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1.
BMC Genomics ; 17(1): 817, 2016 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-27769165

RESUMO

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) are a potentially invaluable resource for regenerative medicine, including the in vitro manufacture of blood products. HiPSC-derived red blood cells are an attractive therapeutic option in hematology, yet exhibit unexplained proliferation and enucleation defects that presently preclude such applications. We hypothesised that substantial differential regulation of gene expression during erythroid development accounts for these important differences between hiPSC-derived cells and those from adult or cord-blood progenitors. We thus cultured erythroblasts from each source for transcriptomic analysis to investigate differential gene expression underlying these functional defects. RESULTS: Our high resolution transcriptional view of definitive erythropoiesis captures the regulation of genes relevant to cell-cycle control and confers statistical power to deploy novel bioinformatics methods. Whilst the dynamics of erythroid program elaboration from adult and cord blood progenitors were very similar, the emerging erythroid transcriptome in hiPSCs revealed radically different program elaboration compared to adult and cord blood cells. We explored the function of differentially expressed genes in hiPSC-specific clusters defined by our novel tunable clustering algorithms (SMART and Bi-CoPaM). HiPSCs show reduced expression of c-KIT and key erythroid transcription factors SOX6, MYB and BCL11A, strong HBZ-induction, and aberrant expression of genes involved in protein degradation, lysosomal clearance and cell-cycle regulation. CONCLUSIONS: Together, these data suggest that hiPSC-derived cells may be specified to a primitive erythroid fate, and implies that definitive specification may more accurately reflect adult development. We have therefore identified, for the first time, distinct gene expression dynamics during erythroblast differentiation from hiPSCs which may cause reduced proliferation and enucleation of hiPSC-derived erythroid cells. The data suggest several mechanistic defects which may partially explain the observed aberrant erythroid differentiation from hiPSCs.


Assuntos
Eritropoese/genética , Sangue Fetal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Diferenciação Celular/genética , Análise por Conglomerados , Eritroblastos/citologia , Eritroblastos/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
2.
Methods ; 59(1): 71-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23079396

RESUMO

The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR.


Assuntos
Perfilação da Expressão Gênica/métodos , Células Progenitoras Linfoides/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Célula Única , Linhagem Celular , Interpretação Estatística de Dados , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Via de Sinalização Wnt
3.
Nat Med ; 29(1): 104-114, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36624315

RESUMO

Affinity-optimized T cell receptors can enhance the potency of adoptive T cell therapy. Afamitresgene autoleucel (afami-cel) is a human leukocyte antigen-restricted autologous T cell therapy targeting melanoma-associated antigen A4 (MAGE-A4), a cancer/testis antigen expressed at varying levels in multiple solid tumors. We conducted a multicenter, dose-escalation, phase 1 trial in patients with relapsed/refractory metastatic solid tumors expressing MAGE-A4, including synovial sarcoma (SS), ovarian cancer and head and neck cancer ( NCT03132922 ). The primary endpoint was safety, and the secondary efficacy endpoints included overall response rate (ORR) and duration of response. All patients (N = 38, nine tumor types) experienced Grade ≥3 hematologic toxicities; 55% of patients (90% Grade ≤2) experienced cytokine release syndrome. ORR (all partial response) was 24% (9/38), 7/16 (44%) for SS and 2/22 (9%) for all other cancers. Median duration of response was 25.6 weeks (95% confidence interval (CI): 12.286, not reached) and 28.1 weeks (95% CI: 12.286, not reached) overall and for SS, respectively. Exploratory analyses showed that afami-cel infiltrates tumors, has an interferon-γ-driven mechanism of action and triggers adaptive immune responses. In addition, afami-cel has an acceptable benefit-risk profile, with early and durable responses, especially in patients with metastatic SS. Although the small trial size limits conclusions that can be drawn, the results warrant further testing in larger studies.


Assuntos
Antígenos de Neoplasias , Neoplasias de Cabeça e Pescoço , Masculino , Humanos , Proteínas de Neoplasias , Antígenos HLA-A , Terapia Baseada em Transplante de Células e Tecidos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos
4.
Blood ; 113(12): 2661-72, 2009 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-19168794

RESUMO

Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G(0) residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34(+)CD38(-)Hoechst(lo)Pyronin Y(lo)) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2-conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21(CIP1) and p27(KIP1) do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2(hi)) failed to contribute to hematopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2(lo) cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.


Assuntos
Ciclo Celular , Fator de Transcrição GATA2/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Estradiol/farmacologia , Sangue Fetal/citologia , Fator de Transcrição GATA2/biossíntese , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica/genética , Genes Sintéticos , Genes cdc , Humanos , Interleucina-3/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/fisiologia , Fase de Repouso do Ciclo Celular , Tamoxifeno/farmacologia , Transcrição Gênica
5.
Blood ; 112(13): 4862-73, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18840712

RESUMO

The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein, we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2(+/-)) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin(-)CD117(+)Sca-1(-)CD34(+)CD16/32(high) granulocyte-macrophage progenitor (GMP) compartment of GATA-2(+/-) marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. Although apoptosis and cell-cycle distribution remained unperturbed in GATA-2(+/-) GMP, quiescent cells from GATA-2(+/-) GMP exhibited altered functionality. Gene expression analysis showed attenuated expression of HES-1 mRNA in GATA-2-deficient GMP. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3, and enforced expression of HES-1 expression in GATA-2(+/-) GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as a novel, pivotal determinant of GMP cell fate.


Assuntos
Fator de Transcrição GATA2/fisiologia , Células Progenitoras de Granulócitos e Macrófagos/citologia , Animais , Linhagem Celular , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Perfilação da Expressão Gênica , Genótipo , Células Progenitoras de Granulócitos e Macrófagos/fisiologia , Camundongos , Camundongos Mutantes , Ligação Proteica , Interferência de RNA
6.
J Immunother Cancer ; 7(1): 276, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31651363

RESUMO

BACKGROUND: Gene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells. METHODS: Four cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay. RESULTS: Responses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients. CONCLUSIONS: Our studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01343043 , Registered 27 April 2011.


Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva , Proteínas de Membrana/imunologia , Sarcoma Sinovial/imunologia , Sarcoma Sinovial/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Biomarcadores , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Citocinas/metabolismo , Citotoxicidade Imunológica , Antígenos HLA-A/imunologia , Humanos , Imuno-Histoquímica , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Sarcoma Sinovial/patologia , Especificidade do Receptor de Antígeno de Linfócitos T , Resultado do Tratamento , Microambiente Tumoral/imunologia
7.
Mol Cell Biol ; 25(23): 10235-50, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16287841

RESUMO

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Eritroides/metabolismo , Eritropoese , Megacariócitos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Células Cultivadas , Células Eritroides/citologia , Regulação da Expressão Gênica , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genética , Transcrição Gênica/genética
8.
Biochem J ; 387(Pt 1): 231-8, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15540985

RESUMO

The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.


Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Benzamidas , Linhagem Celular , Linhagem Celular Tumoral , Citarabina/antagonistas & inibidores , Citarabina/metabolismo , Citarabina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Genes Supressores de Tumor , Humanos , Mesilato de Imatinib , Rim/química , Rim/citologia , Rim/embriologia , Rim/metabolismo , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/farmacologia , Proteína Tumoral p73 , Proteínas Supressoras de Tumor , Células U937/enzimologia , Células U937/metabolismo , Células U937/patologia
9.
Exp Hematol ; 44(5): 399-409.e5, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26876150

RESUMO

Transforming growth factor ß (TGFß) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell function, as an early and direct target gene for TGFß-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFß response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFß signaling effector molecule p57 was found to be upregulated as a secondary response to TGFß. We observed Gata2 binding upstream of the p57 genomic locus, and importantly, loss of Gata2 abolished TGFß-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in hematopoietic stem cell self-renewal and reveal a functionally relevant network, regulating proliferation of primitive hematopoietic cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p57/genética , Fator de Transcrição GATA2/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteína Smad4/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular , Proliferação de Células/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Fator de Transcrição GATA2/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad4/metabolismo
10.
Exp Hematol ; 31(11): 1073-80, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14585372

RESUMO

OBJECTIVE: Resistance to imatinib mesylate monotherapy is clearly a barrier to successful treatment of chronic myeloid leukemia (CML) patients. In some patients, resistance arises due to powerful selective pressure on rare cells that carry amplified copies of the BCR-ABL fusion oncogene or point mutations in the Bcr-Abl tyrosine kinase domain that affect binding of the drug to the oncoprotein. However, in a proportion of patients neither mechanism operates, and resistance appears to be a priori, existing prior to exposure to the drug. These mechanisms of imatinib resistance are poorly understood and may be heterogeneous. MATERIALS AND METHODS: We have previously described such innate resistance to imatinib in subclones of a myeloid leukemia cell line, KCL22, in which imatinib exposure inhibits the activity of Bcr-Abl and yet fails to induce apoptosis. We describe here whole-genome expression analysis of imatinib-sensitive and -resistant cells derived from the original KCL22 line, using Affymetrix microarray analysis. RESULTS: We detected differential expression of 39 genes that correlate with the imatinib-resistant phenotype. The resistant cells overexpress several genes associated with the suppression of apoptosis or with conferral of a transformed phenotype. CONCLUSION: Amongst the differentially-expressed genes correlating with imatinib resistance, several suggest the activation of alternative pathway(s) that maintain viability and growth independently of Bcr-Abl kinase activity. Given the high rate of primary imatinib resistance in blast crisis, the potential of activating such alternative pathways appears to correlate with disease progression.


Assuntos
Antineoplásicos/uso terapêutico , Perfilação da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética
11.
PLoS One ; 10(3): e0119836, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25781011

RESUMO

The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10 ng/ml) or to hemozoin (12.5 µg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.


