The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.
Ionizing radiation can lead to a variety of deleterious effects in humans, most importantly to the induction of cancer. DNA doublestrand breaks (DSBs) are among the most significant genetic lesions introduced by ionizing radiation that can initiate carcinogenesis. We have enumerated ␥-H2AX foci as a measure for DSBs in lymphocytes from individuals undergoing computed tomography examination of the thorax and͞or the abdomen. The number of DSBs induced by computed tomography examination was found to depend linearly on the dose-length product, a radiodiagnostic unit that is proportional to both the local dose delivered and the length of the body exposed. Analysis of lymphocytes sampled up to 1 day postirradiation provided kinetics for the in vivo loss of ␥-H2AX foci that correlated with DSB repair. Interestingly, in contrast to results obtained in vitro, normal individuals repair DSBs to background levels. A patient who had previously shown severe side effects after radiotherapy displayed levels of ␥-H2AX foci at various sampling times postirradiation that were several times higher than those of normal individuals. ␥-H2AX and pulsed-field gel electrophoresis analysis of fibroblasts obtained from this patient confirmed a substantial DSB repair defect. Additionally, these fibroblasts showed significant in vitro radiosensitivity. These data show that the in vivo induction and repair of DSBs can be assessed in individuals exposed to low radiation doses, adding a further dimension to DSB repair studies and providing the opportunity to identify repair-compromised individuals after diagnostic irradiation procedures.␥-H2AX foci ͉ ionizing radiation ͉ low radiation doses ͉ radiosensitivity ͉ blood lymphocytes I onizing radiation (IR) exposure is frequently encountered in a person's life. Sources of natural radiation include cosmic rays that come from outer space and the sun and radioactive substances that exist in earth and inside the human body. In addition to occupational exposure, flights at high altitude, manned space exploration, and radiological terrorism represent significant sources of radiation risk for part of the population. Diagnostic x-ray procedures including screening tests for cancer are the largest man-made source of radiation exposure, accounting for Ϸ14% of the total exposure level worldwide (1-4).Exposure to IR can induce leukemia and other cancers. It is assumed that damage to DNA in the nucleus of a single cell can initiate carcinogenesis. Among the different types of lesions induced, DNA double-strand breaks (DSBs) are considered to be the most relevant of the deleterious effects of IR (5, 6). Remarkably, even a single radiation track and, hence, the smallest possible quantity of IR can produce this kind of damage. Estimates of cancer risk from exposure to IR are based on epidemiological studies of exposed human populations, especially the atomic bomb survivors of Hiroshima and Nagasaki. These studies have provided relatively reliable estimates of risk for moderate to high doses (1, 2). The lowest dose...
The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of ␥-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.
Purpose: Radiotherapy is an effective cancer treatment, but a few patients suffer severe radiation toxicities in neighboring normal tissues.There is increasing evidence that the variable susceptibility to radiation toxicities is caused by the individual genetic predisposition, by subtle mutations, or polymorphisms in genes involved in cellular responses to ionizing radiation. Doublestrand breaks (DSB) are the most deleterious form of radiation-induced DNA damage, and DSB repair deficiencies lead to pronounced radiosensitivity. Using a preclinical mouse model, the highly sensitive gH2AX-foci approach was tested to verify even subtle, genetically determined DSB repair deficiencies known to be associated with increased normal tissue radiosensitivity. Experimental Design: By enumerating gH2AX-foci in blood lymphocytes and normal tissues (brain, lung, heart, and intestine), the induction and repair of DSBs after irradiation with therapeutic doses (0.1-2 Gy) was investigated in repair-proficient and repair-deficient mouse strains in vivo and blood samples irradiated ex vivo. Results: gH2AX-foci analysis allowed to verify the different DSB repair deficiencies; even slight impairments caused by single polymorphisms were detected similarly in both blood lymphocytes and solid tissues, indicating that DSB repair measured in lymphocytes is valid for different and complex organs. Moreover, gH2AX-foci analysis of blood samples irradiated ex vivo was found to reflect repair kinetics measured in vivo and, thus, give reliable information about the individual DSB repair capacity. Conclusions: gH2AX analysis of blood and tissue samples allows to detect even minor genetically defined DSB repair deficiencies, affecting normal tissue radiosensitivity. Future studies will have to evaluate the clinical potential to identify patients more susceptible to radiation toxicities before radiotherapy.
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