Modulation of chromatin conformation by the histone deacetylase inhibitor trichostatin A promotes the removal of radiation-induced lesions in ataxia telangiectasia cell lines.

Ataxia telangiectasia is a rare autosomal recessive genome instability syndrome caused by mutations in the Ataxia Telangiectasia Mutated gene and characterized by a very high sensitivity to agents inducing double strand breaks such as ionizing radiation. In cells derived from ataxia telangiectasia patients a prominent enhancement of chromosomal aberrations is revealed as a consequence of this radiosensitivity characteristic, arising from defective DNA repair for a small fraction of breaks localized in the less accessible heterochromatin. Moreover, the signaling mediated by ataxia telangiectasia protein kinase also modifies chromatin structure. Even if there is a lot of knowledge concerning biochemical aspects of repair of double strand breaks, no conclusive results on radiosensitivity of structurally- and functionally-different chromatin are available, particularly in ataxia telangiectasia cells. Thus, a wild-type cell line and two ataxia telangiectasia patient derived ones could represent a suitable model to study the possible relationship between chromatin conformation and sensitivity to ionizing radiation. In this context, the effects of both cytosine arabinoside, an inhibitor of DNA repair synthesis, and trichostatin A, a histone deacetylase inhibitor, were tested in normal and ataxia telangiectasia lymphoblastoid cell lines carrying different mutation in the Ataxia Telangiectasia Mutated gene. The response to both inhibitors was investigated analyzing two endpoints, namely, chromosomal aberrations and the removal of DNA lesions by Comet assay, after exposure to X-rays. Results obtained suggest that the modulation of chromatin structure by trichostatin A leading to a more open conformation, decreases radiation-induced chromosomal aberrations in ataxia telangiectasia cells. The reduction in chromosomal instability can be attributed to an enhancement in DNA repair occurring in the presence of the histone deacetylase inhibitor, as its abolishment by the known inhibitor of DNA repair synthesis cytosine arabinoside clearly demonstrates. Data obtained could indicate a pivotal role of chromatin conformation in the radiosensitivity of ataxia telangiectasia cells.

Modulation of hypersensitivity to oxidative DNA damage in ATM defective cells induced by potassium bromate by inhibition of the Poly (ADP-ribose) polymerase (PARP).

The ataxia telangiectasia mutated (ATM) protein is a pivotal multifunctional protein kinase predominantly involved in DNA damage response, as well as in maintaining overall functional integrity of the cells. Apart from playing its major role in regulating the cellular response to DNA damage, ATM, when mutated, can additionally determine oxidative stress, metabolic syndrome, mitochondrial dysfunction and neurodegeneration. In the present paper we aim to investigate the levels of oxidative stress potentially induced by the oxidizing rodent renal carcinogen KBrO3 in ATM-defective lymphoblastoid cell lines (LCLs) established from four classical AT patients (with different ATM mutations), one AT variant with reduced hypersensitivity to X rays, obligate AT heterozygotes and wild type intrafamilial control. A possible modulatory involvement of PARP in potentially induced oxidative stress is also evaluated following its inhibition with 3-aminobenzamide (3-AB). Treatments with KBrO3 clearly showed a marked hypersensitivity of the ATM-defective LCLs, including the AT variant. A marked and statistically significant reduction of KBrO3-induced chromosomal damage following inhibition of PARP by 3-AB, was observed in all AT LCLs, but not in those from the AT variant, AT heterozygotes and wild type intrafamilial control. This result is suggestive of a modulatory involvement of PARP in the hypersensitivity of ATM-defective cells to DNA oxidative damage.

Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells.

Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H2O2 (600 µM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H2O2, BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H2O2. In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H2O2 compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H2O2 compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H2O2-induced cytotoxicity. The relevance of this kinase in vivo merits further study.

Atm reactivation reverses ataxia telangiectasia phenotypes in vivo.

