Usher Syndrome Belongs to the Genetic Diseases Associated with Radiosensitivity: Influence of the ATM Protein Kinase.

Usher syndrome (USH) is a rare autosomal recessive disease characterized by the combination of hearing loss, visual impairment due to retinitis pigmentosa, and in some cases vestibular dysfunctions. Studies published in the 1980s reported that USH is associated with cellular radiosensitivity. However, the molecular basis of this particular phenotype has not yet been documented. The aim of this study was therefore to document the radiosensitivity of USH1-a subset of USH-by examining the radiation-induced nucleo-shuttling of ATM (RIANS), as well as the functionality of the repair and signaling pathways of the DNA double-strand breaks (DSBs) in three skin fibroblasts derived from USH1 patients. The clonogenic cell survival, the micronuclei, the nuclear foci formed by the phosphorylated forms of the X variant of the H2A histone (É£H2AX), the phosphorylated forms of the ATM protein (pATM), and the meiotic recombination 11 nuclease (MRE11) were used as cellular and molecular endpoints. The interaction between the ATM and USH1 proteins was also examined by proximity ligation assay. The results showed that USH1 fibroblasts were associated with moderate but significant radiosensitivity, high yield of micronuclei, and impaired DSB recognition but normal DSB repair, likely caused by a delayed RIANS, suggesting a possible sequestration of ATM by some USH1 proteins overexpressed in the cytoplasm. To our knowledge, this report is the first radiobiological characterization of cells from USH1 patients at both molecular and cellular scales.

Quantitative Correlations between Radiosensitivity Biomarkers Show That the ATM Protein Kinase Is Strongly Involved in the Radiotoxicities Observed after Radiotherapy.

Tissue overreactions (OR), whether called adverse effects, radiotoxicity, or radiosensitivity reactions, may occur during or after anti-cancer radiotherapy (RT). They represent a medical, economic, and societal issue and raise the question of individual response to radiation. To predict and prevent them are among the major tasks of radiobiologists. To this aim, radiobiologists have developed a number of predictive assays involving different cellular models and endpoints. To date, while no consensus has been reached to consider one assay as the best predictor of the OR occurrence and severity, radiation oncologists have proposed consensual scales to quantify OR in six different grades of severity, whatever the organ/tissue concerned and their early/late features. This is notably the case with the Common Terminology Criteria for Adverse Events (CTCAE). Few radiobiological studies have used the CTCAE scale as a clinical endpoint to evaluate the statistical robustness of the molecular and cellular predictive assays in the largest range of human radiosensitivity. Here, by using 200 untransformed skin fibroblast cell lines derived from RT-treated cancer patients eliciting OR in the six CTCAE grades range, correlations between CTCAE grades and the major molecular and cellular endpoints proposed to predict OR (namely, cell survival at 2 Gy (SF2), yields of micronuclei, recognized and unrepaired DSBs assessed by immunofluorescence with γH2AX and pATM markers) were examined. To our knowledge, this was the first time that the major radiosensitivity endpoints were compared together with the same cohort and irradiation conditions. Both SF2 and the maximal number of pATM foci reached after 2 Gy appear to be the best predictors of the OR, whatever the CTCAE grades range. All these major radiosensitivity endpoints are mathematically linked in a single mechanistic model of individual response to radiation in which the ATM kinase plays a major role.

Radiation on Earth or in Space: What Does It Change?

After having been an instrument of the Cold War, space exploration has become a major technological, scientific and societal challenge for a number of countries. With new projects to return to the Moon and go to Mars, radiobiologists have been called upon to better assess the risks linked to exposure to radiation emitted from space (IRS), one of the major hazards for astronauts. To this aim, a major task is to identify the specificities of the different sources of IRS that concern astronauts. By considering the probabilities of the impact of IRS against spacecraft shielding, three conclusions can be drawn: (1) The impacts of heavy ions are rare and their contribution to radiation dose may be low during low Earth orbit; (2) secondary particles, including neutrons emitted at low energy from the spacecraft shielding, may be common in deep space and may preferentially target surface tissues such as the eyes and skin; (3) a "bath of radiation" composed of residual rays and fast neutrons inside the spacecraft may present a concern for deep tissues such as bones and the cardiovascular system. Hence, skin melanoma, cataracts, loss of bone mass, and aging of the cardiovascular system are possible, dependent on the dose, dose-rate, and individual factors. This suggests that both radiosusceptibility and radiodegeneration may be concerns related to space exploration. In addition, in the particular case of extreme solar events, radiosensitivity reactions-such as those observed in acute radiation syndrome-may occur and affect blood composition, gastrointestinal and neurologic systems. This review summarizes the specificities of space radiobiology and opens the debate as regards refinements of current radiation protection concepts that will be useful for the better estimation of risks.

Human Radiosensitivity and Radiosusceptibility: What Are the Differences?

