RESUMO
Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop.
Assuntos
Comunicação Autócrina , Linfoma de Células T Periférico/patologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT5/fisiologiaRESUMO
AIMS: Neurogenesis in adult humans occurs in at least two areas of the brain, the subventricular zone of the telencephalon and the subgranular layer of the dentate gyrus in the hippocampal formation. We studied dentate gyrus subgranular layer neurogenesis in patients subjected to tailored antero-mesial temporal resection including amygdalohippocampectomy due to pharmacoresistant temporal lobe epilepsy (TLE) using the in vitro neurosphere assay. METHODS: Sixteen patients were enrolled in the study; mesial temporal sclerosis (MTS) was present in eight patients. Neurogenesis was investigated by ex vivo neurosphere expansion in the presence of mitogens (epidermal growth factor + basic fibroblast growth factor) and spontaneous differentiation after mitogen withdrawal. Growth factor synthesis was investigated by qRT-PCR in neurospheres. RESULTS: We demonstrate that in vitro proliferation of cells derived from dentate gyrus of TLE patients is dependent on disease duration. Moreover, the presence of MTS impairs proliferation. As long as in vitro proliferation occurs, neurogenesis is maintained, and cells expressing a mature neurone phenotype (TuJ1, MAP2, GAD) are spontaneously formed after mitogen withdrawal. Finally, formed neurospheres express mRNAs encoding for growth (vascular endothelial growth factor) as well as neurotrophic factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor). CONCLUSION: We demonstrated that residual neurogenesis in the subgranular layer of the dentate gyrus in TLE is dependent on diseases duration and absent in MTS.
Assuntos
Giro Denteado/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Neurogênese/fisiologia , Neurônios/citologia , Adolescente , Adulto , Proliferação de Células , Giro Denteado/patologia , Epilepsia do Lobo Temporal/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclerose/patologiaRESUMO
Dental pulp is a heterogeneous microenviroment where unipotent progenitor and pluripotent mesenchymal stem cells cohabit. In this study we investigated whether human dental pulp stromal (stem) cells (DP-SCs) committed to the angiogenic fate. DP-SCs showed the specific mesenchymal immunophenotypical profile positive for CD29, CD44, CD73, CD105, CD166 and negative for CD14, CD34, CD45, in accordance with that reported for bone marrow-derived SCs. The Oct-4 expression in DP-SCs, evaluated through RT-PCR analysis, increased in relation with the number of the passages in cell culture and decreased after angiogenic induction. In agreement with their multipotency, DP-SCs differentiated toward osteogenic and adipogenic commitments. In angiogenic experiments, differentiation of DP-SCs, through vascular endothelial growth factor (VEGF) induction, was evaluated by in vitro matrigel assay and by cytometric analysis. Accordingly, endothelial-specific markers like Flt-1 and KDR were basally expressed and they increased after exposure to VEGF together with the occurrence of ICAM-1 and von Willebrand factor positive cells. In addition, VEGF-induced DP-SCs maintained endothelial cell-like features when cultured in a 3-D fibrin mesh, displaying focal organization into capillary-like structures. The DP-SC angiogenic potential may prove a remarkable tool for novel approaches to developing tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.
Assuntos
Células-Tronco Adultas/fisiologia , Polpa Dentária/fisiologia , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica , Células Estromais/fisiologia , Engenharia Tecidual , Adulto , Células-Tronco Adultas/imunologia , Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/imunologia , Polpa Dentária/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Fibrina/metabolismo , Citometria de Fluxo , Humanos , Imunofenotipagem , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: It has been suggested that soluble factors produced by bone marrow (BM) mesenchymal stromal cells (MSC) play a fundamental role in mediating immune modulation. HLA-G antigens (Ag) are major histocompatibility complex (MHC) class Ib molecules characterized by a limited polymorphism and a splicing mechanism that regulates the production of membrane-bound and soluble isoforms. Interleukin-10 (IL-10) cytokine is one of the main up-modulators of soluble HLA-G Ag (sHLA-G) production by CD14+ peripheral blood monocyte cells and increased IL-10 levels are reported to be associated with MSC immune modulation. METHODS: We investigated, by specific enzyme-linked immunosorbent assay (ELISA), the possible role of sHLA-G molecules in the inhibition of the peripheral blood mononuclear cell (PBMC) response to phytohemagglutinin (PHA) mediated by MSC from different sources. RESULTS: There was a significant correlation between the presence of increased levels of sHLA-G and IL-10 in the MSC/PBMC/PHA culture supernatants and lymphoproliferative inhibition. Neutralizing experiments performed with monoclonal Ab directed against HLA-G and IL-10 molecules confirmed the inhibitory ability of sHLA-G Ag. Furthermore, exogenous IL-10 induced sHLA-G molecule secretion by MSC alone in a polymorphic way, while a longitudinal analysis confirmed the loss of MSC inhibitory functions in relation to in vitro MSC aging. DISCUSSION: Overall the results obtained suggest a functional role for sHLA-G molecules in inhibiting the PBMC response mediated by MSC. Moreover, the ability of IL-10 to induce sHLA-G Ag production by MSC alone could be proposed as a marker of MSC functional ability.
