1 promoter. This chain of events can be recapitulated in nonerythroid cells by transfection of the implicated molecular partners, resulting in the expression of the normally silent endogenous TIMP-1 gene. Conversely, TIMP-1 secretion is profoundly decreased in erythroid cells from fetal livers of transgenic knock-in mice homozygous for a GATA(S310A) gene, which encodes a GATA-1 mutant that cannot be phosphorylated at Ser(310). Furthermore, retrovirus-mediated expression of GATA(S310A) into GATA-1(null)-derived embryonic stem cells decreases the rate of hemoglobinization by more than 50% compared to expressed wild-type GATA-1. These findings provide the first example of a chain of coupling mechanisms between the binding of erythropoietin to its receptor and GATA-1-dependent gene expression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Eritroides/metabolismo , Eritropoetina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/química , Células Eritroides/citologia , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/deficiência , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fatores de Transcrição/química , Transcrição Gênica/genética
16.
Cell Rep ; 21(12): 3524-3535, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29262331

RESUMO

GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. Mammals have six GATA and two FOG factors. We recently demonstrated that interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation. We show here that the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Unlike GATA-1, which inhibits cell division, FOG-2 promotes proliferation. Mice with a knockin of a Fog2 gene bearing a mutated LXCXE pRb-binding site are resistant to obesity and display higher rates of white-to-brown fat conversion. Thus, each component of the GATA/FOG complex (GATA-1 and FOG-2) is involved in pRb/E2F regulation, but these molecules have markedly different roles in the control of tissue homeostasis.


Assuntos
Adipogenia , Proteínas de Ligação a DNA/metabolismo , Obesidade/genética , Fatores de Transcrição/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Camundongos , Mutação , Obesidade/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
17.
Cell Signal ; 14(10): 869-78, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12135708

RESUMO

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Membrana Celular/enzimologia , Eritrócitos/enzimologia , Células Precursoras Eritroides/enzimologia , Eritropoetina/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Eritropoetina/genética , Proteínas de Helminto/metabolismo , Humanos , Hidrólise/efeitos dos fármacos , Fosfolipase C gama , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Frações Subcelulares , Transfecção , Tirosina/metabolismo
18.
Blood ; 105(2): 600-8, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15358619

RESUMO

Activation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.


Assuntos
Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores da Eritropoetina/metabolismo , Células Cultivadas , Eritropoetina/metabolismo , Eritropoetina/farmacologia , Humanos , Radioisótopos do Iodo , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Receptores da Eritropoetina/química , Ubiquitina/metabolismo
19.
Blood ; 105(10): 4035-42, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15677568

RESUMO

Myelodysplastic syndromes (MDSs) are characterized by peripheral blood cytopenia including anemia. We have investigated the implication of the extrinsic pathway of apoptosis in MDS-ineffective erythropoiesis by in vitro expansion of erythroid precursors from early stage (low and intermediate-1 International Prognosis Scoring System [IPSS]) MDS, advanced stage (intermediate-2 IPSS) MDS, and control bone marrow samples. We have previously shown that Fas and its ligand were overexpressed in early stage MDS erythroid cells. Here, we show that caspase-8 activity is significantly increased, whereas the expression of death receptors other than Fas, including the type 1 receptor for tumor necrosis factor alpha (TNF-alpha) and the receptors for the TNF-related apoptosis-inducing ligand (TRAIL), DR4 and DR5, was normal. We also observed that the adapter Fas-associated death domain (FADD) was overexpressed in early stage MDS erythroid cells. Transduction of early stage MDS-derived CD34+ progenitors with a FADD-encoding construct increased apoptosis of erythroid cells and dramatically reduced erythroid burst-forming unit (BFU-E) growth. Transduction of a dominant-negative (dn) mutant of FADD inhibited caspase-8 activity and cell death and rescued BFU-E growth without abrogating erythroid differentiation. These results extend the observation that Fas-dependent activation of caspase-8 accounts for apoptosis of early stage MDS erythroid cells and demonstrate for the first time that FADD is a valuable target to correct ineffective erythropoiesis in these syndromes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular , Células Eritroides/metabolismo , Células Eritroides/patologia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Proteínas Reguladoras de Apoptose , Caspase 8 , Caspases/metabolismo , Células Cultivadas , Proteína de Domínio de Morte Associada a Fas , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Dominantes/genética , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Estadiamento de Neoplasias , Células-Tronco/citologia , Células-Tronco/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismo
20.
J Biol Chem ; 277(11): 9139-47, 2002 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11784712

RESUMO

Thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells via activation of the c-Mpl receptor and multiple downstream signal transduction pathways. We used two-hybrid screening to identify new proteins that interacted with the cytoplasmic domain of Mpl, and we found a new family of proteins designated A2D (for Ataxin-2 Domain protein). The A2D are 130-kDa proteins that have three regions similar to those of Ataxin-2, the gene product causing familial type 2 spinocerebellar ataxia. A2D has several isoforms with different C-terminal domains, all produced from a single gene by alternative splicing. Northern blotting indicated that the A2D gene is widely expressed in immortalized cell lines and hematopoietic and fetal tissues. A2D proteins were constitutively associated with Mpl in vivo in human hematopoietic UT7 cells. TPO also caused the release of A2D from the activated receptor, and the phosphorylation of A2D on tyrosines residues was dependent on the Mpl C-terminal domain. Finally, A2D bound to the unstimulated erythropoietin receptor, whereas erythropoietin caused dissociation from the erythropoietin receptor, suggesting that A2D proteins are new components of the cytokine signaling system.


Assuntos
Proteínas de Neoplasias , Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas , Sequência de Aminoácidos , Ataxinas , Células Cultivadas , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Fosforilação , Isoformas de Proteínas , Proteínas/química , Proteínas/metabolismo , RNA Mensageiro/análise , Receptores da Eritropoetina/análise , Receptores de Trombopoetina , Trombopoetina/metabolismo , Trombopoetina/farmacologia , Técnicas do Sistema de Duplo-Híbrido
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