Hereditary deficiencies in DNA damage signaling are invariably associated with cancer predisposition, immunodeficiency, radiation sensitivity, gonadal abnormalities, premature aging, and tissue degeneration. ATM kinase has been established as a central player in DNA double-strand break repair and its deficiency causes ataxia telangiectasia, a rare, multi-system disease with no cure. So ATM represents a highly attractive target for the development of novel types of gene therapy or transplantation strategies. Atm tamoxifen-inducible mouse models were generated to explore whether Atm reconstitution is able to restore Atm function in an Atm-deficient background. Body weight, immunodeficiency, spermatogenesis, and radioresistance were recovered in transgenic mice within 1 month from Atm induction. Notably, life span was doubled after Atm restoration, mice were protected from thymoma and no cerebellar defects were observed. Atm signaling was functional after DNA damage in vivo and in vitro. In summary, we propose a new Atm mouse model to investigate novel therapeutic strategies for ATM activation in ataxia telangiectasia disease.

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells.

Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function.

Longitudinal analysis of the neurological features of ataxia-telangiectasia.

AIM: To assess the relationship between genotype and neurological progression in ataxia-telangiectasia (A-T). METHODS: Clinical and laboratory data were extracted retrospectively from the records of patients attending the UK National Ataxia-Telangiectasia Clinic. Neurological assessments were performed using the A-T Index (Crawford Score) and the A-T Neurological Examination Scale Toolkit (A-T NEST). Variables influencing phenotype were identified by using an information-theoretic approach starting from a maximal model to generate estimates of coefficients for each variable. Per-individual progression was assessed for patients with three or more clinic attendances. RESULTS: The genotype could be determined for 125/135 patients. Crawford and A-T NEST scores were well correlated. For both scoring systems the estimated coefficients were significantly positive for Age x kinase activity but not Age x protein expression. Unlike the per-genotype analysis, the individual progression of neurological scores in the 34 patients that attended on three or more occasions was not smooth and linear (and in some cases improved over time). INTERPRETATION: Residual kinase activity confers a milder phenotype but there is no difference between kinase-dead and protein-null genotypes. The non-linear progression of individual patients' neurological scores may reflect biological complexity, day-to-day variability, limitations of the assessment methods or a combination of all three.

A novel mouse model for ataxia-telangiectasia with a N-terminal mutation displays a behavioral defect and a low incidence of lymphoma but no increased oxidative burden.

Ataxia-telangiectasia (A-T) is a rare multi-system disorder caused by mutations in the ATM gene. Significant heterogeneity exists in the underlying genetic mutations and clinical phenotypes. A number of mouse models have been generated that harbor mutations in the distal region of the gene, and a recent study suggests the presence of residual ATM protein in the brain of one such model. These mice recapitulate many of the characteristics of A-T seen in humans, with the notable exception of neurodegeneration. In order to study how an N-terminal mutation affects the disease phenotype, we generated an inducible Atm mutant mouse model (Atm(tm1Mmpl/tm1Mmpl), referred to as A-T [M]) predicted to express only the first 62 amino acids of Atm. Cells derived from A-T [M] mutant mice exhibited reduced cellular proliferation and an altered DNA damage response, but surprisingly, showed no evidence of an oxidative imbalance. Examination of the A-T [M] animals revealed an altered immunophenotype consistent with A-T. In contrast to mice harboring C-terminal Atm mutations that disproportionately develop thymic lymphomas, A-T [M] mice developed lymphoma at a similar rate as human A-T patients. Morphological analyses of A-T [M] cerebella revealed no substantial cellular defects, similar to other models of A-T, although mice display behavioral defects consistent with cerebellar dysfunction. Overall, these results suggest that loss of Atm is not necessarily associated with an oxidized phenotype as has been previously proposed and that loss of ATM protein is not sufficient to induce cerebellar degeneration in mice.

NADPH oxidase 4 is a critical mediator in Ataxia telangiectasia disease.

Ataxia telangiectasia (A-T), a rare autosomal recessive disorder characterized by progressive cerebellar degeneration and a greatly increased incidence of cancer among other symptoms, is caused by a defective or missing ataxia telangiectasia mutated (ATM) gene. The ATM protein has roles in DNA repair and in the regulation of reactive oxygen species (ROS). Here, we provide, to our knowledge, the first evidence that NADPH oxidase 4 (NOX4) is involved in manifesting A-T disease. We showed that NOX4 expression levels are higher in A-T cells, and that ATM inhibition leads to increased NOX4 expression in normal cells. A-T cells exhibit elevated levels of oxidative DNA damage, DNA double-strand breaks and replicative senescence, all of which are partially abrogated by down-regulation of NOX4 with siRNA. Sections of degenerating cerebelli from A-T patients revealed elevated NOX4 levels. ATM-null mice exhibit A-T disease but they die from cancer before the neurological symptoms are manifested. Injecting Atm-null mice with fulvene-5, a specific inhibitor of NOX4 and NADPH oxidase 2 (NOX2), decreased their elevated cancer incidence to that of the controls. We conclude that, in A-T disease in humans and mice, NOX4 may be critical mediator and targeting it will open up new avenues for therapeutic intervention in neurodegeneration.

Functional and molecular defects of hiPSC-derived neurons from patients with ATM deficiency.

Loss of ataxia telangiectasia mutated (ATM) kinase, a key factor of the DNA damage response (DDR) pathway, causes the cancer predisposing and neurodegenerative syndrome ataxia-telangiectasia (A-T). To investigate the mechanisms of neurodegeneration, we have reprogrammed fibroblasts from ATM-null A-T patients and normal controls to pluripotency (human-induced pluripotent stem cells), and derived from these neural precursor cells able to terminally differentiate into post-mitotic neurons positive to >90% for ß-tubulin III+/microtubule-associated protein 2+. We show that A-T neurons display similar voltage-gated potassium and sodium currents and discharges of action potentials as control neurons, but defective expression of the maturation and synaptic markers SCG10, SYP and PSD95 (postsynaptic density protein 95). A-T neurons exhibited defective repair of DNA double-strand breaks (DSBs) and repressed phosphorylation of ATM substrates (e.g., γH2AX, Smc1-S966, Kap1-S824, Chk2-T68, p53-S15), but normal repair of single-strand breaks, and normal short- and long-patch base excision repair activities. Moreover, A-T neurons were resistant to apoptosis induced by the genotoxic agents camptothecin and trabectedin, but as sensitive as controls to the oxidative agents. Most notably, A-T neurons exhibited abnormal accumulation of topoisomerase 1-DNA covalent complexes (Top1-ccs). These findings reveal that ATM deficiency impairs neuronal maturation, suppresses the response and repair of DNA DSBs, and enhances Top1-cc accumulation. Top1-cc could be a risk factor for neurodegeneration as they may interfere with transcription elongation and promote transcriptional decline.

[The role of ATM kinase in neurodegeneration]. / Znaczenie kinazy ATM w procesach neurodegeneracyjnych.

Neurodegenerative diseases represent a major challenge for modern medicine. Despite many years of research, no effective neuroprotective therapy has been proposed. Ataxia telangiectasia (A-T) is rare disease, which is caused by a mutation of the ATM protein. Cerebellar degeneration is the main symptom of the A-T. The kinase ATM, inter alia is involved in the repair of DNA damage, cell cycle regulation and the control of apoptosis. In recent years the presence of that kinase in the cytoplasm has been demonstrated. This led to the discovery of its participation in the regulation of metabolic processes, homeostasis mitochondrial oxidative stress response or modulation of synaptic function. The pleiotropic effect of ATM kinase requires effective control exercised by, inter alia, proteins having specific binding motifs this kinase, such as ATMIN and NBS1. The regulation of prosurvival processes which are controlled by ATM kinase, may prove an attractive therapeutic strategy in treatment of neurodegenerative diseases.

p38 (MAPK) stress signalling in replicative senescence in fibroblasts from progeroid and genomic instability syndromes.

Werner Syndrome (WS) is a human segmental progeria resulting from mutations in a DNA helicase. WS fibroblasts have a shortened replicative capacity, an aged appearance, and activated p38 MAPK, features that can be modulated by inhibition of the p38 pathway. Loss of the WRNp RecQ helicase has been shown to result in replicative stress, suggesting that a link between faulty DNA repair and stress-induced premature cellular senescence may lead to premature ageing in WS. Other progeroid syndromes that share overlapping pathophysiological features with WS also show defects in DNA processing, raising the possibility that faulty DNA repair, leading to replicative stress and premature cellular senescence, might be a more widespread feature of premature ageing syndromes. We therefore analysed replicative capacity, cellular morphology and p38 activation, and the effects of p38 inhibition, in fibroblasts from a range of progeroid syndromes. In general, populations of young fibroblasts from non-WS progeroid syndromes do not have a high level of cells with an enlarged morphology and F-actin stress fibres, unlike young WS cells, although this varies between strains. p38 activation and phosphorylated HSP27 levels generally correlate well with cellular morphology, and treatment with the p38 inhibitor SB203580 effects cellular morphology only in strains with enlarged cells and phosphorylated HSP27. For some syndromes fibroblast replicative capacity was within the normal range, whereas for others it was significantly shorter (e.g. HGPS and DKC). However, although in most cases SB203580 extended replicative capacity, with the exception of WS and DKC the magnitude of the effect was not significantly different from normal dermal fibroblasts. This suggests that stress-induced premature cellular senescence via p38 activation is restricted to a small subset of progeroid syndromes.