Assuntos
Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunidade/fisiologia , Mesoderma/citologia , Células Estromais/imunologia , Adulto , Animais , Anticorpos/imunologia , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Antígenos HLA-G , Humanos , Imunofenotipagem , Interleucina-10/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Pessoa de Meia-Idade , Células Estromais/citologiaRESUMO
Hyaluronan-based scaffold were used for in vitro commitment of human and rat bone marrow mesenchymal stem cells (MSC). Cells were cultured either in monolayer and in 3D conditions up to 35 days. In order to monitor the differentiating processes molecular biology and morphological studies were performed at different time points. All the reported data supported the evidence that both human and rat MSC grown onto hyaluronan-derived three-dimensional scaffold were able to acquire a unique phenotype of chondrocytes and osteocytes depending on the presence of specific differentiation inducing factors added into the culture medium without significative differences in term of time expression of extracellular matrix proteins.
Assuntos
Células-Tronco Adultas/citologia , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Células da Medula Óssea , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/fisiologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese , Osteonectina/genética , Osteonectina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Release of vascular endothelial growth factor (VEGF) and other candidate angiogenic factors such as basic fibroblast growth factor and transforming growth factor beta, may play a role in sustaining neoplastic cell proliferation and tumor growth. We evaluated VEGF expression and synthesis in the two erythromegakaryocytic cell lines B1647, HEL and one megakaryocytic cell line MO7 expressing erythroid markers. In this study RT-PCR was performed to evaluate VEGF expression and that of its receptor KDR; VEGF production was assayed by Elisa test and western blot analysis; sensitivity to VEGF was tested by thymidine incorporation. VEGF and its receptor KDR were expressed in B1647 and HEL, both as mRNAs and as proteins, while only KDR transcript was found in MO7 cells. Only B1647 and HEL cells showed a strong spontaneous proliferating activity. In fact, measurable amounts of VEGF were present in the unstimulated cell medium, thus suggesting an autocrine production of VEGF by B1647 and HEL cells, but not by MO7, which was inhibited in mRNA-silencing conditions. This production could not be further boosted by other growth factors, whereas it was inhibited by TGF-beta1. Finally, analysis of Shc signal transduction proteins following stimulation with VEGF indicated that only p46 was tyrosine phosphorylated. These data indicate that leukemic cells may be capable of autocrine production of VEGF which, in turn, maintains cell proliferation, possibly mediated by Shc p46 phosphorylation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Megacariócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Megacariócitos/efeitos dos fármacos , Fosforilação , Testes de Precipitina , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Tirosina/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The present study was undertaken to: (i) compare the effect of some hematopoietic growth factors, like interleukine-3, thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, stem cell factor, and reactive oxygen species such as H(2)O(2) on glucose uptake in a human leukemic megakaryocytic cell line, M07; (ii) investigate the changes in kinetic parameters of the transport activity induced by these stimuli; and (iii) evaluate the effect of genistein, a tyrosine kinase inhibitor, on the glucose uptake activation by the cited agents. The results are as follows: (i) exposure of M07 cells to thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, and stem cell factor resulted in a rapid stimulation of glucose transport; interleukine-3-treated cells exhibited no increase in the rate of glucose uptake, although M07 proliferation is interleukine-3 dependent; a rapid glucose transport enhancement was also observed when M07 cells were exposed to low doses of H(2)O(2); (ii) the transport kinetic parameters point out that an important difference exists between the effect of cytokines and that of H(2)O(2): cytokines increased predominantly the affinity for glucose, while H(2)O(2) raised both the V(max) and K(m) values; (iii) the isoflavone genistein, at a very low concentration, inhibited the stem cell factor- or H(2)O(2)-induced stimulation of hexose transport, reversing the variations of K(m) and V(max), but it did not affect the transport activity of granulocyte-megakaryocyte colony-stimulating factor-treated cells; and (iv) catalase completely abolished the stimulatory action of H(2)O(2) on glucose transport and slightly prevented the effect of stem cell factor, while caffeic acid phenethyl ester was only able to affect the activation due to stem cell factor.
