DNA double-strand breaks (DSBs) have been recognized as the most serious lesions in irradiated cells. While several biochemical pathways capable of repairing these lesions have been identified, the mechanisms by which cells select a specific pathway for activation at a given DSB site remain poorly understood. Our knowledge of DSB induction and repair has increased dramatically since the discovery of ionizing radiation-induced foci (IRIFs), initiating the possibility of spatiotemporally monitoring the assembly and disassembly of repair complexes in single cells. IRIF exploration revealed that all post-irradiation processes-DSB formation, repair and misrepair-are strongly dependent on the characteristics of DSB damage and the microarchitecture of the whole affected chromatin domain in addition to the cell status. The microscale features of IRIFs, such as their morphology, mobility, spatiotemporal distribution, and persistence kinetics, have been linked to repair mechanisms. However, the influence of various biochemical and structural factors and their specific combinations on IRIF architecture remains unknown, as does the hierarchy of these factors in the decision-making process for a particular repair mechanism at each individual DSB site. New insights into the relationship between the physical properties of the incident radiation, chromatin architecture, IRIF architecture, and DSB repair mechanisms and repair efficiency are expected from recent developments in optical superresolution microscopy (nanoscopy) techniques that have shifted our ability to analyze chromatin and IRIF architectures towards the nanoscale. In the present review, we discuss this relationship, attempt to correlate still rather isolated nanoscale studies with already better-understood aspects of DSB repair at the microscale, and consider whether newly emerging "correlated multiscale structuromics" can revolutionarily enhance our knowledge in this field.

• Keywords
• DNA damage and repair, DNA double-strand breaks (DSBs), DSB repair pathway choice and hierarchy, chromatin architecture, ionizing radiation, ionizing radiation-induced foci (IRIFs), linear energy transfer (LET), single-molecule localization microscopy (SMLM), superresolution microscopy,
• Publication type
• Journal Article MeSH
• Review MeSH

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.

• MeSH
• Apoptosis MeSH
• In Situ Hybridization, Fluorescence MeSH
• Humans MeSH
• Retinal Neoplasms genetics   pathology   MeSH
• Ploidies MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Flow Cytometry MeSH
• Retinoblastoma genetics   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Cell Nucleus genetics   radiation effects   ultrastructure   MeSH
• Cell Cycle MeSH
• Bone Marrow Cells radiation effects   ultrastructure   MeSH
• Centromere radiation effects   MeSH
• Genes radiation effects   MeSH
• HL-60 Cells MeSH
• In Situ Hybridization, Fluorescence MeSH
• Interphase MeSH
• Cell Compartmentation MeSH
• Leukopoiesis MeSH
• Humans MeSH
• Chromosomes, Human radiation effects   MeSH
• Lymphocytes cytology   radiation effects   ultrastructure   MeSH
• U937 Cells MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH