In this project we assessed the mechanisms that are involved in the formation of ionizing radiation (IR) induced CA starting from initial damage up to the final quantification of CA. Initial damage and its processing might be influenced by chromatin structure and indeed, we found that a fraction of IR induced DSB possible relates to free radicals induced DNA lesions in open chromatin and this fraction is repaired very fast. However, in more general terms it appeared that gene density or chromatin conformation has no significant influence on the formation of CA. Therefore we conclude that these factors are not of importance for retrospective biodosimetry.
In this project two novel findings have been made regarding the formation of CA. Firstly, repair of DNA damage generated by 125I decay leads to CA that involved the damaged chromosome as well as undamaged chromosomes. This indicates that CA can be formed by a single DSB e.g. by lesion-nonlesion interactions. Such a mechanism is quite controversial in view of overwhelming evidence that two hits are required to form CA. Secondly, elucidation of the 3-D distribution of chromosomes in interphase nuclei of mammalian cells revealed that the positioning of gene rich and gene poor chromosomes predicts the outcome of CA in X-irradiated lymphocytes. This provides for the first time evidence for the correctness of the proposed nuclear model. Moreover, analysis of X-ray induced CA also showed non-random interactions between other pairs of chromosomes.
Although we found that IR does not induce global changes in nuclear organisation of chromatin, it induces local changes in chromatin involving heterochromatin. The current hypothesis is that this redistribution of hetero-chromatic regions is part of a stress response. Accumulating evidence was generated that radiosensitivity affects the length of the telomeres (ends of chromosomes) and vice versa, e.g. that shortening of telomeres leads to radiosensitivity. This observation was made both in vitro studies as well as in vivo and mechanistic studies revealed that structural chromosomal alterations and repair defects may affect telomeres stability. Experiments with human lymphocytes provided direct evidence that the frequencies and types of CA after radiation are modulated by cell cycle checkpoints and apoptosis.
Our results point to two different mechanisms that might be of direct relevance to genetic effects of IR. Firstly, DSB might be repaired by lesion-nonlesion interactions suggesting that under certain conditions (e.g. possibly depending on the complexity of the damage) a single DSB may give rise to translocations. Secondly, detailed analysis of the formation of CA provided direct experimental evidence that proximity of chromosomes in the interphase nucleus leads to preferential exchanges between these chromosomes. We consider this as an important direction of research for future radiobiology. The relevance of nuclear organisation with respect to cancer was recently demonstrated by Haigis et al (Nature Genetics 33 (2003), 33) showing that changes in spatial organisation of DNA sequences by translocation, affect the frequency of LOH for tumour suppressor genes. It is obvious that this could be an alternative mechanism by which radiation can lead to cancer.
Regarding both the novel findings as well as the number of publications (in total 68), the project has been very successful and this demonstrates that the high-resolution molecular cytogenetics of metaphase chromosomes in concert with interphase cytogenetics provides powerful tools to address key questions in radiation protection also in the future. Concepts and results of this project will be implicated in future research such as the EU sponsored integrated project RISC-RAD.
