Magdalena Skalníková

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.

The topological organization of chromosomes 9 and 22 in cell nuclei has a determinative role in the induction of t(9,22) translocations and in the pathogenesis of t(9,22) leukemias.

Abstract.The neighborhood relationships of chromosomes can be of great importance for basic cellular processes such as gene expression or translocation induction. In this study, the topological organization of chromosomes 9 and 22 was investigated in cell nuclei of G0-phase lymphocytes. We found that the territories of both chromosomes are predominantly located in the central region of cell nuclei. In addition to this, chromosomes 9 and 22 were frequently associated in pairs detected as false-positive ABL-BCR fusions. Both effects might substantially increase the probability of interaction between chromosomes. Because of this, exchange aberrations were studied in chromosomes 9 and 22 of human lymphocytes irradiated by neutrons. The rate of aberration induction between these chromosomes was 11 times higher than the expected frequency based on the fractional molecular weight of these chromosomes. We show that the increased rate of exchange between chromosomes 9 and 22 induced by neutrons corresponds to the neighborhood relationships of both chromosomes. Similar topological characteristics of ABL and BCR genes were found in several cell lines: T- and B-lymphocytes, HL60 cells and bone marrow cells. This finding suggests that the specific chromatin structure mentioned might be responsible for the high rate of induction of t(9;22)-positive leukemias in the human population.

Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases

The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

Nuclear structure and gene activity in human differentiated cells.

The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing.

Spatial arrangement of genes, centromeres and chromosomes in human blood cell nuclei and its changes during the cell cycle, differentiation and after irradiation.

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.

The influence of the cell cycle, differentiation and irradiation on the nuclear location of the abl, bcr and c-myc genes in human leukemic cells

abl and bcr genes play an important role in the diagnostics of chronic myelogenous leukemia (CML). The translocation of these genes results in an abnormal chromosome 22 called the Philadelphia chromosome (Ph). The chimeric bcr-abl gene is a fundamental phenomenon in the pathogenesis of CML. Malignant transformation of hematopoietic cells is also accompanied by the c-myc gene changes (translocation, amplification). Nuclear topology of the abl, bcr and c-myc genes was determined in differentiated as well as in irradiated HL-60 cells using dual-colour fluorescence in situ hybridisation and image analysis by means of a high resolution cytometer. After the induction of the granulocytic differentiation of HL-60 cells with all trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO), the abl and bcr homologous genes were repositioned closer to the nuclear periphery and the average distances between homologous abl-abl and bcr-bcr genes as well as between heterologous abl-bcr genes were elongated as compared with untreated human leukemic promyelocytic HL-60 cells. Elongated gene-to-gene and centre-to-gene distances were also found for the c-myc gene during granulocytic differentiation. In the case of the monocytic maturation of HL-60 cells treated with phorbol esters (PMA), the abl and bcr homologous genes were repositioned closer to each other and closer to the nuclear centre. The position of the c-myc gene did not change significantly after the PMA stimulus. The proximity of the abl and bcr genes was also found after gamma irradiation using 60Co (5 Gy). Immediately after the gamma irradiation c-myc was repositioned closer to the nuclear centre, but 24 h after radiation exposure the c-myc position returned back to the pretreatment level. The c-myc gene topology after gamma irradiation (when the cells are blocked in G2 phase) was different from that detected in the G2 sorted control population. We suggest that changes in the abl, bcr and c-myc topology in the case of gamma irradiation are not the effects of the cell cycle. It is possible, that differences in the cell cycle of hematopoietic cells after the gamma irradiation and concurrent proximity of the abl, bcr and c-myc genes could be important from the point of view of contingent gene translocations, that are responsible for malignant transformation of cells.

Nuclear topography of the c-myc gene in human leukemic cells

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.

Spatial distribution of selected genetic loci in nuclei of human leukemia cells after irradiation.

Abstract Jirsová, P., Kozubek, S., Bártová, E., Kozubek, M., Lukášová, E., Cafourková, A., Koutná, I. and Skalníková, M. Spatial Distribution of Selected Genetic Loci in Nuclei of Human Leukemia Cells after Irradiation. 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 γ 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.

Chromosomes participating in translocations typical of malignant hemoblastoses are also involved in exchange aberrations induced by fast neutrons.

Repeated triple-color fluorescence in situ hybridization was used for the detection of exchange aberrations among 10 selected chromosomes of human lymphocytes irradiated with three doses of fast neutrons with a mean energy of 7 MeV. In each hybridization two different pairs of chromosomes were stained. Defined stage positions of metaphases on a slide were stored on a hard disk and an automatic scan of images according to these positions was performed after six successive hybridizations. In this way we obtained six different images of the same metaphase with differently stained pairs of chromosomes and centromeres. The comparison of these images enabled the identification of mutual exchanges between chromosomes 1, 2, 3, 4, 8, 9, 12, 14, 18 and 22. The frequencies of exchanges were not linearly proportional to the molecular weight of interacting chromosomes. The most significant were exchanges between chromosomes 14/18, 14/8, 18/8, 8/3, 1/14, 1/8, 3/18, 3/14 and 9/22. The results indicate significant interactions between chromosomes involved in translocations in B-cell non-Hodgkins lymphoma and chronic myeloid leukemia. We propose that the reason for the high frequency of exchanges between these chromosomes is their proximity in the cell nucleus. It may also be one of the reasons for the induction of specific translocations leading to malignant transformation of cells.

Exchange aberrations among 11 chromosomes of human lymphocytes induced by γ-rays

Purpose : To detect the frequencies of interchanges among 11 chromosomes in lymphocytes irradiated with γ-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 γ-rays of 60 Co. 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 γ-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.