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Sample records for a-t mutated atm

  1. ATM mutations for surgeons.

    PubMed

    Mansfield, Sara A; Pilarski, Robert; Agnese, Doreen M

    2016-12-17

    The ataxia-telangiectasia mutated (ATM) gene encodes a protein kinase involved in DNA repair. Heterozygotic carriers are at an increased risk of developing breast cancer. As the use of genetic testing increases, identification of at-risk patients will also increase. The aim of this study is to review two cases of heterozygous ATM mutation carriers and review the literature to clarify the cancer risks and suggested management for breast surgeons who will be intimately involved in the care of these patients.

  2. Underexpression and abnormal localization of ATM products in ataxia telangiectasia patients bearing ATM missense mutations

    PubMed Central

    Jacquemin, Virginie; Rieunier, Guillaume; Jacob, Sandrine; Bellanger, Dorine; d'Enghien, Catherine Dubois; Laugé, Anthony; Stoppa-Lyonnet, Dominique; Stern, Marc-Henri

    2012-01-01

    Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immune defects and predisposition to malignancies. A-T is caused by biallelic inactivation of the ATM gene, in most cases by frameshift or nonsense mutations. More rarely, ATM missense mutations with unknown consequences on ATM function are found, making definitive diagnosis more challenging. In this study, a series of 15 missense mutations, including 11 not previously reported, were identified in 16 patients with clinical diagnosis of A-T belonging to 14 families and 1 patient with atypical clinical features. ATM function was evaluated in patient lymphoblastoid cell lines by measuring H2AX and KAP1 phosphorylation in response to ionizing radiation, confirming the A-T diagnosis for 16 cases. In accordance with previous studies, we showed that missense mutations associated with A-T often lead to ATM protein underexpression (15 out of 16 cases). In addition, we demonstrated that most missense mutations lead to an abnormal cytoplasmic localization of ATM, correlated with its decreased expression. This new finding highlights ATM mislocalization as a new mechanism of ATM dysfunction, which may lead to therapeutic strategies for missense mutation associated A-T. PMID:22071889

  3. Underexpression and abnormal localization of ATM products in ataxia telangiectasia patients bearing ATM missense mutations.

    PubMed

    Jacquemin, Virginie; Rieunier, Guillaume; Jacob, Sandrine; Bellanger, Dorine; d'Enghien, Catherine Dubois; Laugé, Anthony; Stoppa-Lyonnet, Dominique; Stern, Marc-Henri

    2012-03-01

    Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immune defects and predisposition to malignancies. A-T is caused by biallelic inactivation of the ATM gene, in most cases by frameshift or nonsense mutations. More rarely, ATM missense mutations with unknown consequences on ATM function are found, making definitive diagnosis more challenging. In this study, a series of 15 missense mutations, including 11 not previously reported, were identified in 16 patients with clinical diagnosis of A-T belonging to 14 families and 1 patient with atypical clinical features. ATM function was evaluated in patient lymphoblastoid cell lines by measuring H2AX and KAP1 phosphorylation in response to ionizing radiation, confirming the A-T diagnosis for 16 cases. In accordance with previous studies, we showed that missense mutations associated with A-T often lead to ATM protein underexpression (15 out of 16 cases). In addition, we demonstrated that most missense mutations lead to an abnormal cytoplasmic localization of ATM, correlated with its decreased expression. This new finding highlights ATM mislocalization as a new mechanism of ATM dysfunction, which may lead to therapeutic strategies for missense mutation associated A-T.

  4. Twelve novel Atm mutations identified in Chinese ataxia telangiectasia patients.

    PubMed

    Huang, Yu; Yang, Lu; Wang, Jianchun; Yang, Fan; Xiao, Ying; Xia, Rongjun; Yuan, Xianhou; Yan, Mingshan

    2013-09-01

    Ataxia telangiectasia (A-T) is an autosomal recessive disease characterized mainly by progressive cerebellar ataxia, oculocutaneous telangiectasia, and immunodeficiency. This disease is caused by mutations of the ataxia telangiectasia mutated (Atm) gene. More than 500 Atm mutations that are responsible for A-T have been identified so far. However, there have been very few A-T cases reported in China, and only two Chinese A-T patients have undergone Atm gene analysis. In order to systemically investigate A-T in China and map their Atm mutation spectrum, we recruited eight Chinese A-T patients from six unrelated families nationwide. Using direct sequencing of genomic DNA and the multiplex ligation-dependent probe amplification, we identified twelve pathogenic Atm mutations, including one missense, four nonsense, five frameshift, one splicing, and one large genomic deletion. All the Atm mutations we identified were novel, and no homozygous mutation and founder-effect mutation were found. These results suggest that Atm mutations in Chinese populations are diverse and distinct largely from those in other ethnic areas.

  5. ATM Gene Mutation Detection Techniques and Functional Analysis.

    PubMed

    Rieunier, Guillaume; D'Enghien, Catherine Dubois; Fievet, Alice; Bellanger, Dorine; Stoppa-Lyonnet, Dominique; Stern, Marc-Henri

    2017-01-01

    Ataxia Telangiectasia (A-T) is caused by biallelic inactivation of the Ataxia Telangiectasia Mutated (ATM) gene, due to nonsense or missense mutations, small insertions/deletions (indels), splicing alterations, and large genomic rearrangements. After establishing A-T clinical diagnosis, a molecular confirmation is needed, based on the detection of one of these loss-of-function mutations in at least one allele. In most cases, the pathogenicity of the detected mutations is sufficient to make a definitive diagnosis. More rarely, mutations of unknown consequences are identified and direct biological analyses are required to establish their pathogenic characters. In such cases, complementary analyses of ATM expression, localization, and activity allow fine characterization of these mutations and facilitate A-T diagnosis. Here, we present genetic and biochemical protocols currently used in the laboratory that have proven to be highly accurate, reproducible, and quantitative. We also provide additional discussion on the critical points of the techniques presented here.

  6. Novel ATM mutations with ataxia-telangiectasia.

    PubMed

    Liu, Xiao-Li; Wang, Tian; Huang, Xiao-Jun; Zhou, Hai-Yan; Luan, Xing-Hua; Shen, Jun-Yi; Chen, Sheng-Di; Cao, Li

    2016-01-12

    Ataxia telangiectasia is an autosomal recessive multisystem disorder characterized by progressive cerebellar ataxia with onset in childhood, oculocutaneous telangiectasia, increased serum alpha-fetoprotein, immunodeficiency, chromosomal instability, and radiation hypersensitivity. Ataxia-telangiectasia mutated gene (ATM) is one of the known genes to be associated with ataxia telangiectasia. We reported the clinical and genetic findings of three early-onset Chinese patients who demonstrated ataxia, oculomotor apraxia, choreoathetosis, myoclonus and telangiectasia of eyes. Sequence analysis of ATM revealed two known nonsense mutations c.8287C>T and c.9139C>T in the siblings. Though the siblings carried the same mutations, they showed different clinical features involving strephenopodia, exotropia, torsion dystonia, myoclonus and extrapyramidal impairments. The other patient was compound heterozygotes for ATM: c.8911C>T and c.7141_7151delAATGGAAAAAT, both of which were not reported previously and not found in 200 control chromosomes. This study widens the spectrum of mutations and phenotypes in ataxia telangiectasia. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. ATM mutations and phenotypes in ataxia-telangiectasia families in the British Isles: expression of mutant ATM and the risk of leukemia, lymphoma, and breast cancer.

    PubMed Central

    Stankovic, T; Kidd, A M; Sutcliffe, A; McGuire, G M; Robinson, P; Weber, P; Bedenham, T; Bradwell, A R; Easton, D F; Lennox, G G; Haites, N; Byrd, P J; Taylor, A M

    1998-01-01

    We report the spectrum of 59 ATM mutations observed in ataxia-telangiectasia (A-T) patients in the British Isles. Of 51 ATM mutations identified in families native to the British Isles, 11 were founder mutations, and 2 of these 11 conferred a milder clinical phenotype with respect to both cerebellar degeneration and cellular features. We report, in two A-T families, an ATM mutation (7271T-->G) that may be associated with an increased risk of breast cancer in both homozygotes and heterozygotes (relative risk 12.7; P=. 0025), although there is a less severe A-T phenotype in terms of the degree of cerebellar degeneration. This mutation (7271T-->G) also allows expression of full-length ATM protein at a level comparable with that in unaffected individuals. In addition, we have studied 18 A-T patients, in 15 families, who developed leukemia, lymphoma, preleukemic T-cell proliferation, or Hodgkin lymphoma, mostly in childhood. A wide variety of ATM mutation types, including missense mutations and in-frame deletions, were seen in these patients. We also show that 25% of all A-T patients carried in-frame deletions or missense mutations, many of which were also associated with expression of mutant ATM protein. PMID:9463314

  8. Prevalence of deleterious ATM germline mutations in gastric cancer patients.

    PubMed

    Huang, Dong-Sheng; Tao, Hou-Quan; He, Xu-Jun; Long, Ming; Yu, Sheng; Xia, Ying-Jie; Wei, Zhang; Xiong, Zikai; Jones, Sian; He, Yiping; Yan, Hai; Wang, Xiaoyue

    2015-12-01

    Besides CDH1, few hereditary gastric cancer predisposition genes have been previously reported. In this study, we discovered two germline ATM mutations (p.Y1203fs and p.N1223S) in a Chinese family with a history of gastric cancer by screening 83 cancer susceptibility genes. Using a published exome sequencing dataset, we found deleterious germline mutations of ATM in 2.7% of 335 gastric cancer patients of different ethnic origins. The frequency of deleterious ATM mutations in gastric cancer patients is significantly higher than that in general population (p=0.0000435), suggesting an association of ATM mutations with gastric cancer predisposition. We also observed biallelic inactivation of ATM in tumors of two gastric cancer patients. Further evaluation of ATM mutations in hereditary gastric cancer will facilitate genetic testing and risk assessment.

  9. ATM germline mutations in women with familial breast cancer and a relative with haematological malignancy.

    PubMed

    Paglia, Laura La; Laugé, Anthony; Weber, Jérémie; Champ, Jérôme; Cavaciuti, Eve; Russo, Antonio; Viovy, Jean-Louis; Stoppa-Lyonnet, Dominique

    2010-01-01

    Biallelic inactivation of the ATM gene causes ataxia-telangiectasia (A-T), a complex neurological disease associated with a high risk of leukaemias and lymphomas. Mothers of A-T children, obligate ATM heterozygote mutation carriers, have a breast cancer (BC) relative risk of about 3. The frequency of ATM carriers in BC women with a BC family history has been estimated to be 2.70%. To further our clinical understanding of familial BC and examine whether haematological malignancies are predictive of ATM germline mutation, we estimated the frequency of heterozygote mutation carriers in a series of 122 BC women with a family history of both BC and haematological malignancy and without BRCA1/2 mutation. The gene screening was performed with a new high throughput method, EMMA (enhanced mismatch mutation analysis). Amongst 28 different ATM variants, eight mutations have been identified in eight patients: two mutations leading to a putative truncated protein and six being likely deleterious mutations. One of the truncating mutations was initially interpreted as a missense mutation, p.Asp2597Tyr, but is actually a splice mutation (c.7789G>T/p.Asp2597_Lys2643>LysfsX3). The estimated frequency of ATM heterozygote mutation carriers in our series is 6.56% (95% CI: 2.16-10.95), a significantly higher figure than that observed in the general population, estimated to be between 0.3 and 0.6%. Although a trend towards an increased frequency of ATM carriers was observed, it was not different from that observed in a population of familial BC women not selected for haematological malignancy as the frequency of ATM carriers was 2.70%, a value situated in the confidence interval of our study.

  10. Germline ATM mutational analysis in BRCA1/BRCA2 negative hereditary breast cancer families by MALDI-TOF mass spectrometry.

    PubMed

    Graña, B; Fachal, L; Darder, E; Balmaña, J; Ramón Y Cajal, T; Blanco, I; Torres, A; Lázaro, C; Diez, O; Alonso, C; Santamariña, M; Velasco, A; Teulé, A; Lasa, A; Blanco, A; Izquierdo, A; Borràs, J; Gutiérrez-Enríquez, S; Vega, A; Brunet, J

    2011-07-01

    Biallelic inactivation of ATM gene causes the rare autosomal recessive disorder Ataxia-telangiectasia (A-T). Female relatives of A-T patients have a two-fold higher risk of developing breast cancer (BC) compared with the general population. ATM mutation carrier identification is laborious and expensive, therefore, a more rapid and directed strategy for ATM mutation profiling is needed. We designed a case-control study to determine the prevalence of 32 known ATM mutations causing A-T in Spanish population in 323 BRCA1/BRCA2 negative hereditary breast cancer (HBC) cases and 625 matched Spanish controls. For the detection of the 32 ATM mutations we used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique. We identified one patient carrier of the c.8264_8268delATAAG ATM mutation. This mutation was not found in the 625 controls. These results suggest a low frequency of these 32 A-T causing mutations in the HBC cases in our population. Further case-control studies analyzing the entire coding and flanking sequences of the ATM gene are warranted in Spanish BC patients to know its implication in BC predisposition.

  11. ATM gene mutations in sporadic breast cancer patients from Brazil.

    PubMed

    Mangone, Flavia Rotea; Miracca, Elisabete C; Feilotter, Harriet E; Mulligan, Lois M; Nagai, Maria Aparecida

    2015-01-01

    The Ataxia-telangiectasia mutated (ATM) gene encodes a multifunctional kinase, which is linked to important cellular functions. Women heterozygous for ATM mutations have an estimated relative risk of developing breast cancer of 3.8. However, the pattern of ATM mutations and their role in breast cancer etiology has been controversial and remains unclear. In the present study, we investigated the frequency and spectrum of ATM mutations in a series of sporadic breast cancers and controls from the Brazilian population. Using PCR-Single Strand Conformation Polymorphism (SSCP) analysis and direct DNA sequencing, we screened a panel of 100 consecutive, unselected sporadic breast tumors and 100 matched controls for all 62 coding exons and flanking introns of the ATM gene. Several polymorphisms were detected in 12 of the 62 coding exons of the ATM gene. These polymorphisms were observed in both breast cancer patients and the control population. In addition, evidence of potential ATM mutations was observed in 7 of the 100 breast cancer cases analyzed. These potential mutations included six missense variants found in exon 13 (p.L546V), exon 14 (p.P604S), exon 20 (p.T935R), exon 42 (p.G2023R), exon 49 (p.L2307F), and exon 50 (p.L2332P) and one nonsense mutation in exon 39 (p.R1882X), which was predicted to generate a truncated protein. Our results corroborate the hypothesis that sporadic breast tumors may occur in carriers of low penetrance ATM mutant alleles and these mutations confer different levels of breast cancer risk.

  12. ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin

    PubMed Central

    Navrkalova, Veronika; Sebejova, Ludmila; Zemanova, Jana; Kminkova, Jana; Kubesova, Blanka; Malcikova, Jitka; Mraz, Marek; Smardova, Jana; Pavlova, Sarka; Doubek, Michael; Brychtova, Yvona; Potesil, David; Nemethova, Veronika; Mayer, Jiri; Pospisilova, Sarka; Trbusek, Martin

    2013-01-01

    ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin. PMID:23585524

  13. ATM mutations uniformly lead to ATM dysfunction in chronic lymphocytic leukemia: application of functional test using doxorubicin.

    PubMed

    Navrkalova, Veronika; Sebejova, Ludmila; Zemanova, Jana; Kminkova, Jana; Kubesova, Blanka; Malcikova, Jitka; Mraz, Marek; Smardova, Jana; Pavlova, Sarka; Doubek, Michael; Brychtova, Yvona; Potesil, David; Nemethova, Veronika; Mayer, Jiri; Pospisilova, Sarka; Trbusek, Martin

    2013-07-01

    ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

  14. Mutation at intronic repeats of the ataxia-telangiectasia mutated (ATM) gene and ATM protein loss in primary gastric cancer with microsatellite instability.

    PubMed

    Kim, Hee Sung; Choi, Seung Im; Min, Hae Lim; Kim, Min A; Kim, Woo Ho

    2013-01-01

    Ataxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinase that plays a critical role in DNA damage-induced signaling and initiation of cell cycle checkpoint signaling in response to DNA-damaging agents such as ionizing radiation. We have previously reported the ATM protein loss by immunohistochemistry (IHC) in 16% of human gastric cancer (GC) tissue. We hypothesized that ATM gene intron mutations targeted by microsatellite instability (MSI) cause ATM protein loss in a subset of GC. We studied mononucleotide mutations at the intron of ATM gene, ATM IHC and MSI in GC. Ten human gastric cancer cell lines were studied for the ATM gene mutation at introns, RT-PCR, direct sequencing, and immunohistochemistry. GC tissues of 839 patients were analyzed for MSI and ATM IHC. Among them, 604 cases were analyzed for the ATM mutations at introns preceding exon 6, exon 10 and exon 20. Two human GC cell lines (SNU-1 and -638) showed ATM intron mutations, deletion in RT-PCR and direct sequencing, and ATM protein loss by IHC. The frequencies of ATM mutation, MSI, and ATM protein loss were 12.9% (78/604), 9.2% (81/882) and 15.2% (134/839), respectively. Analysis of associations among MSI, ATM gene mutation, and ATM protein loss revealed highly co-existing ATM gene alterations and MSI. ATM intron mutation and ATM protein loss were detected in 69.3% (52/75) and 53.3% (40/75) of MSI positive GC. MSI positivity and ATM protein loss were present in 68.4% (52/76) and 48.7% (37/76) of GC with ATM intron mutation. ATM mutation and ATM protein loss had characteristics of old age, distal location of tumor, large tumor size, and histologic intestinal type. Our study might be interpreted as that ATM gene mutation at intron might be targeted by MSI and lead to ATM protein loss in a selected group of GC.

  15. Mutation at Intronic Repeats of the Ataxia-Telangiectasia Mutated (ATM) Gene and ATM Protein Loss in Primary Gastric Cancer with Microsatellite Instability

    PubMed Central

    Kim, Hee Sung; Choi, Seung Im; Min, Hae Lim; Kim, Min A.; Kim, Woo Ho

    2013-01-01

    Ataxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinase that plays a critical role in DNA damage-induced signaling and initiation of cell cycle checkpoint signaling in response to DNA-damaging agents such as ionizing radiation. We have previously reported the ATM protein loss by immunohistochemistry (IHC) in 16% of human gastric cancer (GC) tissue. We hypothesized that ATM gene intron mutations targeted by microsatellite instability (MSI) cause ATM protein loss in a subset of GC. We studied mononucleotide mutations at the intron of ATM gene, ATM IHC and MSI in GC. Ten human gastric cancer cell lines were studied for the ATM gene mutation at introns, RT-PCR, direct sequencing, and immunohistochemistry. GC tissues of 839 patients were analyzed for MSI and ATM IHC. Among them, 604 cases were analyzed for the ATM mutations at introns preceding exon 6, exon 10 and exon 20. Two human GC cell lines (SNU-1 and -638) showed ATM intron mutations, deletion in RT-PCR and direct sequencing, and ATM protein loss by IHC. The frequencies of ATM mutation, MSI, and ATM protein loss were 12.9% (78/604), 9.2% (81/882) and 15.2% (134/839), respectively. Analysis of associations among MSI, ATM gene mutation, and ATM protein loss revealed highly co-existing ATM gene alterations and MSI. ATM intron mutation and ATM protein loss were detected in 69.3% (52/75) and 53.3% (40/75) of MSI positive GC. MSI positivity and ATM protein loss were present in 68.4% (52/76) and 48.7% (37/76) of GC with ATM intron mutation. ATM mutation and ATM protein loss had characteristics of old age, distal location of tumor, large tumor size, and histologic intestinal type. Our study might be interpreted as that ATM gene mutation at intron might be targeted by MSI and lead to ATM protein loss in a selected group of GC. PMID:24324828

  16. Functional and computational assessment of missense variants in the ataxia-telangiectasia mutated (ATM) gene: mutations with increased cancer risk.

    PubMed

    Mitui, M; Nahas, S A; Du, L T; Yang, Z; Lai, C H; Nakamura, K; Arroyo, S; Scott, S; Purayidom, A; Concannon, P; Lavin, M; Gatti, R A

    2009-01-01

    The functional consequences of missense variants are often difficult to predict. This becomes especially relevant when DNA sequence changes are used to determine a diagnosis or prognosis. To analyze the consequences of 12 missense variants in patients with mild forms of ataxia-telangiectasia (A-T), we employed site-directed mutagenesis of ataxia-telangiectasia mutated (ATM) cDNA followed by stable transfections into a single A-T cell line to isolate the effects of each allele on the cellular phenotype. After induction of the transfected cells with CdCl2, we monitored for successful ATM transcription and subsequently assessed: 1) intracellular ATM protein levels; 2) ionizing radiation (IR)-induced ATM kinase activity; and 3) cellular radiosensitivity. We then calculated SIFT and PolyPhen scores for the missense changes. Nine variants produced little or no correction of the A-T cellular phenotype and were interpreted to be ATM mutations; SIFT/PolyPhen scores supported this. Three variants corrected the cellular phenotype, suggesting that they represented benign variants or polymorphisms. SIFT and PolyPhen scores supported the functional analyses for one of these variants (c.1709T>C); the other two were predicted to be "not tolerated" (c.6188G>A and c.6325T>G) and were classified as "operationally neutral." Genotype/phenotype relationships were compared: three deleterious missense variants were associated with an increased risk of cancer (c.6679C>T, c.7271T>G, and c.8494C>T). In situ mutagenesis represents an effective experimental approach for distinguishing deleterious missense mutations from benign or operationally neutral missense variants. Copyright 2008 Wiley-Liss, Inc.

  17. The role of ATM mutations and 11q deletions in disease progression in chronic lymphocytic leukemia.

    PubMed

    Stankovic, Tatjana; Skowronska, Anna

    2014-06-01

    Abstract ATM gene alteration is a frequent event in pathogenesis of chronic lymphocytic leukemia (CLL) and occurs as monoallelic loss in the form of 11q23 deletion, with and without mutation in the remaining ATM allele. ATM is a principal DNA damage response gene and biallelic ATM alterations lead to ATM functional loss and chemoresistance. The introduction of new therapies, such as intensive chemoimmunotherapy and inhibition of B-cell receptor (BCR) signaling, has changed clinical responses for the majority of CLL tumors including those with 11q deletion, but it remains to be determined whether these strategies can prevent clonal evolution of tumors with biallelic ATM alterations. In this review we discuss ATM function and the consequences of its loss during CLL pathogenesis, differences in clinical behavior of tumors with monoallelic and biallelic ATM alterations, and we outline possible approaches for targeting the ATM null CLL phenotype.

  18. Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

    DTIC Science & Technology

    2006-07-01

    ATM genetic variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for each mutation examined. 15. SUBJECT...women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation ...each ATM variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for mutations identified. Body STATEMENT

  19. A Novel Method to Screen for Dominant Negative ATM Mutations in Familial Breast Cancer

    DTIC Science & Technology

    2005-04-01

    carry dominant negative mutation in ATM due to natural variation amongst LCLs. Microarrays have been performed to determine differences in gene expression... genes that are altered in their expression in ATMmutation carriers. The validation of this data in carriers of different ATM mutation indicated that the...heterozygous carriers of T727 1 G mutation display a gene expression phenotype that appears identical to carriers of protein truncating mutations in

  20. New mutations in the ATM gene and clinical data of 25 AT patients.

    PubMed

    Demuth, Ilja; Dutrannoy, Véronique; Marques, Wilson; Neitzel, Heidemarie; Schindler, Detlev; Dimova, Petja S; Chrzanowska, Krystyna H; Bojinova, Veneta; Gregorek, Hanna; Graul-Neumann, Luitgard M; von Moers, Arpad; Schulze, Ilka; Nicke, Marion; Bora, Elcin; Cankaya, Tufan; Oláh, Éva; Kiss, Csongor; Bessenyei, Beáta; Szakszon, Katalin; Gruber-Sedlmayr, Ursula; Kroisel, Peter Michael; Sodia, Sigrun; Goecke, Timm O; Dörk, Thilo; Digweed, Martin; Sperling, Karl; de Sá, Joaquim; Lourenco, Charles Marques; Varon, Raymonda

    2011-11-01

    Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, oculocutaneous telangiectasias, chromosomal instability, radiosensitivity, and cancer predisposition. The gene mutated in the patients, ATM, encodes a member of the phosphatidylinositol 3-kinase family proteins. The ATM protein has a key role in the cellular response to DNA damage. Truncating and splice site mutations in ATM have been found in most patients with the classical AT phenotype. Here we report of our extensive ATM mutation screening on 25 AT patients from 19 families of different ethnic origin. Previously unknown mutations were identified in six patients including a new homozygous missense mutation, c.8110T>C (p.Cys2704Arg), in a severely affected patient. Comprehensive clinical data are presented for all patients described here along with data on ATM function generated by analysis of cell lines established from a subset of the patients.

  1. Identification of ATM Mutations in Korean Siblings with Ataxia-Telangiectasia

    PubMed Central

    Huh, Hee Jae; Cho, Kyoo-ho; Lee, Ji Eun; Kwon, Min-Jung

    2013-01-01

    Ataxia-telangiectasia (A-T) is a rare autosomal recessive neurodegenerative disorder. It is characterized by early-onset, progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, immunodeficiency, and an increased risk of malignancy. Although A-T is known to be the most common cause of progressive cerebellar ataxia in childhood, there have been no confirmed cases in Korea. We report the clinical and genetic findings of Korean siblings who presented with limb and truncal ataxia, oculomotor apraxia, choreoathetosis, and telangiectasias of the eyes. Sequence analysis of the ataxia-telangiectasia mutated (ATM) gene revealed a known missense mutation (c.8546G>C; p.Arg2849Pro) and a novel intronic variant of intron 17 (c.2639-19_2639-7del13). Reverse-transcription PCR and sequencing analysis revealed that the c.2639-19_2639-7del13 variant causes a splicing aberration that potentiates skipping exon 18. Because A-T is quite rare in Korea, the diagnosis of A-T in Korean patients can be delayed. We recommend that a diagnosis of A-T should be suspected in Korean patients exhibiting the clinical features of A-T. PMID:23667852

  2. Identification of ATM mutations in Korean siblings with ataxia-telangiectasia.

    PubMed

    Huh, Hee Jae; Cho, Kyoo-Ho; Lee, Ji Eun; Kwon, Min-Jung; Ki, Chang-Seok; Lee, Phil Hyu

