RESUMEN
BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance.
Asunto(s)
Citometría de Imagen/métodos , Hibridación Fluorescente in Situ , Microscopía Confocal/métodos , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas , Núcleo Celular/química , Núcleo Celular/genética , Computadores , Genes abl , Humanos , Citometría de Imagen/instrumentación , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal/instrumentación , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcr , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
The structural organisation of chromatin in eukaryotes plays an important role in a number of biological processes. Our results provide a comprehensive insight into the nuclear topography of human peripheral blood granulocytes, mainly neutrophils. The nuclei of granulocytes are characterised by a segmented shape consisting of two to five lobes that are in many cases connected by a thin DNA-containing filament. The segregation of chromosomes into the nuclear lobes was studied using fluorescence in situ hybridisation (FISH). We were able to distinguish different topographic types of granulocytes on the basis of the pattern of segregation. Five topographic types were detected using dual-colour FISH in two-lobed nuclei. The segregation of four sets of genetic structures could be studied with the aid of repeated FISH and a large number of topographic types were observed. In all these experiments a non-random distribution of chromosomes into nuclear lobes was found. The painting of a single type of chromosome in two-lobed nuclei showed the prevalence of symmetric topographic types (on average in 65.5% of cases) with significant variations among individual chromosomes. The results of analysis of five topographic types (defined by two chromosomes in two-lobed nuclei) showed that the symmetric topographic types for both chromosomes are significantly more frequent than predicted. Repeated hybridisation experiments confirmed that the occurrence of certain patterns of chromosome segregation is much higher than that predicted from the combination of probabilities. The frequency of symmetric topographic types for chromosome domains was systematically higher than for genes located on these chromosomes. It appears that the prevalence of symmetric segregation patterns is more probable for large objects such as chromosome domains than for genes located on chromatin loops extending outwards from the surface of the domain defined by specific chromosome paints. This means that one chromosome domain may occur in different lobes of granulocytic nuclei. This observation is supported by the fact that both genes and centromeres were observed on filaments joining different lobes. For all chromosomes, the distances between the membrane and fluorescence gravity centre of the chromosome were measured and correlated with the segregation patterns. A higher percentage of symmetric topographic types was found in those chromosomes that were located closer to the nuclear membrane. Nuclear positioning of all genetic elements in granulocytic nuclei was studied in two-dimensional projection; however, the results were verified using three-dimensional analysis.
Asunto(s)
Cromatina/ultraestructura , Granulocitos/ultraestructura , Neutrófilos/ultraestructura , Granulocitos/fisiología , Células HL-60 , Humanos , Hibridación Fluorescente in Situ , Neutrófilos/fisiologíaRESUMEN
PURPOSE: To detect the frequencies of interchanges among 11 chromosomes in lymphocytes irradiated with gamma-rays and to find out whether these frequencies reflect the proximity of some of these chromosomes within the interphase nucleus. MATERIAL AND METHODS: Exchange aberrations were detected in the first mitosis after irradiation of human lymphocytes with 3 and 5 Gy gamma-rays of 60Co. Two-colour repeated FISH with two differently chemically modified probes in each hybridization was applied. The microscope stage positions of each mitosis were recorded after the first hybridization and used for the automatic scanning of images after all successive experiments. Five images were obtained for each mitosis differing in visualized pairs of chromosomes. Comparing these images, exchanges among 10 chromosomes could be detected. Painting of the p arm of chromosome 21 with the painting probe for chromosome 22 also made it possible to detect exchanges of this chromosome with other chromosomes of the selected group. RESULTS: Frequencies of exchange aberrations induced in chromosomes of the selected group as well as interchanges between many pairs of chromosomes of this group were roughly proportional to the DNA content of chromosomes. Higher frequencies of interchanges than expected according to the model of linear proportionality were found between several chromosomes involved in translocations frequent in different subtypes of leukaemia. CONCLUSIONS: Frequencies of interchanges among 11 chromosomes of human lymphocytes induced by gamma-rays do not indicate as clearly as fast neutrons the non-random arrangement of chromosomes in the cell nucleus. The interaction of a large number of chromosomes in exchange aberrations suggests that the chromatin in the territory of one chromosome is accessible for several other chromosomes.
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Aberraciones Cromosómicas , Linfocitos/efectos de la radiación , Células Cultivadas , ADN/análisis , Rayos gamma , Humanos , Linfocitos/ultraestructuraRESUMEN
Fluorescence in situ hybridization (FISH) combined with high-resolution cytometry was used to determine the topographic characteristics of the centromeric heterochromatin (of the chromosomes 6, 8, 9, 17) and the tumor suppressor gene TP53 (which is located on chromosome 17) in cells of the human leukemia cell lines ML-1 and U937. Analysis was performed on cells that were either untreated or irradiated with gamma rays and incubated for different intervals after exposure. Compared to untreated cells, homologous centromeres and the TP53 genes were found closer to each other and also closer to the nuclear center 2 h after irradiation. The spatial relationship between genetic elements returned to that of the unirradiated controls during the next 2-3 h. Statistical evaluation of our experimental results shows that homologous centromeres and the homologous genes are positioned closer to each other 2 h after irradiation because they are localized closer to the center of the nucleus (probably due to more pronounced decondensation of the chromatin related to repair). This radial movement of genetic loci, however, is not connected with repair of DSBs by processes involving homologous recombination, because the angular distribution of homologous sequences remains random after irradiation.
