RESUMO
Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal B cells arrested in G0/G1 stages that coexist, in different proportions, with proliferative B cells. Understanding the crosstalk between the proliferative subsets and their milieu could provide clues on CLL biology. We previously identified one of these subpopulations in the peripheral blood from unmutated patients that appears to be a hallmark of a progressive disease. Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mRNA and microRNA expression of this leukemic subpopulation, and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microRNA-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that miR-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-Kip1) and overexpression of Survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors.
Assuntos
Linfócitos B/citologia , Proliferação de Células , Leucemia Linfocítica Crônica de Células B/patologia , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Enzimática , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/metabolismo , TranscriptomaRESUMO
Despite significant progress in treatment, chronic lymphocytic leukemia (CLL) remains an incurable disease. Advances have been made to understand the molecular pathogenesis underlying CLL progression and treatment resistance. We here review the available evidences concerning the role of the B-cell receptor (BCR) and the tumor microenvironment interactions in CLL pathogenesis. Antigen likely has a key role in the selection of the tumoral clone, the mutational status of immunoglobulin genes is a strong prognostic predictor and BCR signaling has been postulated to have a role for CLL trafficking and interaction with the stromal microenvironment. There is also important evidence, favoring a role for the microenvironment in CLL pathogenesis. Most, if not all, proliferative events occur in the lymph nodes and bone marrow, where leukemic cells receive through microenvironment interactions survival signals aiming to avoid apoptosis and acquire favorable tumoral growing conditions. In addition, the tumoral microenvironment appears to be the site where the acquisition of additional genetic lesions in the clone occur, which should greatly influence clinical outcome. The advent of new tyrosine kinase inhibitors which seem to be able to modulate microenvironment interactions and circumvent the p53 deletion have generated significant promise by raising the possibility that they could provide significant progress in disease treatment.
RESUMO
Chronic lymphocytic leukaemia is the commonest form of leukaemia in Europe and North America, and mainly, though not exclusively, affects older individuals. It has a very variable course, with survival ranging from months to decades. Major progress has been made in identification of molecular and cellular markers that could predict disease progression in patients with chronic lymphocytic leukaemia. In particular, the mutational profile of immunoglobulin genes and some cytogenetic abnormalities are important predictors of prognosis. However, these advances have raised new questions about the biology, prognosis, and management of chronic lymphocytic leukaemia, some of which are addressed here. In particular, we discuss how better understanding of the function of the B-cell receptor, the nature of genetic lesions, and the balance between proliferation and apoptosis have affected our ability to assess prognosis and to manage chronic lymphocytic leukaemia. Available treatments generally induce remission, although nearly all patients relapse, and chronic lymphocytic leukaemia remains an incurable disease. Advances in molecular biology have enhanced our understanding of the pathophysiology of the disease and, together with development of new therapeutic agents, have made management of chronic lymphocytic leukaemia more rational and more effective than previously. Unfortunately, we know of no way that chronic lymphocytic leukaemia can be prevented. Early detection is practised widely, but seemingly makes no difference to the patient's eventual outcome.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/efeitos adversos , Biomarcadores Tumorais/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Masculino , Prognóstico , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
MicroRNAs (miRNAs) are a novel class of small noncoding RNA molecules that regulate gene expression by inducing degradation or translational inhibition of target mRNAs. There are more than 500 miRNA genes reported in the human genome, constituting one of the largest classes of regulatory genes. Increasing experimental evidence supports the idea of aberrant miRNA expression in cancer pathogenesis. We analyzed the pattern of miRNA expression in chronic lymphocytic leukemia (CLL) cells and our results showed a global reduction in miRNA expression levels in CLL cells associated to a consistent underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are differentially expressed between patients with different clinical outcomes. Five novel miRNA candidates cloned from leukemic cells are reported. Surprisingly, predicted mRNA targets for these novel miRNA revealed a high proportion of targets located in a small region of chromosome 1, which is frequently altered in human cancer. Additionally, several targets were shared by at least two of miRNA candidates. Predicted targets included several genes recently described as tumor suppressors. These data could afford new avenues for exploring innovative pathways in CLL biology and therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Leucemia Linfocítica Crônica de Células B/etiologia , MicroRNAs/fisiologia , Regulação para CimaRESUMO
Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/enzimologia , Óxido Nítrico Sintase/genética , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Vidarabina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Antineoplásicos/farmacologia , Apoptose/fisiologia , Linfócitos B/enzimologia , Sequência de Bases , Caspase 3 , Caspases/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Feminino , Humanos , Interleucina-4/farmacologia , Isoenzimas , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Vidarabina/farmacologiaRESUMO
Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.
Assuntos
Apoptose/efeitos dos fármacos , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Nitratos/farmacologia , Linfócitos T/imunologia , Tirosina/metabolismo , Apoptose/imunologia , Complexo CD3/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Células Cultivadas , Relação Dose-Resposta Imunológica , Humanos , Linfonodos/imunologia , Linfonodos/patologia , Metaloporfirinas/farmacologia , Monócitos/fisiologia , Nitratos/antagonistas & inibidores , Nitratos/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Superóxido Dismutase/farmacologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Fatores de Tempo , Tirosina/análogos & derivadosAssuntos
Leucemia Linfocítica Crônica de Células B , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Transplante de Medula Óssea , Aberrações Cromossômicas , Ensaios Clínicos como Assunto , Diagnóstico Diferencial , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/terapia , Estadiamento de Neoplasias , Oncogenes , Prognóstico , Análise de SobrevidaRESUMO
A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease.