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Marfan syndrome (MFS) is an autosomal dominant connective-tissue disorder affecting multiple systems, such as skeletal, cardiovascular, and ocular systems. MFS is predominantly caused by mutations in the FBN1 gene, which encodes the fibrillin-1 protein, crucial for connective-tissue integrity. FBN1 mutations lead to defective fibrillin, resulting in structurally compromised connective tissues. Additionally, these mutations cause aberrant TGF-β expression, contributing to vascular issues and increased susceptibility to radiation-induced fibrosis. Studies about the potential radiosensitivity of MFS are rare and generally limited to case reports. Here, we aimed to investigate the radiation-induced ATM nucleo-shuttling (RIANS) model to explore the molecular and cellular radiation response in fibroblasts from MFS patients. The results showed that the MFS fibroblast cell lines tested are associated with moderate but significant radiosensitivity, high yield of micronuclei, and impaired recognition of DNA double-strand breaks (DSBs) caused by a diminished RIANS. The diminished RIANS is supported by the sequestration of ATM protein in the cytoplasm not only by mutated FBN1 protein but also by overexpressed TGF-β. This report is the first molecular and cellular characterization of the radiation response of MFS fibroblasts and highlights the importance of the FBN1-TGF-β complex after irradiation. [ABSTRACT FROM AUTHOR]

Simple Summary: By hypothesizing that molecular hyper-recombination and cellular proliferation are among the major features of multi-factorial cancer proneness, by using skin fibroblasts derived from eight major cancer syndromes, a significant correlation was found between the hyper-recombination rate quantified by plasmid assay, proliferation capacity assessed by flow cytometry, and excess of relative cancer risk (ERR). The product of the hyper-recombination rate and capacity of proliferation described a linear function of ERR. Context: Although carcinogenesis is a multi-factorial process, the mutability and the capacity of cells to proliferate are among the major features of the cells that contribute together to the initiation and promotion steps of cancer formation. Particularly, mutability can be quantified by hyper-recombination rate assessed with specific plasmid assay, hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations frequency rate, or MRE11 nuclease activities. Cell proliferation can be assessed by flow cytometry by quantifying G2/M, G1 arrests, or global cellular evasion. Methods: All these assays were applied to skin untransformed fibroblasts derived from eight major cancer syndromes characterized by their excess of relative cancer risk (ERR). Results: Significant correlations with ERR were found between hyper-recombination assessed by the plasmid assay and G2/M arrest and described a third-degree polynomial ERR function and a sigmoidal ERR function, respectively. The product of the hyper-recombination rate and capacity of proliferation described a linear ERR function that permits one to better discriminate each cancer syndrome. Conclusions: Hyper-recombination and cell proliferation were found to obey differential equations that better highlight the intrinsic bases of cancer formation. Further investigations to verify their relevance for cancer proneness induced by exogenous agents are in progress. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common neurodegenerative dementia, for which the molecular origins, genetic predisposition and therapeutic approach are still debated. In the 1980s, cells from AD patients were reported to be sensitive to ionizing radiation. In order to examine the molecular basis of this radiosensitivity, the ATM-dependent DNA double-strand breaks (DSB) signaling and repair were investigated by applying an approach based on the radiation-induced ataxia telangiectasia-mutated (ATM) protein nucleoshuttling (RIANS) model. Early after irradiation, all ten AD fibroblast cell lines tested showed impaired DSB recognition and delayed RIANS. AD fibroblasts specifically showed spontaneous perinuclear localization of phosphorylated ATM (pATM) forms. To our knowledge, such observation has never been reported before, and by considering the role of the ATM kinase in the stress response, it may introduce a novel interpretation of accelerated aging. Our data and a mathematical approach through a brand-new model suggest that, in response to a progressive and cumulative stress, cytoplasmic ATM monomers phosphorylate the APOE protein (pAPOE) close to the nuclear membrane and aggregate around the nucleus, preventing their entry in the nucleus and thus the recognition and repair of spontaneous DSB, which contributes to the aging process. Our findings suggest that pATM and/or pAPOE may serve as biomarkers for an early reliable diagnosis of AD on any fibroblast sample. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: A review of the molecular and cellular features of the major cancer syndromes associated with radiosensitivity revealed the importance of the ATM protein, either as an impaired kinase in the nucleus or as a complex in the cytoplasm, with the mutated protein responsible for the syndrome. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Retinoblastoma (Rb) is a rare form of pediatric cancer that develops from retina cells. Bilateral and some unilateral forms of Rb are associated with heterozygous germline mutations of the (retinoblastoma 1) RB1 gene. RB1 mutations are also associated with a significant risk of secondary malignancy like head and neck tumors. Hence, to date, even if Rb patients are less subjected to radiotherapy to treat their primary ocular tumors, their healthy tissues may be exposed to significant doses of ionizing radiation during the treatment against their secondary malignancies with a significant risk of adverse tissue reactions (radiosensitivity) and/or radiation-induced cancer (radiosusceptibility). However, the biological role of the Rb protein in response to radiation remains misunderstood. Since the ataxia telangiectasia mutated (ATM) protein is a key protein of radiation response and since untransformed skin fibroblasts are a current model to quantify cellular radiosensitivity, we investigated here for the first time the functionality of the ATM-dependent signaling and repair pathway of the radiation-induced DNA double-strand breaks (DSB) in irradiated skin fibroblasts derived from Rb patients The major biomarkers of the DSB repair and signaling, namely clonogenic cell survival, micronuclei, nuclear foci of the phosphorylated forms of the X variant of the H2A histone (γH2AX), the phosphorylated forms of the ATM protein (pATM) and the meiotic recombination 11 nuclease (MRE11) were assessed in untransformed skin fibroblasts derived from three Rb patients. Skin fibroblasts from Rb patients showed significant cellular radiosensitivity, incomplete DSB recognition, delay in the ATM nucleo-shuttling and exacerbated MRE11 nuclease activity. Treatment with statin and bisphosphonates led to significant complementation of these impairments. Our findings strongly suggest the involvement of the ATM kinase in the radiosensitivity/radiosusceptibility phenotype observed in Rb cases. [ABSTRACT FROM AUTHOR]

MacCune–Albright syndrome (MAS) is a rare autosomal dominant osteo-hormonal disorder. MAS is characterized by a severe form of polyostotic fibrous dysplasia, 'café-au-lait' pigmentation of the skin and multiple endocrinopathies. MAS was shown to be caused by mosaic missense somatic mutations in the GNAS gene coding for the alpha-subunit of the stimulatory G-protein. MAS is also associated with radiation-induced malignant tumors, like osteosarcoma, fibrosarcoma and chondrosarcoma but their origin remains misunderstood. In parallel, bisphosphonates treatment was shown to improve the MAS patients' outcome, notably by increasing bone density but, again, the molecular mechanisms supporting these observations remain misunderstood. Here, by using fibroblast and osteoblast cell lines derived from 2 MAS patients, the major radiobiological features of MAS were investigated. Notably, the clonogenic cell survival, the micronuclei and the γH2AX, pATM and MRE11 immunofluorescence assays were applied to MAS cells. It appears that cells from the 2 MAS patients are associated with a moderate but significant radiosensitivity, a delayed radiation-induced nucleoshuttling of the ATM kinase likely caused by its sequestration in cytoplasm, suggesting impaired DNA double-strand breaks (DSB) repair and signaling in both fibroblasts and osteoblasts. Such delay may be partially corrected by using bisphosphonates combined with statins, which renders cells more radioresistant. Our findings represent the first radiobiological characterization of fibroblasts and osteoblasts providing from MAS patients. Although the number of studied cases is reduced, our findings suggest that the MAS cells tested belong to the group of syndromes associated with moderate but significant radiosensitivity. Further investigations are however required to secure the clinical transfer of the combination of bisphosphonates and statins, to reduce the disease progression and to better evaluate the potential risks linked to radiation exposure. [ABSTRACT FROM AUTHOR]

Purpose: Xeroderma Pigmentosum (XP) is a rare, recessive genetic disease associated with photosensitivity, skin cancer proneness, neurological abnormalities and impaired nucleotide excision repair of the UV-induced DNA damage. Less frequently, XP can be associated with sensitivity to ionizing radiation (IR). Here, a complete radiobiological characterization was performed on a panel of fibroblasts derived from XP-group D patients (XPD). Materials and methods: Cellular radiosensitivity and the functionality of the recognition and repair of chromosome breaks and DNA double-strand breaks (DSB) was evaluated by different techniques including clonogenic cell survival, micronuclei, premature chromosome condensation, pulsed-field gel electrophoresis, chromatin decondensation and immunofluorescence assays. Quantitative correlations between each endpoint were analyzed systematically. Results: Among the seven fibroblast cell lines tested, those derived from three non-relative patients holding the p.[Arg683Trp];[Arg616Pro] XPD mutations showed significant cellular radiosensitivity, high yield of residual micronuclei, incomplete DSB recognition, DSB and chromosome repair defects, impaired ATM, MRE11 relocalization, significant chromatin decondensation. Interestingly, XPD transduction and treatment with statins and bisphosphonates known to accelerate the radiation-induced ATM nucleoshuttling led to significant complementation of these impairments. Conclusions: Our findings suggest that some subsets of XPD patients may be at risk of radiosensitivity reactions and treatment with statins and bisphosphonates may be an interesting approach of radioprotection countermeasure. Different mechanistic models were discussed to better understand the potential specificity of the p.[Arg683Trp];[Arg616Pro] XPD mutations. [ABSTRACT FROM AUTHOR]

A mechanistic model from radiobiology has emerged by pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition, the repair of DNA double-strand breaks (DSB), and the final response to genotoxic stress. More recently, we provided evidence in this journal that the RIANS model is also relevant for exposure to metal ions. To document the role of the ATM-dependent DSB repair and signaling after pesticide exposure, we applied six current pesticides of domestic and environmental interest (lindane, atrazine, glyphosate, permethrin, pentachlorophenol and thiabendazole) to human skin fibroblast and brain cells. Our findings suggest that each pesticide tested may induce DSB at a rate that depends on the pesticide concentration and the RIANS status of cells. At specific concentration ranges, the nucleo-shuttling of ATM can be delayed, which impairs DSB recognition and repair, and contributes to toxicity. Interestingly, the combination of copper sulfate and thiabendazole or glyphosate was found to have additive or supra-additive effects on DSB recognition and/or repair. A general mechanistic model of the biological response to metal and/or pesticide is proposed to define quantitative endpoints for toxicity. [ABSTRACT FROM AUTHOR]

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 (ɣH2AX), 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. [ABSTRACT FROM AUTHOR]

Despite a considerable amount of data, the molecular and cellular bases of the toxicity due to metal exposure remain unknown. Recent mechanistic models from radiobiology have emerged, pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition and the repair of DNA double-strand breaks (DSB) and the final response to genotoxic stress. In order to document the role of ATM-dependent DSB repair and signalling after metal exposure, we applied twelve different metal species representing nine elements (Al, Cu, Zn Ni, Pd, Cd, Pb, Cr, and Fe) to human skin, mammary, and brain cells. Our findings suggest that metals may directly or indirectly induce DSB at a rate that depends on the metal properties and concentration, and tissue type. At specific metal concentration ranges, the nucleo-shuttling of ATM can be delayed which impairs DSB recognition and repair and contributes to toxicity and carcinogenicity. Interestingly, as observed after low doses of ionizing radiation, some phenomena equivalent to the biological response observed at high metal concentrations may occur at lower concentrations. A general mechanistic model of the biological response to metal exposure based on the nucleo-shuttling of ATM is proposed to describe the metal-induced stress response and to define quantitative endpoints for toxicity and carcinogenicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Whereas post-radiation therapy overreactions (OR) represent a clinical and societal issue, there is still no consensual radiobiological endpoint to predict clinical radiosensitivity. Since 2003, skin biopsy specimens have been collected from patients treated by radiation therapy against different tumor localizations and showing a wide range of OR. Here, we aimed to establish quantitative links between radiobiological factors and OR severity grades that would be relevant to radioresistant and genetic hyperradiosensitive cases.Methods and Materials: Immunofluorescence experiments were performed on a collection of skin fibroblasts from 12 radioresistant, 5 hyperradiosensitive, and 100 OR patients irradiated at 2 Gy. The numbers of micronuclei, γH2AX, and pATM foci that reflect different steps of DNA double-strand breaks (DSB) recognition and repair were assessed from 10 minutes to 24 hours after irradiation and plotted against the severity grades established by the Common Terminology Criteria for Adverse Events and the Radiation Therapy Oncology Group.Results: OR patients did not necessarily show a gross DSB repair defect but a systematic delay in the nucleoshuttling of the ATM protein required for complete DSB recognition. Among the radiobiological factors, the maximal number of pATM foci provided the best discrimination among OR patients and a significant correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions.Conclusions: Our results are consistent with a general classification of human radiosensitivity based on 3 groups: radioresistance (group I); moderate radiosensitivity caused by delay of nucleoshuttling of ATM, which includes OR patients (group II); and hyperradiosensitivity caused by a gross DSB repair defect, which includes fatal cases (group III). [ABSTRACT FROM AUTHOR]