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For the understanding of radiation action on biological systems like cellular macromolecules (e.g., DNA in its higher structures) a synergistic approach of experiments and quantitative modelling of working hypotheses is necessary. Further on, the influence on calculated results of certain assumptions in such working hypotheses must critically be evaluated. In the present work, this issue is highlighted in two aspects for the case of DNA damage in single cells. First, yields of double-strand breaks and frequency distributions of DNA fragment lengths after ion irradiation were calculated using different assumptions on the DNA target model. Compared to a former target model now a moderate effect due to the inclusion of a spherical chromatin domain model has been found. Second, the influence of assumptions on particular geometric chromosome models on calculated chromosome aberration data is illustrated with two target-modelling approaches for this end point.

A quantitative computer model was applied to simulate the three-dimensional (3D) spatial organization of chromatin in human cell nuclei under defined conditions of virtual irradiation to explore the implications of spatial organization on chromosome aberrations. To calibrate the virtual irradiation algorithm, a dose-dependent spectrum of radiation-induced chromosome aberrations such as dicentrics, translocations and centric rings was calculated for low-LET radiation doses ranging from 0.5 to 5 Gy. This was compared with the results from experimental studies. While the dose-response curves calculated from model simulations agree well with experimental dose-response curves for dicentrics and translocations, centric rings are significantly more frequent in the model simulation than in experiments despite taking into account exclusive arm territories in the applied Spherical 1 Mbp Chromatin Domain (SCD) computer model explicitly. Taking into account the non-random positioning of chromosome territories observed in lymphocyte cell nuclei (a so-called gene density-correlated arrangement of chromosome territories), aberration frequencies were calculated with the calibrated irradiation algorithm to investigate the impact of chromosome territory neighborhood effects (proximity effects). The absolute frequencies of pairwise exchanges agree well with those found in an experimental study. In conclusion, the results obtained using the computer model approach presented here based on only a few adjustable parameters correlated well with those of experimental studies of chromosome aberration frequencies. Thus the model may be a useful tool in radiation-induced cancer risk estimates in combination with epidemiological studies.

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into 'neighborhoods' comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.

To understand the influence of geometrical constraints in the spatial distribution of cancer correlated "Double Minute chromosomes (DMs)" in human cell nuclei, we applied computer simulations of the nuclear 3D structure in combination with a voxel based segmentation algorithm. With this approach we determined the overlap volumes and intensities of the DMs with the chromatin free space in the simulated nucleus. For this purpose, beginning from a start configuration, simulated linear chromosome chains together with the DMs were relaxed according to the Monte Carlo process. The simulations predict a preferential positioning of DMs within the "peripheral" "Inter Chromatin Domain (ICD)" space.

Various models for the nuclear architecture in interphase cell nuclei have been presented, proposing a territorial or a non-territorial organization of chromosomes. To better understand the correlation between nuclear architecture and the formation of chromosomal aberrations, we applied computer simulations to model the extent of radiation induced chromosome damage under certain geometrical constraints. For this purpose, chromosomes were described by different models, which approximate the chromatin fiber by a polymer chain, folded in different ways. Corresponding to the different condensation levels, a territorial or a non-territorial organization of chromosomes was obtained. To determine the relative frequencies of radiation induced damage, the effects of isotropic ionizing radiation and of a focused laser UV-beam were studied. For isotropic ionizing radiation, the calculated translocation frequencies showed no differences between territorial and non-territorial models except for one special case. For localized irradiation, the results of both organizations were clearly different, with respect to the total number of damaged chromosomes per cell. The predictions agreed well with the experimental data available., (Copyright 1998 Elsevier Science B.V.)

A new fluorescence in situ hybridization (FISH) technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available alpha-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, and microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

Progress in fluorescence in situ hybridization, three dimensional microscopy and image analysis has provided the means to study the three-dimensional structure and distribution of chromosome territories within the cell nucleus. In this contribution, we summarize the present state of knowledge of the territorial organization of interphase chromosomes and their topological relationships with other macromolecular domains in the human cell nucleus, and present data from computer simulations of chromosome territory distributions. On this basis, we discuss models of chromosome territory and nuclear architecture and topological consequences for the formation of chromosome exchanges.

For cytogenetics of pig (Sus scrofa domestica) and the influence of chromosome aberrations on pig production, high interest exists in flow sorted chromosomes for gene mapping, to establish DNA-libraries, or to produce DNA-probes. Flow karyotyping and sorting as well as slit scan flow analysis of metaphase chromosomes of an abnormal cell type carrying a translocation marker chromosome 6/15 are described. Flow sorting of the largest chromosomes of these cells was performed. After sorting the chromosomes still had a well preserved morphology and were identified microscopically by G-banding. The quality of the band pattern of the sorted chromosomes was compatible to that of isolated chromosomes not subjected to flow cytometry. The sorted fraction showed an enrichment of chromosome 6/15 and chromosome 1 which have quantitatively about the same integrated fluorescence intensity. Slit scan flow analysis was performed to discriminate these two chromosomes. Metacentric and submetacentric chromosomes were analyzed according to their bimodal slit scan profiles. Profiles of the largest chromosomes were distinguished by their different centromeric indices. Two groups were interpreted as the normal chromosome 1 and the translocation chromosome 6/15.

The multiprocessor NERV and its application to slit scan flow cytometry is described. Up to 320 processors and 640 MBytes of RAM may be used in one VME crate, providing a computing power of less than or equal to 1300 MIPS. The multiprocessor is controlled by a host computer that provides a friendly user interface and comfortable program development tools. All hardware and software has been tested on a prototype NERV system with 5 processors. For a real-time classification/detection of normal and aberrant chromosomes, the centromeric index or the number of centromeres are computed or specifically labeled DNA sequences are detected. The program is partitioned into 60 tasks that can be executed concurrently. A total analysis time of less than 600 microseconds including system overhead will be achieved according to timing measurements which have been done for all individual tasks.

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Synchronized and asynchronously growing cells of a V79 sub-line of the Chinese hamster were either whole-cell irradiated ( gamma, 254 nm) or laser-UV-microirradiated ( gamma, 257 nm). Post-incubation with caffeine (1-2 mM) often resulted in chromosome shattering, which was a rare event in the absence of this compound. In experiments with caffeine, the following results were obtained. Shattering of all the chromosomes of a cell (generalized chromosome shattering, GCS) was induced by whole-cell irradiation at the first post-irradiation mitosis when the UV fluence exceeded a "threshold" value in the sensitive phases of the cell cycle (G1 and S). GCS was also induced by laser-UV-microirradiation of a small part of the nucleus in G1 or S whereas microirradiation of cytoplasm beside the nucleus was not effective. An upper limit of the UV fluence in the non-irradiated nuclear part due to scattering of the microbeam was experimentally obtained. This UV fluence was significantly below the threshold fluence necessary to induce GCS in whole-cell irradiation experiments. In other cells, partial nuclear irradiation resulted in shattering of a few chromosomes only, while the majority remained intact (partial chromosome shattering, PCS). G1/early S was the most sensitive phase for induction of GCS by whole-cell and partial nuclear irradiation. The frequency of PCS was observed to increase when partial nuclear irradiation was performed either at lower incident doses or at later stages of S. We suggest that PCS and GCS indicate 2 levels of chromosome damage which can be produced by the synergistic action of UV irradiation and caffeine. PCS may be restricted to microirradiated chromatin whereas GCS involves both irradiated and unirradiated chromosomes in the microirradiated nucleus.