Alpha-Particle Exposure Induces Mainly Unstable Complex Chromosome Aberrations which do not Contribute to Radiation-Associated Cytogenetic Risk

The mechanism underlying the carcinogenic potential of α radiation is not fully understood, considering that cell inactivation (e.g., mitotic cell death) as a main consequence of exposure efficiently counteracts the spreading of heritable DNA damage. The aim of this study is to improve our understanding of the effectiveness of α particles in inducing different types of chromosomal aberrations, to determine the respective values of the relative biological effectiveness (RBE) and to interpret the results with respect to exposure risk. Human peripheral blood lymphocytes (PBLs) from a single donor were exposed ex vivo to doses of 0–6 Gy X rays or 0–2 Gy α particles. Cells were harvested at two different times after irradiation to account for the mitotic delay of heavily damaged cells, which is known to occur after exposure to high-LET radiation (including α particles). Analysis of the kinetics of cells reaching first or second (and higher) mitosis after irradiation and aberration data obtained by the multiplex fluorescence in situ hybridization (mFISH) technique are used to determine of the cytogenetic risk, i.e., the probability for transmissible aberrations in surviving lymphocytes. The analysis shows that the cytogenetic risk after α exposure is lower than after X rays. This indicates that the actually observed higher carcinogenic effect of α radiation is likely to stem from small scale mutations that are induced effectively by high-LET radiation but cannot be resolved by mFISH analysis.

Transcriptomic analysis links hepatocellular carcinoma (HCC) in HZE ion irradiated mice to a human HCC subtype with favorable outcomes

High-charge, high-energy ion particle (HZE) radiations are extraterrestrial in origin and characterized by high linear energy transfer (high-LET), which causes more severe cell damage than low-LET radiations like γ-rays or photons. High-LET radiation poses potential cancer risks for astronauts on deep space missions, but the studies of its carcinogenic effects have relied heavily on animal models. It remains uncertain whether such data are applicable to human disease. Here, we used genomics approaches to directly compare high-LET radiation-induced, low-LET radiation-induced and spontaneous hepatocellular carcinoma (HCC) in mice with a human HCC cohort from The Cancer Genome Atlas (TCGA). We identified common molecular pathways between mouse and human HCC and discovered a subset of orthologous genes (mR-HCC) that associated high-LET radiation-induced mouse HCC with a subgroup (mrHCC2) of the TCGA cohort. The mrHCC2 TCGA cohort was more enriched with tumor-suppressing immune cells and showed a better prognostic outcome than other patient subgroups.

USP9X Is Required to Maintain Cell Survival in Response to High-LET Radiation

Ionizing radiation (IR) principally acts through induction of DNA damage that promotes cell death, although the biological effects of IR are more broad ranging. In fact, the impact of IR of higher-linear energy transfer (LET) on cell biology is generally not well understood. Critically, therefore, the cellular enzymes and mechanisms responsible for enhancing cell survival following high-LET IR are unclear. To this effect, we have recently performed siRNA screening to identify deubiquitylating enzymes that control cell survival specifically in response to high-LET α-particles and protons, in comparison to low-LET X-rays and protons. From this screening, we have now thoroughly validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using small interfering RNA (siRNA), leads to significantly decreased survival of cells after high-LET radiation. We consequently investigated the mechanism through which this occurs, and demonstrate that an absence of USP9X has no impact on DNA damage repair post-irradiation nor on apoptosis, autophagy, or senescence. We discovered that USP9X is required to stabilize key proteins (CEP55 and CEP131) involved in centrosome and cilia formation and plays an important role in controlling pericentrin-rich foci, particularly in response to high-LET protons. This was also confirmed directly by demonstrating that depletion of CEP55/CEP131 led to both enhanced radiosensitivity of cells to high-LET protons and amplification of pericentrin-rich foci. Our evidence supports the importance of USP9X in maintaining centrosome function and biogenesis and which is crucial particularly in the cellular response to high-LET radiation.

Strong Shift to ATR-Dependent Regulation of the G2-Checkpoint after Exposure to High-LET Radiation

The utilization of high linear-energy-transfer (LET) ionizing radiation (IR) modalities is rapidly growing worldwide, causing excitement but also raising concerns, because our understanding of their biological effects is incomplete. Charged particles such as protons and heavy ions have increasing potential in cancer therapy, due to their advantageous physical properties over X-rays (photons), but are also present in the space environment, adding to the health risks of space missions. Therapy improvements and the protection of humans during space travel will benefit from a better understanding of the mechanisms underpinning the biological effects of high-LET IR. There is evidence that high-LET IR induces DNA double-strand breaks (DSBs) of increasing complexity, causing enhanced cell killing, owing, at least partly, to the frequent engagement of a low-fidelity DSB-repair pathway: alternative end-joining (alt-EJ), which is known to frequently induce severe structural chromosomal abnormalities (SCAs). Here, we evaluate the radiosensitivity of A549 lung adenocarcinoma cells to X-rays, α-particles and 56Fe ions, as well as of HCT116 colorectal cancer cells to X-rays and α-particles. We observe the expected increase in cell killing following high-LET irradiation that correlates with the increased formation of SCAs as detected by mFISH. Furthermore, we report that cells exposed to low doses of α-particles and 56Fe ions show an enhanced G2-checkpoint response which is mainly regulated by ATR, rather than the coordinated ATM/ATR-dependent regulation observed after exposure to low doses of X-rays. These observations advance our understanding of the mechanisms underpinning high-LET IR effects, and suggest the potential utility for ATR inhibitors in high-LET radiation therapy.

