RESUMEN
Myc (c-Myc) counteracts p27 effects, and low p27 usually correlates with high Myc expression in human cancer. However there is no information on the co-expression of both genes in chronic lymphocytic leukemia (CLL). We found a lack of correlation between RNA and protein levels of p27 and Myc in CLL cells, so we determined the protein levels by immunoblot in 107 cases of CLL. We observed a high p27 protein expression in CLL compared to normal B cells. Ectopic p27 expression in a CLL-derived cell line resulted in cell death resistance. Surprisingly, Myc expression was very low or undetectable in most CLL cases analyzed, with a clear correlation between high p27 and low Myc protein levels. This was associated with low Skp2 expression, which is consistent with the Skp2 role in p27 degradation and with SKP2 being a Myc target gene. High Myc expression did not correlate with leukemia progression, despite that cell cycle-related Myc target genes were upregulated. However, biochemical analysis showed that the high p27 levels inhibited cyclin-Cdk complexes even in Myc expressing CLL cells. Our data suggest that the combination of high p27 and low Myc is a marker of CLL cells which is mediated by Skp2.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Ciclinas/genética , Ciclinas/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Immunoblotting , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-myb/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas Asociadas a Fase-S/genéticaRESUMEN
It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Mitosis/genética , Fase S/genética , Transcripción Genética , Línea Celular , Análisis por Conglomerados , Biología Computacional/métodos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/química , Ciclinas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células K562 , Queratinocitos/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.
Asunto(s)
Regulación hacia Abajo , Regulación Leucémica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Fase G1/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células K562 , Leucemia/genética , Leucemia/patología , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Elementos de Respuesta/genética , Fase S/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21(Cip1), initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21(Cip1) expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21(Cip1) in the response to imatinib in CML.