Activation of AMP-activated protein kinase in cerebella of Atm-/- mice is attributable to accumulation of reactive oxygen species.

Ataxia telangiectasia (A-T) is an inherited disease, the most prominent feature of which is ataxia caused by degeneration of cerebellar neurons and synapses. The mechanisms underlying A-T neurodegeneration are still unclear, and many factors are likely to be involved. AMP-activated protein kinase (AMPK) is a sensor of energy balance, and research on its function in neural cells has gained momentum in the last decade. The dual roles of AMPK in neuroprotection and neurodegeneration are complex, and they need to be identified and characterized. Using an Atm (ataxia telangiectasia mutated) gene deficient mouse model, we showed here that: (a) upregulation of AMPK phosphorylation and elevation of reactive oxygen species (ROS) coordinately occur in the cerebella of Atm-/- mice; (b) hydrogen peroxide induces AMPK phosphorylation in primary mouse cerebellar astrocytes in an Atm-independent manner; (c) administration of the novel antioxidant monosodium luminol (MSL) to Atm-/- mice attenuates the upregulation of both phosphorylated-AMPK (p-AMPK) and ROS, and corrects the neuromotor deficits in these animals. Together, our results suggest that oxidative activation of AMPK in the cerebellum may contribute to the neurodegeneration in Atm-/- mice, and that ROS and AMPK signaling pathways are promising therapeutic targets for treatment of A-T and other neurodegenerative diseases.

Lymphoid tumours and breast cancer in ataxia telangiectasia; substantial protective effect of residual ATM kinase activity against childhood tumours.

BACKGROUND: Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T. METHODS: From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity. RESULTS: In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype. CONCLUSION: Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age.

Stable brain ATM message and residual kinase-active ATM protein in ataxia-telangiectasia.

The gene that is mutated in ataxia-telangiectasia (A-T), ATM, is catalytically activated in response to DNA damage. Yet a full accounting for the CNS deficits in human A-T or its mouse models remains elusive. We have analyzed the CNS phenotypes of two mouse Atm alleles--Atm(tm1Bal) (Bal) and Atm(tm1Awb) (Awb). Neither mutant has detectable mRNA or protein in peripheral tissues. In brain, although Bal/Bal mice have no ATM protein, they have nearly normal amounts of Atm mRNA. Bal/Bal neurons exhibit extensive cell cycle reentry and degeneration in both cortex and cerebellum. Unexpectedly, in Awb/Awb mice a novel mRNA is found in which the engineered mutation is excised. This mRNA is apparently translated and produces a catalytically active ATM protein that responds to DNA damage by phosphorylating p53 and Chk2. Prompted by these results, we examined eight cases of human A-T and found evidence for residual ATM protein in seven of them. These findings offer important new insights into the human disease and the role of brain ATM activity in the severity of the neurological symptoms of A-T.

Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum.

Antisense morpholino oligonucleotides (AMOs) can reprogram pre-mRNA splicing by complementary binding to a target site and regulating splice site selection, thereby offering a potential therapeutic tool for genetic disorders. However, the application of this technology into a clinical scenario has been limited by the low correction efficiency in vivo and inability of AMOs to efficiently cross the blood brain barrier and target brain cells when applied to neurogenetic disorders such as ataxia-telangiecatasia (A-T). We previously used AMOs to correct subtypes of ATM splicing mutations in A-T cells; AMOs restored up to 20% of the ATM protein and corrected the A-T cellular phenotype. In this study, we demonstrate that an arginine-rich cell-penetrating peptide, (RXRRBR)(2)XB, dramatically improved ATM splicing correction efficiency when conjugated with AMOs, and almost fully corrected aberrant splicing. The restored ATM protein was close to normal levels in cells with homozygous splicing mutations, and a gene dose effect was observed in cells with heterozygous mutations. A significant amount of the ATM protein was still detected 21 days after a single 5 µm treatment. Systemic administration of an fluorescein isothiocyanate-labeled (RXRRBR)(2)XB-AMO in mice showed efficient uptake in the brain. Fluorescence was evident in Purkinje cells after a single intravenous injection of 60 mg/kg. Furthermore, multiple injections significantly increased uptake in all areas of the brain, notably in cerebellum and Purkinje cells, and showed no apparent signs of toxicity. Taken together, these results highlight the therapeutic potential of (RXRRBR)(2)XB-AMOs in A-T and other neurogenetic disorders.

