RESUMO
BACKGROUND: Despite the discovery of the p.V617F in JAK2, the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617FJAK2-negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes (CBL, CBLB and CBLC) in a selected group of patients with myeloproliferative neoplasms. We also included V617FJAK2-positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. DESIGN AND METHODS: Using denaturing high performance liquid chromatography, we screened for mutations in CBL, CBLB and CBLC in a group of 172 V617FJAK2-negative and 232 V617FJAK2-positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. RESULTS: An initial screening of all coding exons of CBL, CBLB and CBLC in 44 V617FJAK2-negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617FJAK2-negative and 232 V617FJAK2-positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617FJAK2-positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. CONCLUSIONS: Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617FJAK2-positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.
Assuntos
Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sequência Conservada , Éxons , Proteínas de Fusão bcr-abl/deficiência , Proteínas de Fusão bcr-abl/genética , Expressão Gênica , Ordem dos Genes , Humanos , Interleucina-3/farmacologia , Janus Quinase 2/metabolismo , Camundongos , Dados de Sequência Molecular , Transtornos Mieloproliferativos/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismoAssuntos
Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Crônica/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Idade de Início , Dioxigenases , Frequência do Gene , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mielomonocítica Crônica/diagnóstico , Leucemia Mielomonocítica Crônica/epidemiologia , Mutação/fisiologia , Síndromes Mielodisplásicas/epidemiologia , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/epidemiologia , Transtornos Mieloproliferativos/genéticaRESUMO
A search for genes potentially regulated by STAT5 identified leukemia inhibitory factor (LIF) as a good candidate. Using various experimental approaches, we have validated LIF as a direct transcriptional target of STAT5 in myeloid cell lines: STAT5 binds to LIF promoter, and LIF expression is increased after activation of the JAK2/STAT5 pathway. We also found that LIF expression is significantly increased in patients with chronic myeloproliferative neoplasms with and without activating mutations of the pathway, indicating that LIF might play an important role in STAT5-mediated oncogenesis.
RESUMO
Hematological malignancies with eosinophilia are often associated with fusions in PDGFRA, PDGFRB, or FGFR1 genes. RT-PCR has proved to be useful for finding new PDGFRA gene fusions, but some studies have shown overexpression of the TK domain which cannot be explained by the existence of such aberrations. This fact could be related to the expression of alternative PDGFRA transcripts. We show that quantification of the expression of three different PDGFRA fragments discriminates between PDGFRA alternative transcripts and fusion genes, and we have tested this novel methodological approach in a group of eosinophilia cases. Our data show that alternative PDGFRA transcripts should be taken into account when screening for PDGFRA aberrations, such as gene fusions, by RT-PCR. Expression from an internal PDGFRA promoter seems to be a frequent event, in both normal and leukemic samples, and is probably related to physiological conditions, but it could have a role in other tumors. Even so, we show that our RQ-PCR methodology can discriminate expression of alternative transcripts from the presence of X-PDGFRA fusion genes.
Assuntos
Processamento Alternativo/genética , Eosinofilia/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/genética , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Eosinofilia/etiologia , Eosinofilia/patologia , Feminino , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/patologia , Humanos , Hibridização in Situ Fluorescente , Masculino , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BCR/ABL1-negative chronic myeloproliferative neoplasms (CMPNs) are a heterogeneous group of clonal hematological malignancies. Over recent years, some genetic events in tyrosine kinase (TK) genes have been described as causal events of these diseases. To identify new genetic aberrations underlying these diseases, we used denaturing high performance liquid chromatography and fluorescence in situ hybridization (FISH) to analyze 17 genes from two receptor-TK families (III and IV) and from three cytoplasmic-TK families (Syk, Abl, and Jak) on samples from 44 BCR/ABL1-negative and JAK2(V617F)-negative CMPN patients with different clinical phenotypes. Although screening by FISH did not reveal novel chromosomal aberrations, several sequence changes were detected. None of them were frequent events, but we identified a new potential activating mutation in the FERM domain of JAK2(R340Q). None of the germline JAK2(V617F) single-nucleotide polymorphisms detected differed in distribution between patients and control subjects. In summary, data presented here show that these genes are not frequently mutated or rearranged in CMPNs, suggesting that molecular events causing these disorders must be located in other genes.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Janus Quinase 2/química , Janus Quinase 2/genética , Mutação/genética , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Oncogenes/genética , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Doença Crônica , Éxons/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de ProteínaAssuntos
Metilação de DNA , Neoplasias Hematológicas/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Ilhas de CpG , Proteínas de Fusão bcr-abl/genética , Humanos , Janus Quinase 2/genética , Células Mieloides/patologia , Mutação Puntual , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de CitocinasRESUMO
Hepatitis C virus (HCV) is a highly prevalent virus and one of the major agents of chronic hepatitis. Since HCV NS3 protease is essential for the processing of HCV polyprotein, this protease is a target of choice to control HCV replication. Peptide inhibitors of NS3 were developed by selective amino acid replacement of six leader sequences, corresponding to regions of HCV polyprotein that are cleaved by NS3. The large numbers of potential 14-mer and 16-mer peptide inhibitors thus obtained were tested against NS3 using the fluorescent probe RETS1 and peptide cofactor SVVIVGRIILSGRA from NS4A protein. This afforded several peptide inhibitors with an IC50 of around 2 microM. These peptides may be good leading compounds for the development of peptidomimetics to control HCV replication in the treatment of chronic hepatitis C.
Assuntos
Oligopeptídeos/química , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Hepatite C/tratamento farmacológico , Humanos , Hidrólise , Concentração Inibidora 50 , Cinética , Mimetismo Molecular , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
Different mechanisms, such as chromosomal rearrangements, deletions, mutations, and methylation/demethylation of the promoter regions of genes, have been shown to be involved in acute lymphoblastic leukaemia (ALL). These genetic and epigenetic alterations lead to the activation of protooncogenes or to inactivation of tumour suppressor genes promoting cell proliferation. One of the most frequently inactivated tumour suppressor genes is TP53, which is altered in 50% of human tumours. In this study, we have analysed: (1) the complete coding region, all intron-exon junctions and noncoding regions of exons 1-11 of TP53 by lexon-DGGE; (2) the methylation status of the 5' region of TP53 and (3) the deletion of one or both alleles of the gene by fluorescence in situ hybridisation (FISH) in 57 ALL patients. Using these techniques, we have found promoter methylation in 32% of the cases, missense mutations in 8.8%, and deletion of one allele in 7.5% of the samples, with TP53 being altered in 40% of the ALL samples studied in this series.
Assuntos
Metilação de DNA , Deleção de Genes , Genes p53/fisiologia , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiões Promotoras Genéticas/genética , Ilhas de CpG , DNA de Neoplasias/genética , Éxons , Regulação Leucêmica da Expressão Gênica/genética , Inativação Gênica , Humanos , Hibridização in Situ Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMO
It has been shown that methylation of CpG dinucleotides located in the promoter region of TP53 is associated with low expression levels of this gene. We have analysed the methylation status of one CpG dinucleotide and of three CCWGG motifs, also located in the promoter region of the gene, in bone marrow samples obtained from patients with acute lymphoblastic leukemia (ALL). Eight out of 25 samples analysed showed methylation of either the CpG dinucleotide, the CCWGG motifs or both. Relative to nonmethylated leukemia samples, TP53 expression levels were decreased in all methylated samples in which TP53 expression could be measured. Methylation of CpG and CCWGG motifs in the promoter of TP53 could represent a novel mechanism leading to functional impairment of this tumor suppressor gene in ALL.