Assuntos
Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Glucose/agonistas , Glucose/farmacocinética , Substâncias de Crescimento/metabolismo , Peróxido de Hidrogênio/metabolismo , Álcool Feniletílico/análogos & derivados , Ácidos Cafeicos/farmacologia , Catalase/metabolismo , Catalase/farmacologia , Desoxiglucose/agonistas , Desoxiglucose/farmacocinética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Interleucina-3/metabolismo , Interleucina-3/farmacologia , Cinética , Leucemia Megacarioblástica Aguda/metabolismo , Álcool Feniletílico/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologia , Trombopoetina/metabolismo , Trombopoetina/farmacologia , Células Tumorais Cultivadas/metabolismoRESUMO
Over 200 adenomatous polyposis coli (APC) gene mutations have been described in familial adenomatous polyposis (FAP) patients. Recent single-strand conformation polymorphism (SSCP) screening methods have introduced minigel runs, simple ethidium bromide staining and external temperature control without any loss of sensitivity (cold-SSCP). In order to test the effectiveness in APC mutation detection, cold-SSCP was employed following polymerase chain reaction (PCR) amplification in three patients with FAP. Different running parameter combinations were compared. The three mutations already known were all diagnosed by cold-SSCP. The gel concentration was found to be essential in detecting the single-base substitution in fragments of different lengths. The observation of deletions was not affected by gel concentrations and heteroduplex bands were always produced. The temperature or glycerol addition did not significantly affect sensitivity. This modified cold-SSCP method provides a simple and effective way for detecting several known Apc gene mutations without any loss of sensitivity and could be useful for large-scale molecular diagnosis of FAP.
Assuntos
Análise Mutacional de DNA/métodos , Polimorfismo Conformacional de Fita Simples , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/genética , Temperatura Baixa , DNA/química , DNA/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Mutação , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , TemperaturaRESUMO
B19 parvovirus can replicate in erythroid progenitor cells and in a small number of human blast cell lines. To better understand and analyze the B19 virus replicative cycle, we performed and compared the infection of bone marrow cells and of different blast cell lines with erythroblastoid and megakaryoblastoid phenotypic characteristics (UT-7, TF-1, M-07, and B1647). Following in vitro infection, B19-specific nucleic acids were characterized with regard to the genome-replicative intermediates, the transcription pattern, and the localization of virus-specific nucleic acids inside infected cells. While all cell lines tested proved to be susceptible to B19 virus infection, two different patterns of restriction to replication of B19 virus were observed. In the first restriction pattern, observed in UT-7 cells, the single-stranded viral DNA was converted to double-stranded replicative intermediates, identical to those found in bone marrow cells, and a full set of viral transcripts were observed. However, replication and transcription were restricted to a small subset of cells, and production of capsid proteins was not detected. In the second restriction pattern, observed in TF-1, M-07, and B1647 cells, the single-stranded viral DNA was not converted to double-stranded replicative intermediates.