Assuntos
Eritrócitos/citologia , Eritropoese/fisiologia , Doenças Hematológicas/etiologia , Hemeproteínas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hemeproteínas/metabolismo , Hemeproteínas/fisiologia , Humanos , Imunidade Celular , Interferons/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia
12.
Semin Hematol ; 40(2 Suppl 2): 83-91, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12783381

RESUMO

Imatinib mesylate (Gleevec) (formerly STI571) has secured a definitive role in the treatment of chronic myeloid leukemia (CML) due to its specificity and efficacy. Although some patients become resistant to the drug, it may still be possible to control the leukemia with imatinib-containing regimens. Front-line treatment with such combinations may indeed minimize the risk that resistance, and hence relapse, occurs. In this review, we discuss the published data on in vitro studies that address this question in a variety of models, and attempt to predict efficacious combinations for future clinical trials. These represent regimens where imatinib is combined with conventional chemotherapeutic drugs or with inhibitors of other key signal transduction molecules that may be preferentially activated in CML cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Benzamidas , Interações Medicamentosas , Humanos , Mesilato de Imatinib , Transdução de Sinais/efeitos dos fármacos
13.
Nat Biotechnol ; 31(8): 748-52, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23873083

RESUMO

Gene expression in multiple individual cells from a tissue or culture sample varies according to cell-cycle, genetic, epigenetic and stochastic differences between the cells. However, single-cell differences have been largely neglected in the analysis of the functional consequences of genetic variation. Here we measure the expression of 92 genes affected by Wnt signaling in 1,440 single cells from 15 individuals to associate single-nucleotide polymorphisms (SNPs) with gene-expression phenotypes, while accounting for stochastic and cell-cycle differences between cells. We provide evidence that many heritable variations in gene function--such as burst size, burst frequency, cell cycle-specific expression and expression correlation/noise between cells--are masked when expression is averaged over many cells. Our results demonstrate how single-cell analyses provide insights into the mechanistic and network effects of genetic variability, with improved statistical power to model these effects on gene expression.


Assuntos
Expressão Gênica , Estudos de Associação Genética , Locos de Características Quantitativas/genética , Via de Sinalização Wnt/genética , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Análise de Célula Única
14.
Cell Stem Cell ; 13(6): 754-68, 2013 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-24120743

RESUMO

We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.


Assuntos
Linhagem da Célula/genética , Regulação da Expressão Gênica , Genoma/genética , Hematopoese/genética , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Células Eritroides/citologia , Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo
15.
Int J Biochem Cell Biol ; 44(3): 457-60, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22192845

RESUMO

Unremitting blood cell production throughout the lifetime of an organism is reliant on hematopoietic stem cells (HSCs). A rare and relatively quiescent cell type, HSCs are, on entry into cell cycle fated to self-renew, undergo apoptosis or differentiate to progenitors (HPCs) that eventually yield specific classes of blood cells. Disruption of these HSC fate decisions is considered to be fundamental to the development of leukemia. Much effort has therefore been placed on understanding the molecular pathways that regulate HSC fate decisions and how these processes are undermined in leukemia. Transcription factors have emerged as critical regulators in this respect. Here we review the participation of zinc finger transcription factor GATA-2 in regulating normal hematopoietic stem and progenitor cell functionality, myelodysplasia and myeloid leukemia.


Assuntos
Fator de Transcrição GATA2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/patologia , Síndromes Mielodisplásicas/patologia , Animais , Diferenciação Celular/genética , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Fator de Transcrição GATA2/genética , Células-Tronco Hematopoéticas/patologia , Humanos , Mutação/genética
16.
Nat Cell Biol ; 14(3): 287-94, 2012 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-22344032

RESUMO

How the molecular programs of differentiated cells develop as cells transit from multipotency through lineage commitment remains unexplored. This reflects the inability to access cells undergoing commitment or located in the immediate vicinity of commitment boundaries. It remains unclear whether commitment constitutes a gradual process, or else represents a discrete transition. Analyses of in vitro self-renewing multipotent systems have revealed cellular heterogeneity with individual cells transiently exhibiting distinct biases for lineage commitment. Such systems can be used to molecularly interrogate early stages of lineage affiliation and infer rules of lineage commitment. In haematopoiesis, population-based studies have indicated that lineage choice is governed by global transcriptional noise, with self-renewing multipotent cells reversibly activating transcriptome-wide lineage-affiliated programs. We examine this hypothesis through functional and molecular analysis of individual blood cells captured from self-renewal cultures, during cytokine-driven differentiation and from primary stem and progenitor bone marrow compartments. We show dissociation between self-renewal potential and transcriptome-wide activation of lineage programs, and instead suggest that multipotent cells experience independent activation of individual regulators resulting in a low probability of transition to the committed state.


Assuntos
Linhagem da Célula/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Multipotentes/metabolismo , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Transformada , Células Cultivadas , Análise por Conglomerados , Citocinas/farmacologia , Células Eritroides/metabolismo , Perfilação da Expressão Gênica/métodos , Immunoblotting , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcriptoma
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