Towards parameter-free nanodosimetric quantities in the impact of highly ionizing radiation

<p>Cosmic Rays, in particular the high charge and high energy (HZE) particles and eventual secondary low energy protons, are high Linear Energy Transfer (LET) radiation, i.e. they transfer a high amount of energy to the target per unit path length travelled in the target itself, leaving behind a dense track of ionization and atomic excitations. Understanding the radiation physics and the biology induced by the impact of high LET radiation is of importance for different fields of research, such as radiation therapy with charged particles, space radiation protection of astronauts and of human explorers on Mars and eventually also survival of any bacterial, plant cell on other planetary/small bodies. While data for low LET radiation  such as X-ray have been studied in the survivors of the atomic-bombs, medical patients and nuclear reactor workers, for high LET radiation there is no relevant collection of human data for risk estimates, and experiments with nuclei created at accelerators are necessary.</p><p>At present we still do not have an understanding of how the  radiation  interaction  with a  single nanometric  target (units of DNA), the so-called track  structure [1],  should  decide  the  fate  of  the  irradiated cell. Monte Carlo (MC) track structure codes essentially work only with the physics given by impact cross sections on the sole water, there is no real consideration of the electronic/chemical characteristics of the hosted biomolecule [2]. Limitations given by such an approach have been highlighted [3], but on the positive side a massive effort is being done to follow the different steps of radiation effects up to biological damage [4].</p><p>In this contribution we would like to highlight how a chain of models from different communities could be of help to study the radiation effects on biomolecules. In particular, we will present how ab-initio (parameter-free) approaches from the chemical-physics community can be used to derive in detail the energy loss of the impacting ions/secondary electrons on water and small biological units [5,6], either following in real time the ion or based on perturbative theories for low energy electrons, and how the derived quantity can be given  as input to Monte Carlo track structure codes, extending their capabilities to different relevant targets. Given the physical limitations and high costs of irradiation experiments, such calculations offer an efficient approach that can boost the understanding of radiation physics and consolidate existing MC track structure codes.</p><p>This work is initiated in the context of the EU H2020 project ESC2RAD, Grant 776410.</p><p>[1] H. Nikjoo, S. Uehara, W.E. Wilson, et al, International Journal of Radiation Biology 73, 355 (1998)</p><p>[2] H. Palmans, H Rabus, A L Belchior, et al, Br. J. Radiol. 88, 20140392 (2015)</p><p>[3] H. Rabus and H. Nettelback, Radiation Measurements 46, 1522 (2011)</p><p>[4] M. Karamitros, S. Luan, M.A. Bernal, et al,  Journal of Computational Physics 274,  841 (2014)</p><p>[5] B. Gu, B. Cunningham D. Munoz-Santiburcio, F. Da Pieve, E. Artacho and J. Kohanoff, J. Chem. Phys. 153, 034113 (2020)</p><p>[6] N. Koval, J. Kohanoff, E. Artacho et al, in preparation</p>

Protective Effects of p53 Regulatory Agents Against High-LET Radiation-Induced Injury in Mice

Radiation damage to normal tissues is one of the most serious concerns in radiation therapy, and the tolerance dose of the normal tissues limits the therapeutic dose to the patients. p53 is well known as a transcription factor closely associated with radiation-induced cell death. We recently demonstrated the protective effects of several p53 regulatory agents against low-LET X- or γ-ray-induced damage. Although it was reported that high-LET heavy ion radiation (>85 keV/μm) could cause p53-independent cell death in some cancer cell lines, whether there is any radioprotective effect of the p53 regulatory agents against the high-LET radiation injury in vivo is still unclear. In the present study, we verified the efficacy of these agents on bone marrow and intestinal damages induced by high-LET heavy-ion irradiation in mice. We used a carbon-beam (14 keV/μm) that was shown to induce a p53-dependent effect and an iron-beam (189 keV/μm) that was shown to induce a p53-independent effect in a previous study. Vanadate significantly improved 60-day survival rate in mice treated with total-body carbon-ion (p < 0.0001) or iron-ion (p < 0.05) irradiation, indicating its effective protection of the hematopoietic system from radiation injury after high-LET irradiation over 85 keV/μm. 5CHQ also significantly increased the survival rate after abdominal carbon-ion (p < 0.02), but not iron-ion irradiation, suggesting the moderate relief of the intestinal damage. These results demonstrated the effectiveness of p53 regulators on acute radiation syndrome induced by high-LET radiation.