Biological consequences of complex DNA damage induced by high-LET ionizing radiations

Abstract

Radiation interactions with biological matter release energy through processes that increase the entropy of the system often to a limit that the organisms cannot tolerate, resulting in the deleterious effects of radiation. Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly harmful since radiation can perturb all important components of the cell, from the membrane to the nucleus. Depending on the source, diverse DNA lesions can be induced including nucleotide alterations, bulky adducts, base lesions, single strand breaks (SSBs) and double strand breaks (DSBs). Such types of DNA damage can occur separately or in conjunction with each other forming clustered lesions (complex DNA damage). Clustered DNA damage consisting of a combination of DNA lesions is considered as one of the most important biological endpoints regarding IR exposure and the prime factor behind cell lethality with increasing radiation linear energy transfer (LET). The biological significance of such damages relates to the inability of cells to process them efficiently, and the outcome in case of erroneous repair can vary from mutations up to genomic instability, cell death and cancer. Even though there is accumulating evidence on the pivotal role of complex DNA damage on the determination of the final biological (or even clinical) outcome after exposure to IR, detection challenges still pose a serious obstacle in establishing definite associations between IR-induced damage and prediction of biological responses. Here, state-of-the art approaches are used to provide evidence of the formation of clustered DNA damage after exposure to different radiations. The in situ detection of clustered DNA damage induced by high-LET radiations (a clinically operating proton beam and different HZE particle beams simulating cosmic rays found in space) is pursued using elaborate methodologies of immunofluorescence imaging and colocalisation analysis introduced by DNA Damage Lab in NTUA. Such research areas are of great importance, since high-LET IR is the most frequent mode of irradiation for the average human, is an emerging treatment modality for cancer and is relevant to future human aspirations to deep space exploration. A hallmark of high-LET radiation is the DSB clustering. DSB clusters have been considered as particularly consequential in several mathematical models, but experimental demonstration of their relevance to the adverse IR effects, as well as information on the exact mechanisms underpinning their severity as DNA lesions is still lacking. Here, the biological consequences of DSB clustering are being investigated using a genome engineering-based model system, aiming to offer a mechanistic explanation for the increased efficacy of high-LET radiation. For this purpose cell lines with specially designed, multiply integrated constructs modeling defined combinations of DSB-clusters through appropriately engineered I-SceI meganuclease recognition sites are being employed. Using this model system, efficient activation of the DNA damage response (DDR) is clearly demonstrated, as well as a markedly increased potential of DSB clusters compared to single-DSBs, to kill cells and cause chromosomal aberrations. Overall, results presented here point to the significance of IR-induced clustered DNA lesions, providing clear evidence of their formation after exposure to different radiations of high-LET and of their detrimental biological effects.