Induction and repair of DNA double-strand breaks in human cells exposed to different radiation qualities

Sammanfattning: Radiation therapy is used in attempting to kill tumor cells by inducing DNA double-strand breaks (DSB), the most critical DNA lesions. Recent and planned radiation therapy strategies use high-linear energy transfer (LET) radiation to effectively treat malignant tumors. The aim of this thesis was to investigate the mechanisms of DNA DSB induction and repair after exposure to ionizing radiation of different ionization densities, with focus on the role of DNA damage clustering and chromatin structure, as well as the activation of repair-related proteins at clustered damage sites in human cells. DSB induction was assessed after exposure to accelerated ions of different ionization densities with LET ranging from 40 eV/nm to 300 eV/nm. Exposure of human cells to high-LET radiation resulted in clustering of DSB, leading to an excess of small DNA fragments (< 1 Mbp), as measured by pulsed-field gel electrophoresis (PFGE). Importantly, it was found that DNA organization into chromatin fibers and higher-order structures is responsible for most of DSB clustering induced by high-LET radiation. A newly developed cold lysis protocol for preparation of genomic DNA, which avoids release of heat-labile sites (HLS) into DSB at elevated temperatures, was used to accurately measure the DSB number. DSB yields and relative biological effectiveness (RBE) values were strongly influenced by chromatin compactness. Furthermore, the presence of HLS had a substantial impact on DSB induction yields and DSB rejoining rates measured by PFGE. The lesions involved in the release of HLS into DSB were repaired independent of DNA-PKcs, XRCC1 or PARP-1. Moreover, cells with defect or inhibited function of DNA-PKcs did not show any fast rejoining of DSB when HLS were eliminated. The substructure and spatial dynamics of DNA damage and repair along high-LET particle tracks was monitored by immunofluorescence. Interestingly, high-LET ion track irradiation revealed one of the earliest responses to ionizing radiation, ATM phosphorylation at Ser1981, as pATM foci that clearly correlated with gamma-H2AX foci within particle tracks, as well as punctuated/diffuse staining dispersed throughout the whole nucleoplasm, far away from DSB. In addition, the number of gamma-H2AX foci detected at early time-points was independent of the number of DSB, indicating that a single gamma-H2AX focus contains clusters of several DSB within 1-2 Mbp of chromatin. In summary, the presented data show that DSB yields and distributions are greatly influenced by ionization density and chromatin compactness. Clustering of DSB and other DNA lesions may affect the reparability. Furthermore, detection of protein activation in single cells after ion track irradiation suggested that chromatin changes or other signalling processes might take place at distance from DSB. This thesis provides new insights on the importance of the chromatin organization and the repair of clustered DNA damage sites, as well as the role of repair-associated proteins in DNA damage recognition after high-LET radiation.