RESUMO
Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição STAT5/genética , Fatores de Transcrição/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Indóis , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Luciferases/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirróis/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Transformation of hematopoietic cells by the BCR/ABL oncogene is caused by perturbation of signal transduction pathways leading to altered patterns of gene expression and activity. By oligonucleotide microarray hybridization of polysomal RNA of untreated and STI571-treated 32D-BCR/ABL cells, we identified the beta-chemokine CCL9 as a gene regulated by BCR/ABL in a tyrosine kinase-dependent manner. BCR/ABL repressed CCL9 expression at the transcriptional level by mechanisms involving suppression of p38 MAP kinase, and modulation of the activity of CDP/cut and C/EBPalpha, two transcription regulators of myeloid differentiation. However, repression of C/EBP-dependent transcription did not prevent the induction of CCL9 expression by STI571, suggesting that C/EBPalpha is involved in maintaining rather than in inducing CCL9 expression. Restoration of CCL9 expression in 32D-BCR/ABL cells had no effect on the in vitro proliferation of these cells, but reduced their leukemogenic potential in vivo, possibly by recruitment of CD3-positive immune cells. Together, these findings suggest that downregulation of chemokine expression may be involved in BCR/ABL-dependent leukemogenesis by altering the relationship between transformed cells and the microenvironment.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mieloide/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Animais , Benzamidas , Células da Medula Óssea/patologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Testes de Carcinogenicidade , Proliferação de Células , Quimiocinas CC , Regulação para Baixo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesilato de Imatinib , Leucemia Mieloide/patologia , Proteínas Inflamatórias de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The extensive characterization of the replicative human DNA ligase I (LigI) undertaken in the last decade demonstrated that the level of this protein strongly correlates with the rate of cell proliferation. This may allow to expand the repertoire of clinical biomarkers for the analysis of cell proliferation. We have produced a new monoclonal antibody (5H5) against LigI and exploited it as cell proliferation marker in Western blotting and immunofluorescence as well as in immunohistochemistry on paraffin tissue sections. The Western blot analysis showed that the LigI level detected by 5H5 antibody is high in all proliferating cells. On the contrary the protein is down regulated in resting human fibroblast and peripheral blood lymphocytes. Immunofluorescence analysis on cultured HeLa cells showed that 5H5 antibody labels all proliferating cells and displays the same staining pattern of BrdU in S-phase nuclei. Finally the analysis of serial sections of inflamed tonsils and NHL lymph nodes (either frozen or paraffin embedded) demonstrated that 5H5 marks the same population of cells as the Ki-67 antibody. Our results demonstrate that 5H5 antibody is a valuable tool for labeling proliferating cells that can be conveniently used in Western blotting, immunocytochemistry and immunohistochemistry.