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Núcleo Celular/ultraestructura , Cromosomas Humanos/ultraestructura , Genes/efectos de la radiación , Leucemia/patología , Células Madre Neoplásicas/ultraestructura , Células U937/ultraestructura , Núcleo Celular/química , Núcleo Celular/efectos de la radiación , Centrómero/química , Centrómero/efectos de la radiación , Centrómero/ultraestructura , Cromosomas Humanos/química , Cromosomas Humanos/efectos de la radiación , Daño del ADN , Reparación del ADN , Rayos gamma , Heterocromatina/química , Heterocromatina/efectos de la radiación , Heterocromatina/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Leucemia/genética , Células Madre Neoplásicas/química , Células Madre Neoplásicas/efectos de la radiación , Recombinación Genética , Células U937/química , Células U937/efectos de la radiaciónRESUMEN
Higher-order compartments of nuclear chromatin have been defined according to the replication timing, transcriptional activity, and information content (Ferreira et al. 1997, Sadoni et al. 1999). The results presented in this work contribute to this model of nuclear organization. Using different human blood cells, nuclear positioning of genes, centromeres, and whole chromosomes was investigated. Genes are located mostly in the interior of cell nuclei; centromeres are located near the nuclear periphery in agreement with the definition of the higher-order compartments. Genetic loci are found in specific subregions of cell nuclei which form distinct layers at defined centre-of-nucleus to locus distances. Inside these layers, the genetic loci are distributed randomly. Some chromosomes are polarized with genes located in the inner parts of the nucleus and centromere located on the nuclear periphery; polar organization was not found for some other chromosomes. The internal structure of the higher-order compartments as well as the polar and non-polar organization of chromosomes are basically conserved in different cell types and at various stages of the cell cycle. Some features of the nuclear structure are conserved even in differentiated cells and during cellular repair after irradiation, although shifted positioning of genetic loci was systematically observed during these processes.
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Núcleo Celular/ultraestructura , Linfocitos/ultraestructura , Células de la Médula Ósea/efectos de la radiación , Células de la Médula Ósea/ultraestructura , Compartimento Celular , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Centrómero/efectos de la radiación , Cromosomas Humanos/efectos de la radiación , Genes/efectos de la radiación , Células HL-60 , Humanos , Hibridación Fluorescente in Situ , Interfase , Leucopoyesis , Linfocitos/citología , Linfocitos/efectos de la radiación , Células U937RESUMEN
The c-myc gene plays an essential role in the regulation of the cell cycle and differentiation. Therefore, changes of the c-myc positioning during differentiation are of great interest. As a model system of cell differentiation, the HL-60 and U-937 human leukemic cell lines were used in our experiments. These cells can be induced to differentiation into granulocytes that represent one of the pathways of blood cell maturation. In this study, changes of the topographic characteristics of the c-myc gene (8q24), centromeric region of chromosome 8 and chromosome 8 domain during differentiation of HL-60 and U-937 cells were detected using fluorescence in-situ hybridisation (FISH). FISH techniques and fluorescence microscopy combined with image acquisition and analysis (high-resolution cytometry) were used in order to detect the topographic features of nuclear chromatin. Increased centre of nucleus-to-gene and gene-to-gene distances of c-myc genes, centromeric region of chromosome 8 and chromosome 8 domains were found early after the induction of granulocytic differentiation by dimethyl sulfoxide (DMSO) or retinoic acid (RA); the size of the chromosome 8 domains was rapidly reduced. In differentiated cells, c-myc is located at greater distances from the centromeric regions of chromosome 8. These results support the idea that relocation of the c-myc gene to the nuclear periphery and the condensation of the chromosome 8 domain might be associated with the c-myc gene expression due to common kinetics during granulocytic differentiation.
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Núcleo Celular/metabolismo , Genes myc/genética , Ciclo Celular , Diferenciación Celular , Núcleo Celular/genética , Centrómero/genética , Centrómero/metabolismo , Cromosomas Humanos Par 8/genética , Cromosomas Humanos Par 8/metabolismo , Granulocitos/citología , Granulocitos/metabolismo , Células HL-60 , Humanos , Hibridación Fluorescente in Situ , Leucemia/genética , Leucemia/patología , Antígeno de Macrófago-1/metabolismo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismoRESUMEN
Using single and dual colour fluorescence in situ hybridisation (FISH) combined with image analysis techniques the topographic characteristics of genes and centromeres in nuclei of human colon tissue cells were investigated. The distributions of distances from the centre-of-nucleus to genes (centromeres) and from genes to genes (centromeres to centromeres) were studied in normal colon tissue cells found in the neighbourhood of tumour samples, in tumour cell line HT-29 and in promyelocytic HL-60 cell line for comparison. Our results show that the topography of genetic loci determined in 3D-fixed cell tissue corresponds to that obtained for 2D-fixed cells separated from the tissue. The distributions of the centre-of-nucleus to gene (centromere) distances and gene to gene (centromere to centromere) distances and their average values are different for various genetic loci but similar for normal colon tissue cells, HT-29 colon tumour cell line and HL-60 promyelocytic cell line. It suggests that the arrangement of genetic loci in cell nucleus is conserved in different types of human cells. The investigations of trisomic loci in HT-29 cells revealed that the location of the third genetic element is not different from the location of two homologues in diploid cells. We have shown that the topographic parameters used in our experiments for different genetic elements are not tissue or tumour specific. In order to validate high-resolution cytometry for oncology, further investigations should include more precise parameters reflecting the state of chromatin in the neighbourhood of critical oncogenes or tumour suppresser genes.