Assuntos
Células da Medula Óssea/virologia , Células Precursoras Eritroides/virologia , Megacariócitos/virologia , Parvovirus B19 Humano/fisiologia , Replicação Viral , Células da Medula Óssea/citologia , Linhagem Celular , Células Cultivadas , DNA Viral/genética , Células Precursoras Eritroides/citologia , Genoma Viral , Humanos , Hibridização In Situ , Megacariócitos/citologia , Parvovirus B19 Humano/genética , RNA Viral/genética , Transcrição GênicaRESUMO
Ewing's sarcoma is a primitive highly malignant tumor of bone and soft tissues usually metastasizing to bone, bone marrow, and lung. Growth factor receptors and their ligands may be involved in its growth and dissemination. We analyzed the expression of c-kit and its ligand stem cell factor (SCF) in a panel of six Ewing's sarcoma cell lines. All cell lines exhibited substantial levels of surface c-kit expression, and five of six displayed transmembrane SCF on the cell surface. Expression of c-kit was down-modulated in all lines by exposure to exogenous SCF. The SCF treatment was able to confer to cells a growth advantage in vitro, due both to an increase in cell proliferation and to a reduction in the apoptotic rate. When used in the lower compartment of a migration chamber, SCF acted as a strong chemoattractant for Ewing's sarcoma cells. The pretreatment of cells with SCF reduced their chemotactic response to SCF. In athymic nude mice, Ewing's sarcoma cells injected intravenously metastasized to the lung and to a variety of extrapulmonary sites, including bone and bone marrow. Metastatic sites resembled those observed in Ewing's sarcoma patients and corresponded to SCF-rich microenvironments. The in vitro pretreatment of cells with SCF strongly reduced the metastatic ability of Ewing's sarcoma cells, both to the lung and to extrapulmonary sites. This could be dependent on the down-modulation of c-kit expression observed in SCF-pretreated cells, leading to a reduced sensitivity to the chemotactic and proliferative actions of SCF. Our results indicate that the response to SCF mediated by c-kit may be involved in growth, migration, and metastatic ability of Ewing's sarcoma cells.
Assuntos
Neoplasias Ósseas/patologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Sarcoma de Ewing/secundário , Fator de Células-Tronco/fisiologia , Animais , Neoplasias Ósseas/secundário , Feminino , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Transplante de Neoplasias , Sarcoma de Ewing/patologia , Neoplasias da Coluna Vertebral/secundário , Fator de Células-Tronco/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Diamond-Blackfan anemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation and physical malformations. Most cases are sporadic, but dominant or, more rarely, recessive inheritance is observed in 10% of patients. Mutations in the gene encoding ribosomal protein (RP) S19 have recently been found in 25% of patients with either the dominant or the sporadic form. DBA is the first human disease due to mutations in a ribosomal structural protein. Families unlinked to this locus have also been reported. In an investigation of 23 individuals, we identified eight different mutations in 9 patients. These include five missense, one frameshift, one splice site defect, and one 4-bp insertion in the regulatory sequence. Seven mutations are new; one has so far been found in 8 patients and is a relatively common de novo event. Two mutations are predicted to generate a truncated protein. We also report the prevalence of RPS 19 mutations in the Italian DBA population, as shown by an analysis of 56 patients. No genotype-phenotype correlation was found between patients with the same mutation. The main clinical applications for molecular analysis are clinical diagnosis of patients with an incomplete form of DBA and testing of siblings of a patient with a severe form so as to avoid using those who carry a mutation and a silent phenotype as allogeneic stem cell donors.
Assuntos
Anemia de Fanconi/genética , Proteínas Ribossômicas/genética , Adulto , Substituição de Aminoácidos , Sequência de Bases , Criança , Estudos de Coortes , DNA/química , DNA/genética , Análise Mutacional de DNA , Anemia de Fanconi/patologia , Feminino , Heterogeneidade Genética , Genótipo , Humanos , Itália , Masculino , Mutagênese Insercional , Mutação , Fenótipo , Mutação Puntual , Deleção de SequênciaRESUMO
Congenital amegakaryocytic thrombocytopenia (CAMT) without physical anomalies is a rare disease, presenting isolated thrombocytopenia and megakaryocytopenia in infancy, which can evolve into aplastic anemia and leukemia. Recently, two heterozygous truncating mutations of the thrombopoietin (TPO) receptor MPL, coded by the c-mpl gene, were identified in a 10-year-old Japanese patient with CAMT transmitted in an autosomal recessive manner. Here, we report for the first time two different MPL amino-acid substitutions in a 2-year-old Italian boy with CAMT and compound heterozygosis for two (c-mpl point mutations. C-to-T transitions were detected on exons 5 and 12 at the 769 and 1904 cDNA nucleotide positions, respectively. The mutation in exon 5 substitutes an arginine with a cysteine (R257C) in the extracellular domain, 11 amino acids distant from the WSXWS motif conserved in the cytokine-receptor superfamily. The mutation in exon 12 substitutes a proline with a leucine (P635L) in the last amino acid of the C-terminal intracellular domain, responsible for signal transduction. As in the Japanese family, the mutations were both transmitted from the parents. TPO plasma levels were highly increased in the patient. The patient's 7-year-old brother, who was a candidate donor for allografting, turned out to be an asymptomatic heterozygous carrier of P635L and showed defective megakaryocyte colony formation from bone-marrow progenitor cells. The present study provides important confirmation that CAMT can be associated with (c-mpl) mutations.
Assuntos
Megacariócitos , Proteínas de Neoplasias , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Receptores de Citocinas , Trombocitopenia/congênito , Sequência de Aminoácidos , Células da Medula Óssea , Pré-Escolar , Feminino , Heterozigoto , Humanos , Itália , Masculino , Dados de Sequência Molecular , Linhagem , Receptores de Trombopoetina , Homologia de Sequência de AminoácidosRESUMO
Hereditary thrombocytopenias represent heterogeneous clinical and genetic syndromes. They include a consistent group of families which are considered as a separate clinical entity, characterized by autosomal dominant transmission, incomplete penetrance in females, chronic thrombocytopenia with early age of onset and frequently increased platelet volume, without any other hematologic abnormality. The molecular defect in these families is still unknown. We describe 2 families in 3 generations (10 patients), and report the first study of the TPO/c-mpl system in autosomal dominant thrombocytopenia. We performed mutational screening of c-mpl coding, flanking introns and promoter regions in 2 probands from the two families by DNA sequencing. The results do not provide evidence of c-mpl sequence alterations in either of the 2 families investigated. Moreover, the normal TPO serum levels detected in 5 patients from each family leads us to exclude the possibility of a defect in TPO production in our families. Finally, the involvement of both c-mpl and TPO genes in the pathogenesis of thrombocytopenia in these two families was excluded by negative results of linkage analysis.
Assuntos
Plaquetas/citologia , Proteínas de Neoplasias , Receptores de Citocinas , Trombocitopenia/etiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise Mutacional de DNA , Saúde da Família , Feminino , Frequência do Gene , Ligação Genética , Testes Genéticos , Hematopoese , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Linhagem , Mutação Puntual , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Trombopoetina , Trombocitopenia/genética , Trombopoetina/sangue , Trombopoetina/genética , Regiões não TraduzidasRESUMO
In vitro hemopoiesis and hemopoietic cytokines production were evaluated in 9 centenarians (median age 100.5 years, age range: 100-104 years), 10 old people (median age: 71 years, age range: 66-73 years), and 10 young people (median age: 35 years, age range: 30-45 years), all carefully selected for their healthy status. The main findings were the following: (i) a trend towards a decreased absolute number of CD34+ progenitor cells in the peripheral blood of old people and centenarians, in comparison to young subjects; (ii) a well-preserved capability of CD34+ cells from old people and centenarians to respond to hemopoietic cytokines, and to form erythroid (BFU-E), granulocyte-macrophagic (CFU-GM), and mixed colonies (CFU-GEMM) in a way (number, size, and morphology) indistinguishable from that of young subjects; (iii) an age-related decreased in vitro production of granulocyte-macrophagic colony-stimulating factor (GM-CSF) and a decreased production of interleukin-3 (IL-3) in centenarians by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC); (iv) a linear increase of the serum level of stem cell factor (SCF), measured in the above-mentioned subjects and in 65 additional subjects, including 4 centenarians. These data suggest that basal hematopoietic potential is well preserved in healthy centenarians, and that the hemopoietic cytokine network undergoes a complex remodeling with age.
