Investigation of Spatial Organization of Chromosome Territories in Chromosome Exchange Aberrations After Ionizing Radiation Exposure.

Higher-order organization of the human genome is well established with chromosomes occupying distinct domains or territories in the interphase nucleus. Spatial organization of chromosome territories in the interphase nucleus occurs in a cell-type-specific manner. Since both stable and unstable aberrations induced by ionizing radiation involve the exchange of material between two or more chromosomes, this study investigated the role of spatial organization of chromosome domains in ionizing-radiation-induced chromosome translocation events. Using multicolor fluorescence in situ hybridization, the study characterized the positioning of each human chromosome relative to its neighborhood territories in the interphase nucleus of lymphocytes and B-lymphoblastoid cells before ionizing radiation and compared this interphase positioning with the spectrum of exchanges observed after ionizing radiation in the metaphase chromosomes. In addition to multicolor fluorescence in situ hybridization, the genome-wide chromosome conformation capture technique (Hi-C) was also performed in mock and x-ray-irradiated human B-lymphoblastoid and fibroblast cells to characterize the interactions among chromosomes and to assess the genome reorganization changes, if any, after ionizing radiation exposure. On average, 35-50% of the total translocations induced by x rays and neutrons correlated with proximity of chromosome territories detected by multicolor fluorescence in situ hybridization in both lymphocytes and lymphoblastoid cells. The translocation rate observed in proximally positioned chromosome territories was consistently higher than distally located territories and was found to be statistically significant (p = 0.01) in human lymphoblastoid cells after x rays. The interchromosome interaction frequencies detected by Hi-C correlate fairly well with ionizing-radiation-induced translocations detected by multicolor fluorescence in situ hybridization, suggesting the importance of chromosome proximity effects in ionizing-radiation-induced chromosomal translocation events.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells.

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.

Volume increase and spatial shifts of chromosome territories in nuclei of radiation-induced polyploidizing tumour cells.

The exposure of tumour cells to high doses of ionizing radiation can induce endopolyploidization as an escape route from cell death. This strategy generally results in mitotic catastrophe during the first few days after irradiation. However, some cells escape mitotic catastrophe, polyploidize and attempt to undergo genome reduction and de-polyploidization in order to create new, viable para-diploid tumour cell sub-clones. In search for the consequences of ionizing radiation induced endopolyploidization, genome and chromosome architecture in nuclei of polyploid tumour cells, and sub-nuclei after division of bi- or multi-nucleated cells were investigated during 7 days following irradiation. Polyploidization was induced in p53-function deficient HeLa cells by exposure to 10Gy of X-irradiation. Chromosome territories #1, #4, #12 and centromeres of chromosomes #6, #10, #X were labelled by FISH and analysed for chromosome numbers, volumes and spatial distribution during 7 days post irradiation. The numbers of interphase chromosome territories or centromeres, respectively, the positions of the most peripherally and centrally located chromosome territories, and the territory volumes were compared to non-irradiated controls over this time course. Nuclei with three copies of several chromosomes (#1, #6, #10, #12, #X) were found in the irradiated as well as non-irradiated specimens. From day 2 to day 5 post irradiation, chromosome territories (#1, #4, #12) shifted towards the nuclear periphery and their volumes increased 16- to 25-fold. Consequently, chromosome territories returned towards the nuclear centre during day 6 and 7 post irradiation. In comparison to non-irradiated cells (∼500µm(3)), the nuclear volume of irradiated cells was increased 8-fold (to ∼4000µm(3)) at day 7 post irradiation. Additionally, smaller cell nuclei with an average volume of about ∼255µm(3) were detected on day 7. The data suggest a radiation-induced generation of large intra-nuclear chromosome territories and their repositioning prior to genome reduction.