RESUMO
BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.
Assuntos
Núcleo Celular/química , Proteínas de Ligação a DNA/genética , Genes , Imageamento Tridimensional , Hibridização in Situ Fluorescente/métodos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Linhagem Celular Transformada/química , Linhagem Celular Transformada/ultraestrutura , Linhagem Celular Tumoral/química , Linhagem Celular Tumoral/ultraestrutura , Núcleo Celular/ultraestrutura , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 19/ultraestrutura , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/ultraestrutura , Células HL-60/química , Células HL-60/ultraestrutura , Herpesvirus Humano 4 , Histona-Lisina N-Metiltransferase , Humanos , Interfase , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/patologia , Masculino , Modelos Genéticos , Mieloma Múltiplo/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Fatores de Elongação da Transcrição , Translocação GenéticaRESUMO
BACKGROUND: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved three-dimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function. METHODS: 3D-FISH protocol, confocal microscopy, and digital image analysis were used. RESULTS: New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4',6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of spot loci in the nucleus and inter-spot distance. Results from a pilot experiment using Smart 3D-FISH on ENL, MLL, and AF4 genes in two lymphoblastic cell lines were satisfactory and consistent with data published in the literature. CONCLUSION: Smart 3D-FISH should greatly facilitate image processing and analysis of 3D-FISH images by providing a useful tool to overcome the laborious task of image segmentation based on user-defined parameters and decrease subjectivity in data analysis. It is available as a set of plugins for ImageJ software.