DNA-dependent protein kinase and ataxia telangiectasia mutated (ATM) promote cell survival in response to NK314, a topoisomerase IIα inhibitor.

4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα, leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314, to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314, and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA, a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs, as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit, Ku80, ataxia telangiectasia mutated (ATM), BRCA2, or XRCC3 compared with wild-type cells, indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore, both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314.

Two unrelated patients with MRE11A mutations and Nijmegen breakage syndrome-like severe microcephaly.

MRE11 and NBS1 function together as components of a MRE11/RAD50/NBS1 protein complex, however deficiency of either protein does not result in the same clinical features. Mutations in the NBN gene underlie Nijmegen breakage syndrome (NBS), a chromosomal instability syndrome characterized by microcephaly, bird-like faces, growth and mental retardation, and cellular radiosensitivity. Additionally, mutations in the MRE11A gene are known to lead to an ataxia-telangiectasia-like disorder (ATLD), a late-onset, slowly progressive variant of ataxia-telangiectasia without microcephaly. Here we describe two unrelated patients with NBS-like severe microcephaly (head circumference -10.2 SD and -12.8 SD) and mutations in the MRE11A gene. Both patients were compound heterozygotes for a truncating or missense mutation and carried a translationally silent mutation. The truncating and missense mutations were assumed to be functionally debilitating. The translationally silent mutation common to both patients had an effect on splicing efficiency resulting in reduced but normal MRE11 protein. Their levels of radiation-induced activation of ATM were higher than those in ATLD cells.

Inhibition of ATM kinase activity does not phenocopy ATM protein disruption: implications for the clinical utility of ATM kinase inhibitors.

Biallelic mutations in ataxia-telangiectasia mutated (ATM), which encodes for a protein kinase, cause ataxia telangiectasia (A-T). A-T is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation (IR). A-T patients typically lack both detectable ATM protein and ATM kinase activity, and small molecule inhibitors of ATM kinase activity have been developed as strategies to improve radiotherapy for the treatment of cancers. As predicted, inhibition of ATM kinase activity is sufficient to radiosensitize cells. However, we recently showed that inhibition of ATM kinase activity disrupts DNA damage-induced sister chromatid exchange (SCE). This result was unanticipated since SCE is normal in A-T cells that lack detectable ATM protein. In these studies, we showed, for the first time, that the consequences of inhibition of ATM kinase activity and adaptation to ATM protein disruption are distinct. Here, we discuss the mechanistic implications of this finding for the function of ATM at the replication fork and the clinical utility of ATM kinase inhibitors.

ATM activation by oxidative stress.

The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide-cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells.

Hepatitis C virus inhibits DNA damage repair through reactive oxygen and nitrogen species and by interfering with the ATM-NBS1/Mre11/Rad50 DNA repair pathway in monocytes and hepatocytes.

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and putatively also non-Hodgkin's B cell lymphoma. In this study, we demonstrated that PBMCs obtained from HCV-infected patients showed frequent chromosomal aberrations and that HCV infection of B cells in vitro induced enhanced chromosomal breaks and sister chromatid exchanges. HCV infection hypersensitized cells to ionizing radiation and bleomycin and inhibited nonhomologous end-joining repair. The viral core and nonstructural protein 3 proteins were shown to be responsible for the inhibition of DNA repair, mediated by NO and reactive oxygen species. Stable expression of core protein induced frequent chromosome translocations in cultured cells and in transgenic mice. HCV core protein binds to the NBS1 protein and inhibits the formation of the Mre11/NBS1/Rad50 complex, thereby affecting ATM activation and inhibiting DNA binding of repair enzymes. Taken together, these data indicate that HCV infection inhibits multiple DNA repair processes to potentiate chromosome instability in both monocytes and hepatocytes. These effects may explain the oncogenicity and immunological perturbation of HCV infection.