RESUMEN
Deletion of 11q22-q23 is associated with an aggressive course of B-cell chronic lymphocytic leukaemia (B-CLL). Since only in a subset of these cases biallelic inactivation of ATM was observed, we sought to identify other disease-associated genes within 11q22-q23 by analysing NPAT (cell-cycle regulation), CUL5 (ubiquitin-dependent apoptosis regulation) and PPP2R1B (component of the cell-cycle and apoptosis regulating PP2A) for point mutations and their expression in B-CLL by single-strand conformation polymorphism/sequence analysis of the transcripts and real-time polymerase chain reaction. Though none of the genes were affected by deleterious mutations, we observed a significant down-regulation of NPAT in B-CLL versus CD19+ B cells and of CUL5 in 11q deletion versus non-deletion B-CLL samples and measured reduced PPP2R1B transcript levels in a subset of B-CLL cases. Alternative splicing of PPP2R1B transcripts (skipping of exons 2/3, 3, 9) was associated with a reduced activity of protein phosphatase 2A. Together, these results implicate deregulation of the cell-cycle and apoptosis regulators NPAT, CUL5 and PPP2R1B and a role for these genes in the pathogenesis of B-CLL.
Asunto(s)
Proteínas de Ciclo Celular/genética , Deleción Cromosómica , Cromosomas Humanos Par 11/genética , Proteínas Cullin/genética , Leucemia Linfocítica Crónica de Células B/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Bases/genética , Análisis Mutacional de ADN , Regulación hacia Abajo , Humanos , Mutación/genética , Reacción en Cadena de la Polimerasa/métodos , Proteína Fosfatasa 2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Knowledge about the functional impact of the topological organization of DNA sequences within interphase chromosome territories is still sparse. Of the few analyzed single copy genomic DNA sequences, the majority had been found to localize preferentially at the chromosome periphery or to loop out from chromosome territories. By means of dual-color fluorescence in situ hybridization (FISH), immunolabeling, confocal microscopy, and three-dimensional (3D) image analysis, we analyzed the intraterritorial and nuclear localization of 10 genomic fragments of different sequence classes in four different human cell types. The localization of three muscle-specific genes FLNA, NEB, and TTN, the oncogene BCL2, the tumor suppressor gene MADH4, and five putatively nontranscribed genomic sequences was predominantly in the periphery of the respective chromosome territories, independent from transcriptional status and from GC content. In interphase nuclei, the noncoding sequences were only rarely found associated with heterochromatic sites marked by the satellite III DNA D1Z1 or clusters of mammalian heterochromatin proteins (HP1alpha, HP1beta, HP1gamma). However, the nontranscribed sequences were found predominantly at the nuclear periphery or at the nucleoli, whereas genes tended to localize on chromosome surfaces exposed to the nuclear interior.