Assuntos
Envelhecimento/fisiologia , Citocinas/biossíntese , Hematopoese/fisiologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/sangue , Envelhecimento/metabolismo , Antígenos CD34/análise , Contagem de Células , Tamanho Celular , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Nível de Saúde , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-3/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Pessoa de Meia-Idade , Fito-Hemaglutininas/farmacologia , Fator de Células-Tronco/sangueRESUMO
The hemopoietin stem cell factor (SCF) and its receptor c-kit are expressed in some tumoral cells, including neuroblastoma (NB) cells. We have investigated the effect of retinoic acid (RA), one of the most active differentiating agents on human NB cells, on the SCF production by human neuroblastoma cell lines. SCF concentration was determined by immunoenzymatic assay in the supernatants of seven neuroblastoma cell lines. All cell lines except one showed detectable amounts of SCF in the supernatant in basal culture conditions. A progressive increase pattern of the SCF concentration over time, was common to all SCF secreting cell lines, both unstimulated and RA-stimulated. Moreover, after 48 and 72 hours-exposure to RA, SCF concentrations were higher than in the untreated controls (p < 0.01). Membrane SCF mRNA isoform was also detected by reverse-transcription polymerase chain reaction. These effects demonstrated that RA, besides inducing neuronal differentiation, enhanced SCF production in neuroblastoma cell lines.
Assuntos
Antineoplásicos/farmacologia , Ceratolíticos/farmacologia , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Tretinoína/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Fatores de Tempo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Inherited thrombocytopenias are a heterogenous group of disorders. Different criteria have been suggested to classify the forms, such as the inheritance mechanism and the platelet volume as well as the number and morphology of megakaryocytes. However, the classification is often descriptive, and the precise mechanism of thrombocytopenia still remains unknown. We describe the clinical, biologic, and molecular findings of an autosomal dominant thrombocytopenia in a large family. The 17 patients had normocellular bone marrow and normal platelet volume. Platelets also showed a normal aggregation test and normal response to ADP and thrombopoietin (TPO). In the affected subjects, the mean +/- SD levels of platelet count and plasma TPO were 62+/-25 and 258+/-151, respectively. Comparative analysis showed that the patients with platelet count <70000 had higher plasma TPO concentration. The data are consistent with a mild clinical form of the disease associated with only a few episodes of bleeding. To exclude the possible role of TPO and its receptor c-mpl in the etiology of this condition, linkage analysis was performed using microsatellite markers close to the TPO and c-mpl genes on chromosomes 3q26.3-q27 and 1p34, respectively. The absence of cosegregation within the affected family indicated that these genes, as well as two other candidate loci on chromosomes 11 and 21, are not responsible for this hereditary dominant form of thrombocytopenia. A genome-wide search and subsequent identification of the gene will provide new insight into the pathogenesis of this disorder.
Assuntos
Plaquetas/patologia , Células da Medula Óssea/fisiologia , Genes Dominantes , Proteínas de Neoplasias , Receptores de Citocinas , Trombocitopenia/genética , Tamanho Celular , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 21 , Ligação Genética , Humanos , Linhagem , Testes de Função Plaquetária , Proteínas Proto-Oncogênicas/genética , Receptores de Trombopoetina , Trombopoetina/genéticaRESUMO
The c-mpl ligand, thrombopoietin (TPO), is a physiologic regulator of platelet and megakaryocytic production, acting synergistically on thrombopoiesis with the growth factors interleukin 11 (IL-11), stem cell factor, interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte-macrophage colony-stimulating factor. Because some of these growth factors, especially TPO and IL-11, are now being evaluated clinically to reduce chemotherapy-associated thrombocytopenia in cancer patients, we evaluated 25 acute myeloid leukemia (AML) samples to test whether TPO, IL-11, and other early-acting megakaryocyte growth factors can affect leukemic cell proliferation, cell cycle activation, and programmed cell death (PCD) protection. TPO induced proliferation in the majority of AML samples from an overall mean proportion of S-phase cells of 7.8% +/-1.5% to 14.5% +/- 2.1% (p = 0.0006). Concurrent G0 cell depletion was found in 47.3% of AML samples. TPO-supported leukemic cell precursor (CFU-L) proliferation was reported in 5 of 17 (29.4%) of the samples with a mean colony number of 21.4 +/- 9.6 x 10(5) cells plated. In 13 of 19 samples, a significant protection from PCD (from an overall mean value of 13% +/-0.7% to 8.8% +/- 