Cytogenetics and cytology of retinoblastomas.

PURPOSE: Chromosomal aberrations and the nuclear topography of retinoblastoma tumour cells as well as lymphocytes of patients suffering from the familiar or sporadic form of retinoblastoma were studied. METHODS: Fluorescence in situ hybridisation (FISH) on fresh, paraffin-embedded tumour tissues and on peripheral blood leukocytes was used for cytogenetic analysis. The cell cycle profile and induction of apoptosis was studied by flow cytometry and gene expression changes were detected by RT-PCR. RESULTS: Using the repeated FISH technique, the average distances between the nuclear membrane and the fluorescence gravity centre (FGC) of seven selected chromosomes were determined in the same tumour population and three other cell types. Chromosome order in positioning from the nuclear membrane was similar in all cell populations investigated. Our experimental studies were focused on specific genetic loci relevant for retinoblastoma tumour pathogenesis. We revealed a certain heterogeneity in the copy number of the Rb1, N-myc, and TP53 gene loci in tumour cells. In addition, in lymphocytes isolated from peripheral blood of the patients, a high degree of copy number heterogeneity was also detected. In 60% of analysed retinoblastomas we observed numerical aberration involving the centromeric region of chromosome 6. In these tumours, apoptotic bodies were found irrespective of clinical therapy. Chromosome instability seems to be a typical feature of primary retinoblastomas as well as of the human pseudodiploid cell line Y79. These cells, of a hereditary form of retinoblastoma (Y79), were irradiated by gamma rays and exposed to anti-tumour drugs such as etoposide, vincristine, and cisplatin. These treatments induced apoptosis, changes in the cell cycle profile, and specific modifications in the nuclear topography of selected loci. Treatment with a non-lethal concentration of hydroxyurea was shown to induce the loss of the amplified N-myc gene involved in the homogenously staining region (HSR) that was found to be associated with the nuclear membrane of retinoblastoma Y79 cells. CONCLUSIONS: We assume that not only cytological and cytogenetic parameters but also aberrant chromatin structures and their nuclear topography can be useful tools for optimal tumour marker specification.

Arrangement of chromosome 11 and 22 territories, EWSR1 and FLI1 genes, and other genetic elements of these chromosomes in human lymphocytes and Ewing sarcoma cells.

Standard and repeated fluorescence in situ hybridization and high-resolution cytometry were used to study topographical parameters of chromosome 11 and 22 territories, EWSR1 and FLI1 genes, and other genetic elements of these chromosomes in human lymphocytes and Ewing sarcoma cells. HSA 11 and its elements (BCL1, FLI1, centromere) were found, on average, more peripherally in comparison with HSA 22 and investigated elements (BCR, EWSR1, centromere). After the elimination of fluctuations of chromosome territories in nuclear volume, it was found that genetic elements in most cases adhered to their territories. The investigated genetic elements of HSA 11 were found close to each other relative to the large molecular lengths among them. This finding indicates a higher degree of chromatin condensation of at least a part of HSA 11 compared with HSA 22. In general, there is no correlation between the physical and molecular distance of two loci of the same chromosome territory. The topographical parameters of the EWSR1 and FLI1 genes do not differ substantially for G(0)-lymphocytes, stimulated lymphocytes and Ewing sarcoma cells. The fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway position between the native EWSR1 and FLI1 genes. Comparing results obtained for the EWSR1/FLI1 and ABL1/BCR genes in samples of patients suffering from Ewing sarcoma or chronic myelogenous leukaemia, it can be concluded that the mean positions of the fusion genes are determined by the final structure of the chimeric chromosomes and do not depend on the location of the translocation event.

3D Structure of the human genome: order in randomness.

A complex study of the spatial arrangement of different genetic elements (genes, centromeres and chromosomal domains) in the cell nucleus is presented and the principles of this arrangement are discussed. We show that the radial location of genetic elements in the three-dimensional (3D) space between the center of the nucleus and the nuclear membrane is element specific and dependent on the position of the element on the chromosome. In contrast, mutual angular positioning of both homologous and heterologous genetic elements is, in the majority of cases, random. In several cases, tethering of heterologous genetic elements was observed. This close proximity of specific loci may be responsible for their mutual rearrangement and the development of cancer. Comparison of our results with transcriptome maps shows that the nuclear location of chromosomal domains with highly expressed genes is more central when compared with chromosomes with low expression. The higher-order chromatin structure is strikingly similar in various human cell types, which correlates with the fact that the profiles of gene expression are also similar.

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.