Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control, In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein, Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Spl decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells, Mutation in two Spl consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the;basal level of Spl, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in,A-T cells. On the other hand full-length ATM cDNA increased the basal level of Spl binding in A-T cells, and in response to EGF Spl binding decreased, confirming that this is an ATR I-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Spl binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Spl regulation.

Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress, Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha -lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Use of the comet test to assess DNA damage in children with ataxia-telangiectasia and their relatives.

The electrophoresis of cells in alkaline medium (comet assay) is a valid technique for quantifying DNA damage in patients with ataxia-telangiectasia and their relatives.

ERS statement on the multidisciplinary respiratory management of ataxia telangiectasia.

Ataxia telangiectasia (A-T) is a rare, progressive, multisystem disease that has a large number of complex and diverse manifestations which vary with age. Patients with A-T die prematurely with the leading causes of death being respiratory diseases and cancer. Respiratory manifestations include immune dysfunction leading to recurrent upper and lower respiratory infections; aspiration resulting from dysfunctional swallowing due to neurodegenerative deficits; inefficient cough; and interstitial lung disease/pulmonary fibrosis. Malnutrition is a significant comorbidity. The increased radiosensitivity and increased risk of cancer should be borne in mind when requesting radiological investigations. Aggressive proactive monitoring and treatment of these various aspects of lung disease under multidisciplinary expertise in the experience of national multidisciplinary clinics internationally forms the basis of this statement on the management of lung disease in A-T. Neurological management is outwith the scope of this document.

Pleiotropic defects in ataxia-telangiectasia protein-deficient mice

We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm-deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm-deficient mice then exhibit many of the same symptoms found in ataxia-telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.

The impact of carboxy nitroxide antioxidants on irradiated ataxia telangiectasia cells

Three water-soluble carboxy nitroxide antioxidants, 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl, 4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl, and 3-carboxy-2,2,5,5-tetramethylpyrrolidin-1-yloxyl, show significant impact on the postirradiation survival rates of ataxia telangiectasia (A-T) cells compared to normal cells, an assay which represents a model for understanding the impact of ROS damage on the A-T phenotype. The effects of these antioxidants are much more significant than those of vitamin E or Trolox (a water-soluble vitamin E analog), studied using the same cell survival model. (C) 2004 Elsevier Inc. All rights reserved.

Genetic interactions of the Ashergillus nidulans atmA(ATM) homolog with different components of the DNA damage response pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for Delta atmA, Delta uvsB, Delta chkA, and Delta chkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APCI), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

Chk2 activation dependence on Nbs1 after DNA damage

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G, arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells, interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1, Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.