RESUMO
Dendritic cells (DCs) perform a basic role in the immune system by allowing the initiation of the primary T-cell-dependent immune response. Given previous indirect evidence that DC maturation and function are impaired by HIV, we have developed an in vitro culture system in order to verify the effect of HIV infection on DC function during the development from hematopoietic progenitors. Considering that monocytic (Mo) differentiating cells efficiently replicate monocytotropic HIV, we examined whether HIV-infected monocytic precursors (MoP) were able to generate functional DCs. CD34+ hematopoietic progenitor cells (HPCs) were induced along Mo differentiative pathway in liquid cultures and at an early stage of culture, MoP were infected with M-tropic BaL HIV strain, and after 2 days they were switched to DC differentiation with GM-CSF and IL-4. Derived DCs were actively infected, as detected by HIV-p24 production. HIV did not significantly affect cell viability, but induced a reduction in cell proliferation and an inefficient functional activity in terms of uptake capability and stimulation of allogenic T cells. These results indicate that HIV-infected MoP lost the capacity to generate functional DCs, and this may represent one of the many mechanisms of immunosuppression exploited by HIV.
Assuntos
Antígenos CD34/metabolismo , Diferenciação Celular , Células Dendríticas/virologia , HIV-1/patogenicidade , Células-Tronco Hematopoéticas/virologia , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hospedeiro Imunocomprometido , Interleucina-4/metabolismo , Linfócitos T/imunologia , Fatores de TempoRESUMO
Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.
Assuntos
Megacariócitos/citologia , Megacariócitos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Técnicas In Vitro , Masculino , Megacariócitos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Trombopoese/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
We have developed a new culture system whereby human hematopoietic progenitors purified from adult peripheral blood extensively proliferate and gradually differentiate into >95% pure monocytic (Mo) cells. At all developmental stages treatment with interleukin (IL)-4+granulocyte-macrophage colony-stimulating factor or IL-4+c-Kit-ligand+FLT-3 ligand switched the Mo precursors into dendritic cells (DCs). The switching capacity decreased only at the end of the culture, when most Mo cells matured to macrophages. Moreover, the Mo precursors were highly susceptible to transduction with lentiviral vectors: once switched to DCs, they maintained the transgene expression, as well as the phenotype and function of the DC lineage. Our results provide new insight into the potential role of the Mo lineage as a reservoir of DCs in vivo. Furthermore, the methodology for transduction of Mo precursors provides a tool to generate genetically modified, normally functioning DCs potentially useful for immunotherapy.
Assuntos
Citocinas/farmacologia , Células Dendríticas/citologia , Células-Tronco Hematopoéticas/citologia , Monócitos/citologia , Mielopoese/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/fisiologia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Imunoterapia , Interleucina-4/farmacologia , Lentivirus/genética , Proteínas de Membrana/farmacologia , Monócitos/química , Monócitos/efeitos dos fármacos , Fenótipo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/análise , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Fator de Células-Tronco/farmacologia , Transdução Genética , TransgenesRESUMO
Ets-1 is a widely expressed transcription factor implicated in development, tumorigenesis and hematopoiesis. We analyzed Ets-1 gene expression during human erythroid and megakaryocytic (MK) differentiation in unilineage cultures of CD34+ progenitor cells. During erythroid maturation, Ets-1 is downmodulated and exported from the nucleus into the cytoplasm through an active mechanism mediated by a leucine-rich nuclear export signal. In contrast, during megakaryocytopoiesis Ets-1 increases and remains localized in the nucleus up to terminal maturation. Overexpression of Ets-1 in erythroid cells blocks maturation at the polychromatophilic stage, increases GATA-2 and decreases both GATA-1 and erythropoietin receptor expression. Conversely, Ets-1 overexpressing megakaryocytes are characterized by enhanced differentiation and maturation, coupled with upmodulation of GATA-2 and megakaryocyte-specific genes. We show that Ets-1 binds to and activates the GATA-2 promoter, in vitro and in vivo, indicating that one of the pathways through which Ets-1 blocks erythroid and promotes MK differentiation is via upmodulation of GATA-2 expression.
Assuntos
Diferenciação Celular/fisiologia , Células Eritroides/citologia , Megacariócitos/citologia , Proteína Proto-Oncogênica c-ets-1/fisiologia , Transporte Ativo do Núcleo Celular , Adulto , Animais , Antígenos CD34/metabolismo , Western Blotting , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Megacariócitos/metabolismo , Camundongos , Microscopia Confocal , Sinais de Exportação Nuclear/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Homeobox genes encode transcription factors known to be important morphogenic regulators during embryonic development. An increasing body of work implies a role for homeobox genes in both hematopoiesis and leukemogenesis. In the present study we have analyzed the role of the homeobox gene, HOXB6, in the program of differentiation of the myeloid cell lines, NB4 and HL60. HOXB6 expression is transiently induced during normal granulocytopoiesis and monocytopoiesis, with an initial induction during the early phases of differentiation, followed by a blockade of expression at early maturation. The enforced expression of HOXB6 in promyelocytic NB4 cells or in myeloblastic HL60 cells elicited inhibition of the granulocytic or monocytic maturation, respectively. Furthermore, HOXB6 was frequently expressed (18 out of 49 cases) in AMLs lacking major translocations while it was expressed at very low frequency (two out of 47 cases) in AMLs characterized by PML/RAR-alpha, AML-1/ETO, CBFbeta/MYH11 fusion and rearrangements of the MLL gene at 11q23. According to these observations, we suggest that a regulated pattern of HOXB6 expression is required for normal granulopoiesis and monocytopoiesis. Abnormalities of the HOXB6 expression may contribute to the development of the leukemic phenotype.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Leucemia Mieloide/genética , Doença Aguda , Regulação Neoplásica da Expressão Gênica , Granulócitos/patologia , Células HL-60 , Humanos , Leucemia Mieloide/patologia , Leucopoese/genética , Monócitos/patologiaRESUMO
The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RAR alpha fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RAR alpha mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RAR alpha alters the early hematopoietic program through N-CoR-dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells. (Blood. 2000;96:1531-1537)
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Leucopoese/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Doença Aguda , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Leucemia Mieloide/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Fusão Oncogênica/biossínteseRESUMO
We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.