RESUMEN
The c-Myb gene encodes the p75(c-Myb) isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p(c-Mybex9b), which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein-protein interactions and negative regulation. In hematopoietic cells, expression of p(c-Mybex9b) accounts for 10-15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p(c-Mybex9b) and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75(c-Myb), p(c-Mybex9b) is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p(c-Mybex9b) enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p(c-Mybex9b) reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34(+) cells, without affecting the levels of p75(c-Myb). Together, these studies indicate that expression of the low-abundance p(c-Mybex9b) isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells.
RESUMEN
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.
Asunto(s)
Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factor de Transcripción STAT5/genética , Factores de Transcripción/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Inmunoprecipitación de Cromatina , Ensayo de Unidades Formadoras de Colonias , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Indoles , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Luciferasas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirroles/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
The c-myb gene encodes a transcription factor required for proliferation, differentiation and survival of normal and leukemic hematopoietic cells. c-Myb has a longer half-life in BCR/ABL-expressing than in normal cells, a feature which depends, in part, on PI-3K/Akt-dependent regulation of proteins interacting with the leucine zipper/negative regulatory region of c-Myb. Thus, we asked whether the stability of c-Myb in leukemic cells might be enhanced by mutations interfering with its degradation. We analyzed the c-myb gene in 133 chronic myeloid leukemia (CML) patients in chronic phase and/or blast crisis by denaturing-high performance liquid chromatography (D-HPLC) and sequence analysis of PCR products corresponding to the entire coding sequence and each exon-intron boundary. No mutations were found. We found four single nucleotide polymorphisms (SNPs) and identified an alternatively spliced transcript lacking exon 5, but SNPs frequency and expression of the alternatively spliced transcript were identical in normal and CML cells. Thus, the enhanced stability of c-Myb in CML blast crisis cells and perhaps in other types of leukemia is not caused by a genetic mechanism.