1.8%;p = 0.05) was detected after TPO exposure. Conversely, IL-11-induced cell cycle changes (recruitment from G0 to S phase) were detected in only 2 of 14 samples (14.2%). In addition, IL-11 showed little, if any, effect on CFU-L growth (mean colony number = 17.5 9.5) or apoptosis. Combination of TPO with IL-11 resulted in only a slight increase in the number of CFU-L, whereas IL-3 and stem cell factor significantly raised the mean colony numbers up to 119.2 +/- 68.3 and 52.9 +/- 22.1 x 10(5) cells plated, respectively. We conclude that TPO induces cell cycle activation in a significant proportion of cases and generally protects the majority of AML blast cells from PCD. On the other hand, IL-11 has little effect on the cell cycle or PCD. Combination of both TPO and IL-11 is rarely synergistic in stimulating AML clonogenic growth. These findings may be useful for designing clinical studies aimed at reducing chemotherapy-associated thrombocytopenia in AML patients.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Interleucina-11/farmacologia , Leucemia Mieloide/patologia , Trombopoetina/farmacologia , Doença Aguda , Interações Medicamentosas , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaio Tumoral de Célula-TroncoRESUMO
The hematopoietic defect of Diamond-Blackfan anemia (DBA) results in selective failure of erythropoiesis. Thus far, it is not known whether this defect originates from an intrinsic impediment of hematopoietic progenitors to move forward along the erythroid pathway or to the impaired capacity of the bone marrow (BM) microenvironment to support proliferation and differentiation of hematopoietic cells. Reduced longevity of long-term bone marrow cultures, the most physiologic in vitro system to study the interactions of hematopoietic progenitors and hematopoietic microenvironment, is consistent with a defect of an early hematopoietic progenitor in DBA. However, stromal adherent layers from DBA patients generated in a long-term culture system, the in vitro counterpart of BM microenvironment, did not show evidence of any morphologic, phenotypic, or functional abnormality. Our major finding was an impaired capacity of enriched CD34+ BM cell fraction from DBA patients, cultured in the presence of normal BM stromal cells, to proliferate and differentiate along the erythroid pathway. A similar impairment was observed in some DBA patients along the granulomacrophage pathway. Our result points to an intrinsic defect of a hematopoietic progenitor with bilineage potential that is earlier than previously suspected as a relevant pathogenetic mechanism of the disease. The finding of impaired granulopoiesis in some DBA patients underlines the heterogeneity of this rare disorder.
Assuntos
Células da Medula Óssea/patologia , Anemia de Fanconi/patologia , Granulócitos/patologia , Células-Tronco Hematopoéticas/patologia , Macrófagos/patologia , Adolescente , Adulto , Antígenos CD34/análise , Células da Medula Óssea/imunologia , Células da Medula Óssea/fisiologia , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Criança , Pré-Escolar , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Feminino , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Lactente , Masculino , RNA Mensageiro/biossíntese , Células Estromais/fisiologia , Fatores de TempoRESUMO
Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c-mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c-mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.
Assuntos
Mutação , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/genética , Rádio (Anatomia)/anormalidades , Receptores de Citocinas , Trombocitopenia/congênito , Trombocitopenia/genética , Adolescente , Criança , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase , Receptores de Trombopoetina , SíndromeRESUMO
Human rhabdomyosarcoma cells produce autocrine and paracrine growth factors that can sustain their growth and malignancy. Here we report constitutive production of stem cell factor (SCF) by 5 of 5 human rhabdomyosarcoma cell lines both of alveolar and embryonal histotype. SCF production, ranging from 30 to 162 pg/ml, was independent from the degree of myogenic differentiation and was not modulated by exogenous addition of retinoic acid (RA) or tumor necrosis factor-alpha (TNF-alpha). Four of 5 rhabdomyosarcoma cell lines expressed the mRNA for SCF receptor c-kit, while the 5th cell line became weakly positive for c-kit mRNA only after stimulation with retinoic acid. On the cell surface, c-kit protein was detectable at very low levels in only 1 of 5 rhabdomyosarcoma cell lines and was not up-regulated by RA or TNF-alpha. Addition of anti-c-kit and anti-SCF blocking antibodies, or of exogenous SCF did not alter the in vitro growth ability of rhabdomyosarcoma cells. In conclusion, our data show that rhabdomyosarcoma cells produce consistent amounts of SCF but did not demonstrate autocrine growth modulation. SCF secretion may thus have a paracrine, rather than an autocrine activity in this tumor.