RESUMO
The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.
Assuntos
Antígenos CD34/imunologia , Proteínas de Ligação a DNA/genética , Terapia Genética/métodos , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia/genética , Leucemia/terapia , Metaloproteínas/genética , Receptores de Interleucina-2/genética , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/imunologia , Substâncias de Crescimento/farmacologia , Humanos , Proteínas com Domínio LIM , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/terapia , Mutagênese Insercional , Proteínas Proto-Oncogênicas , RetroviridaeRESUMO
Many acute lymphoblastic leukemias can be considered as malignant counterparts of cells in the various stages of normal lymphoid development in bone marrow and thymus. T-cell development in the thymus is an ordered and tightly controlled process. Two evolutionary conserved signaling pathways, which were first discovered in Drosophila, control the earliest steps of T-cell development. These are the Notch and Wnt-signaling routes, which both are deregulated in several types of leukemias. In this review we discuss both pathways, with respect to their signaling mechanisms, functions during T-cell development and their roles in development of leukemias, especially T-cell acute lymphoblastic leukemia.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Proteínas Wnt/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores Notch/imunologia , Linfócitos T/patologia , Proteínas Wnt/imunologiaRESUMO
Deregulated Notch signaling occurs in the majority of human T-ALL. During normal lymphoid development, activation of the Notch signaling pathway poses a T-cell fate on hematopoietic progenitors. However, the transcriptional targets of the Notch pathway are largely unknown. We sought to identify Notch target genes by inducing Notch signaling in human hematopoietic progenitors using two different methods: an intracellular signal through transfection of activated Notch and a Notch-receptor dependent signal by interaction with its ligand Delta1. Gene expression profiles were generated and evaluated with respect to expression profiles of immature thymic subpopulations. We confirmed HES1, NOTCH1 and NRARP as Notch target genes, but other reported Notch targets, including the genes for Deltex1, pre-T-cell receptor alpha and E2A, were not found to be differentially expressed. Remarkably, no induction of T-cell receptor gene rearrangements or transcription of known T-cell specific genes was found after activation of the Notch pathway. A number of novel Notch target genes, including the transcription factor TCFL5 and the HOXA cluster, were identified and functionally tested. Apparently, Notch signaling is essential to open the T-cell pathway, but does not initiate the T-cell program itself.
Assuntos
Linhagem da Célula/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Receptor Notch1/metabolismo , Linfócitos T/fisiologia , Animais , Animais não Endogâmicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fosfoproteínas/genética , Proteínas/genética , Proteínas/metabolismo , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Estromais , Linfócitos T/citologia , Fatores de Transcrição HES-1 , Fatores de Transcrição , Transfecção , Triglicerídeos/farmacologia , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/farmacologiaRESUMO
Interpretation of laboratory parameters in cases of haemochromatosis can be difficult. Here, we describe two patients with markedly elevated transferrin saturation and high ferritin levels. The first patient is a 51-year-old woman who had been complaining of fatigue, abdominal pain and arthritis for three years. Her liver enzymes were mildly elevated. Secondary causes of iron overload had been excluded. DNA investigation found a homozygous p.Cys282Tyr mutation in the HFE protein, consistent with hereditary haemochromatosis. The second patient is a 58-year-old man with general malaise and cholestatic liver injury. The p.Cys282Tyr and p.His63Asp mutations in the HFE protein could not be detected. Ultrasound of the liver revealed steatosis. The patient was a heavy drinker and a diagnosis of iron overload caused by alcoholic liver disease was made. Based on these case reports, we discuss the strategy to diagnose haemochromatosis and the background of associated laboratory tests.