The individual response to ionizing radiation (IR) raises a number of medical, scientific, and societal issues. While the term "radiosensitivity" was used by the pioneers at the beginning of the 20st century to describe only the radiation-induced adverse tissue reactions related to cell death, a confusion emerged in the literature from the 1930s, as "radiosensitivity" was indifferently used to describe the toxic, cancerous, or aging effect of IR. In parallel, the predisposition to radiation-induced adverse tissue reactions (radiosensitivity), notably observed after radiotherapy appears to be caused by different mechanisms than those linked to predisposition to radiation-induced cancer (radiosusceptibility). This review aims to document these differences in order to better estimate the different radiation-induced risks. It reveals that there are very few syndromes associated with the loss of biological functions involved directly in DNA damage recognition and repair as their role is absolutely necessary for cell viability. By contrast, some cytoplasmic proteins whose functions are independent of genome surveillance may also act as phosphorylation substrates of the ATM protein to regulate the molecular response to IR. The role of the ATM protein may help classify the genetic syndromes associated with radiosensitivity and/or radiosusceptibility.

When DNA Mutations Interplay with Cellular Proliferation: A Narrative History of Theories of Carcinogenesis.

While cancer is one of the most documented diseases, how normal cells become cancerous is still debated. To address this question, in the first part of this review, we investigated the long succession of theories of carcinogenesis since antiquity. Initiated by Hippocrates, Aristotle, and Galen, the humoral theory interpreted cancer as an excess of acid, the black bile. The discovery of the circulation of blood by Harvey in 1628 destroyed the basis of the humoral theory but revived the spontaneous generation hypothesis which was also promoted by Aristotle. In 1859, the theory of microbes promoted by Pasteur demonstrated the irrelevance of this last theory and contributed to the emergence of the germ cancer theory, opposed to the cellular theory of cancer, in which cancer was supposed to be caused by microbes or transformed cells, respectively. These theories were progressively refined by the notions of initiation, promotion, and progression thanks to advances in mutagenesis and cellular proliferation. In the second part of this review, recent discoveries and paradigms in carcinogenesis, notably the role of the protein ATM, a major actor of the stress response involved in both mutagenesis and cellular proliferation, were discussed to better understand the current state of the art of carcinogenesis.

When Chromatin Decondensation Affects Nuclear γH2AX Foci Pattern and Kinetics and Biases the Assessment of DNA Double-Strand Breaks by Immunofluorescence.

Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks (DSBs). Unfortunately, the pattern of γH2AX foci depends upon a number of parameters (nature of stress, number of foci, radiation dose, repair time, cell cycle phase, gene mutations, etc…) whose one of the common points is chromatin condensation/decondensation. Here, we endeavored to demonstrate how chromatin conformation affects γH2AX foci pattern and influences immunofluorescence signal. DSBs induced in non-transformed human fibroblasts were analyzed by γH2AX immunofluorescence with sodium butyrate treatment of chromatin applied after the irradiation that decondenses chromatin but does not induce DNA breaks. Our data showed that the pattern of γH2AX foci may drastically change with the experimental protocols in terms of size and brightness. Notably, some γH2AX minifoci resulting from the dispersion of the main signal due to chromatin decondensation may bias the quantification of the number of DSBs. We proposed a model called "Christmas light models" to tentatively explain this diversity of γH2AX foci pattern that may also be considered for any DNA damage marker that relocalizes as nuclear foci.

Response of Fibroblasts from Menkes' and Wilson's Copper Metabolism-Related Disorders to Ionizing Radiation: Influence of the Nucleo-Shuttling of the ATM Protein Kinase.

Menkes' disease (MD) and Wilson's disease (WD) are two major copper (Cu) metabolism-related disorders caused by mutations of the ATP7A and ATP7B ATPase gene, respectively. While Cu is involved in DNA strand breaks signaling and repair, the response of cells from both diseases to ionizing radiation, a common DNA strand breaks inducer, has not been investigated yet. To this aim, three MD and two WD skin fibroblasts lines were irradiated at two Gy X-rays and clonogenic cell survival, micronuclei, anti-γH2AX, -pATM, and -MRE11 immunofluorescence assays were applied to evaluate the DNA double-strand breaks (DSB) recognition and repair. MD and WD cells appeared moderately radiosensitive with a delay in the radiation-induced ATM nucleo-shuttling (RIANS) associated with impairments in the DSB recognition. Such delayed RIANS was notably caused in both MD and WD cells by a highly expressed ATP7B protein that forms complexes with ATM monomers in cytoplasm. Interestingly, a Cu pre-treatment of cells may influence the activity of the MRE11 nuclease and modulate the radiobiological phenotype. Lastly, some high-passage MD cells cultured in routine may transform spontaneously becoming immortalized. Altogether, our findings suggest that exposure to ionizing radiation may impact on clinical features of MD and WD, which requires cautiousness when affected patients are submitted to radiodiagnosis and, eventually, radiotherapy.

The Radiobiological Characterization of Human and Porcine Lens Cells Suggests the Importance of the ATM Kinase in Radiation-Induced Cataractogenesis.

Studies about radiation-induced human cataractogenesis are generally limited by (1) the poor number of epithelial lens cell lines available (likely because of the difficulties of cell sampling and amplification) and (2) the lack of reliable biomarkers of the radiation-induced aging process. We have developed a mechanistic model of the individual response to radiation based on the nucleoshuttling of the ATM protein (RIANS). Recently, in the frame of the RIANS model, we have shown that, to respond to permanent endo- and exogenous stress, the ATM protein progressively agglutinates around the nucleus attracted by overexpressed perinuclear ATM-substrate protein. As a result, perinuclear ATM crowns appear to be an interesting biomarker of aging. The radiobiological characterization of the two human epithelial lens cell lines available and the four porcine epithelial lens cell lines that we have established showed delayed RIANS. The BFSP2 protein, found specifically overexpressed around the lens cell nucleus and interacting with ATM, may be a specific ATM-substrate protein facilitating the formation of perinuclear ATM crowns in lens cells. The perinuclear ATM crowns were observed inasmuch as the number of culture passages is high. Interestingly, 2 Gy X-rays lead to the transient disappearance of the perinuclear ATM crowns. Altogether, our findings suggest a strong influence of the ATM protein in radiation-induced cataractogenesis.

Molecular Influence of the ATM Protein in the Treatment of Human Cells with Different Radioprotective Drugs: Comparisons between Antioxidative and Pro-Episkevic Strategies.

The radiation protection strategy with chemical agents has long been based on an antioxidative approach consisting in reducing the number of radical oxygen and nitrogen species responsible for the formation of the radiation-induced (RI) DNA damage, notably the DNA double-strand breaks (DSB), whose subset participates in the RI lethal effect as unrepairable damage. Conversely, a DSB repair-stimulating strategy that may be called the "pro-episkevic" approach (from the ancient Greek episkeve, meaning repair) can be proposed. The pro-episkevic approach directly derives from a mechanistic model based on the RI nucleoshuttling of the ATM protein (RIANS) and contributes to increase the number of DSB managed by NHEJ, the most predominant DSB repair and signaling pathway in mammalians. Here, three radioresistant and three radiosensitive human fibroblast cell lines were pretreated with antioxidative agents (N-acetylcysteine or amifostine) or to two pro-episkevic agents (zoledronate or pravastatin or both (ZOPRA)) before X-ray irradiation. The fate of the RI DSB was analyzed by using γH2AX and pATM immunofluorescence. While amifostine pretreatment appeared to be the most efficient antioxidative process, ZOPRA shows the most powerful radiation protection, suggesting that the pro-episkevic strategy may be an alternative to the antioxidative one. Additional investigations are needed to develop some new drugs that may elicit both antioxidative and pro-episkevic properties and to quantify the radiation protection action of both types of drugs applied concomitantly.

X-rays-Induced Bystander Effect Consists in the Formation of DNA Breaks in a Calcium-Dependent Manner: Influence of the Experimental Procedure and the Individual Factor.

Radiation-induced bystander effects (RIBE) describe the biological events occurring in non-targeted cells in the vicinity of irradiated ones. Various experimental procedures have been used to investigate RIBE. Interestingly, most micro-irradiation experiments have been performed with alpha particles, whereas most medium transfers have been done with X-rays. With their high fluence, synchrotron X-rays represent a real opportunity to study RIBE by applying these two approaches with the same radiation type. The RIBE induced in human fibroblasts by the medium transfer approach resulted in a generation of DNA double-strand breaks (DSB) occurring from 10 min to 4 h post-irradiation. Such RIBE was found to be dependent on dose and on the number of donor cells. The RIBE induced with the micro-irradiation approach produced DSB with the same temporal occurrence. Culture media containing high concentrations of phosphates were found to inhibit RIBE, while media rich in calcium increased it. The contribution of the RIBE to the biological dose was evaluated after synchrotron X-rays, media transfer, micro-irradiation, and 6 MeV photon irradiation mimicking a standard radiotherapy session: the RIBE may represent less than 1%, about 5%, and about 20% of the initial dose, respectively. However, RIBE may result in beneficial or otherwise deleterious effects in surrounding tissues according to their radiosensitivity status and their capacity to release Ca2+ ions in response to radiation.

Cancer and Radiosensitivity Syndromes: Is Impaired Nuclear ATM Kinase Activity the Primum Movens?

There are a number of genetic syndromes associated with both high cancer risk and clinical radiosensitivity. However, the link between these two notions remains unknown. Particularly, some cancer syndromes are caused by mutations in genes involved in DNA damage signaling and repair. How are the DNA sequence errors propagated and amplified to cause cell transformation? Conversely, some cancer syndromes are caused by mutations in genes involved in cell cycle checkpoint control. How is misrepaired DNA damage produced? Lastly, certain genes, considered as tumor suppressors, are not involved in DNA damage signaling and repair or in cell cycle checkpoint control. The mechanistic model based on radiation-induced nucleoshuttling of the ATM kinase (RIANS), a major actor of the response to ionizing radiation, may help in providing a unified explanation of the link between cancer proneness and radiosensitivity. In the frame of this model, a given protein may ensure its own specific function but may also play additional biological role(s) as an ATM phosphorylation substrate in cytoplasm. It appears that the mutated proteins that cause the major cancer and radiosensitivity syndromes are all ATM phosphorylation substrates, and they generally localize in the cytoplasm when mutated. The relevance of the RIANS model is discussed by considering different categories of the cancer syndromes.