Assuntos
Hemocromatose/diagnóstico , Sobrecarga de Ferro/diagnóstico , Hepatopatias Alcoólicas/complicações , Feminino , Hemocromatose/etiologia , Hemocromatose/genética , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/genética , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , MutaçãoRESUMO
Class I homeobox (HOX) genes comprise a large family of transcription factors that have been implicated in normal and malignant hematopoiesis. However, data on their expression or function during T-cell development is limited. Using degenerated RT-PCR and Affymetrix microarray analysis, we analyzed the expression pattern of this gene family in human multipotent stem cells from fetal liver (FL) and adult bone marrow (ABM), and in T-cell progenitors from child thymus. We show that FL and ABM stem cells are similar in terms of HOX gene expression, but significant differences were observed between these two cell types and child thymocytes. As the most immature thymocytes are derived from immigrated FL and ABM stem cells, this indicates a drastic change in HOX gene expression upon entry into the thymus. Further analysis of HOX-A7, HOX-A9, HOX-A10, and HOX-A11 expression with specific RT-PCR in all thymocyte differentiation stages showed a sequential loss of 3' region HOX-A cluster genes during intrathymic T-cell development and an unexpected expression of HOX-A11, previously not recognized to play a role in hematopoiesis. Also HOX-B3 and HOX-C4 were expressed throughout thymocyte development. Overall, these data provide novel evidence for an important role of certain HOX genes in human T-cell development.
Assuntos
Expressão Gênica , Genes Homeobox/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Multipotentes/metabolismo , Células-Tronco/metabolismo , Linfócitos T/metabolismo , Adulto , Diferenciação Celular/genética , Linhagem da Célula/genética , Criança , Primers do DNA/química , Feto , Perfilação da Expressão Gênica , Humanos , Fígado/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/citologiaRESUMO
INTRODUCTION: Microscopic differential analysis of leukocytes is a time-consuming activity for routine diagnostic laboratories. The criteria used to decide whether a manual differential should be performed should therefore be as strict as possible. The goal of this investigation was to give recommendations for the use of the left shift (LS) 1+ flag, which signals the presence of band neutrophils. METHODS: The LS1+ flag of the ADVIA 120 and 2120 hematology analyzers was evaluated in 6 peripheral hospital laboratories in the Netherlands. In 2683 samples with exclusively a LS1+ flag, the percentage of band neutrophils were determined microscopically. A set of photographs of neutrophils were used to examine the differences between laboratories in the assessment of band cells. RESULTS: In 18% of all samples with only a LS1+ flag, 5% or more band neutrophils were found. However, this percentage differed greatly between laboratories, as did the proportion of samples that received a LS1+ flag. Several factors were found to influence the amount and accuracy of the LS1+ alarm, i.e. band neutrophil counting by microscopists, specificity of request for leukocyte differentials, percentage of general practitioners requesting a leukocyte differential, and sample storage. Based on these findings, a number of recommendations were formulated. CONCLUSION: Critical control of the factors influencing the LS1+ flag can significantly decrease the number of microscopic samples to be reviewed and may be valuable for every laboratory performing routine differentials, using any type of hematology analyzer.
Assuntos
Técnicas de Laboratório Clínico/instrumentação , Testes Hematológicos/instrumentação , Laboratórios Hospitalares , Neutrófilos/citologia , Automação Laboratorial/instrumentação , Automação Laboratorial/métodos , Técnicas de Laboratório Clínico/métodos , Testes Hematológicos/métodos , Humanos , Contagem de Leucócitos/instrumentação , Contagem de Leucócitos/métodos , Reprodutibilidade dos TestesRESUMO
BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within approximately 4 h, and can be run in parallel to routine immunophenotyping.
Assuntos
Citometria de Fluxo/métodos , Proteínas de Fusão bcr-abl/análise , Imunoensaio/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Anticorpos Monoclonais , Citometria de Fluxo/normas , Humanos , Imunoensaio/normas , Reação em Cadeia da Polimerase , Inibidores de Proteases , Sensibilidade e EspecificidadeRESUMO
Initiation of gene transcription by transcription factors (TFs) is an important regulatory step in many developmental processes. The differentiation of T cell progenitors in the thymus is tightly controlled by signaling molecules, ultimately activating nuclear TFs that regulate the expression of T lineage-specific genes. During the last 2 years, significant progress has been made in our understanding of the signaling routes and TFs operating during the earliest stages of thymic differentiation at the CD4(-)CD8(-) double negative stage. Here we will review the TF families that play an important role in differentiation of thymocytes, particularly focusing on recent new information with respect to the Tcf, bHLH, GATA, and CBF/HES TF families.