    2013-05-01

    Ataxia-telangiectasia (A-T) is a rare autosomal recessive neurodegenerative disorder. It is characterized by early-onset, progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, immunodeficiency, and an increased risk of malignancy. Although A-T is known to be the most common cause of progressive cerebellar ataxia in childhood, there have been no confirmed cases in Korea. We report the clinical and genetic findings of Korean siblings who presented with limb and truncal ataxia, oculomotor apraxia, choreoathetosis, and telangiectasias of the eyes. Sequence analysis of the ataxia-telangiectasia mutated (ATM) gene revealed a known missense mutation (c.8546G>C; p.Arg2849Pro) and a novel intronic variant of intron 17 (c.2639-19_2639-7del13). Reverse-transcription PCR and sequencing analysis revealed that the c.2639-19_2639-7del13 variant causes a splicing aberration that potentiates skipping exon 18. Because A-T is quite rare in Korea, the diagnosis of A-T in Korean patients can be delayed. We recommend that a diagnosis of A-T should be suspected in Korean patients exhibiting the clinical features of A-T.

  3. The effects of Atm haploinsufficiency on mutation rate in the mouse germ line and somatic tissue.

    PubMed

    Ahuja, Akshay K; Barber, Ruth C; Hardwick, Robert J; Weil, Michael M; Genik, Paula C; Brenner, David J; Dubrova, Yuri E

    2008-09-01

    Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm(+/-)) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm(+/-) males did not significantly differ from that in wild-type BALB/c mice. Acute exposure to 1 Gy of gamma-rays did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm(+/-) and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analysed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

  4. ATM Mutations and the Development of Severe Radiation-Induced Morbidity Following Radiotherapy for Breast Cancer

    DTIC Science & Technology

    2005-07-01

    repair of radiation-induced damage. Furthermore, cells possessing a mutated copy of this gene are more radiosensitive than cells from individuals with...AD Award Number: DAMD17-02-1-0503 TITLE: ATM Mutations and the Development of Severe Radiation-Induced Morbidity Following Radiotherapy for Breast...2005 Annual 1 Jul 2004 - 30 Jun 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER ATM Mutations and the Development of Severe Radiation-Induced Morbidity

  5. Large genomic mutations within the ATM gene detected by MLPA, including a duplication of 41 kb from exon 4 to 20.

    PubMed

    Cavalieri, Simona; Funaro, Ada; Pappi, Patrizia; Migone, Nicola; Gatti, Richard A; Brusco, Alfredo

    2008-01-01

    Mutation detection remains problematic for large genes, primarily because PCR-based methodology fails to detect heterozygous deletions and any duplication. In the ATM gene only a handful of multi-exon deletions have been described to date, and this type of mutation has been considered rare. To address this issue we tested a new MLPA (Multiplex Ligation Probe Amplification) kit that covers 33 of the 66 ATM exons, using for controls two previously characterized genomic deletions in addition to three A-T patients, taken from a survey of nine, who had missing four mutations unidentified after conventional mutation screening. We identified for the first time: 1) a approximately 41 kb genomic duplication spanning exons 4-20 (c.-30_2816dup41kb)(a.k.a., ATM dup 41 kb); 2) a novel genomic deletion including exon 31, and 3) in hemizygosis a point mutation in the non-deleted exon 31. In this study we extended mutation detection to nine new Italian A-T patients, using a combined approach of haplotype analysis, DHPLC and MLPA. Overall we achieved a mutation detection rate of >97%, and can now define a spectrum of ATM mutations based on twenty-one consecutive Italian families with A-T.

  6. A high frequency of distinct ATM gene mutations in ataxia-telangiectasia

    SciTech Connect

    Wright, J.; Teraoka, S.; Concannon, P.

    1996-10-01

    The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families. 33 refs., 1 fig., 1 tab.

  7. ATM function and its relationship with ATM gene mutations in chronic lymphocytic leukemia with the recurrent deletion (11q22.3-23.2).

    PubMed

    Jiang, Y; Chen, H-C; Su, X; Thompson, P A; Liu, X; Do, K-A; Wierda, W; Keating, M J; Plunkett, W

    2016-09-02

    Approximately 10-20% of chronic lymphocytic leukemia (CLL) patients exhibit del(11q22-23) before treatment, this cohort increases to over 40% upon progression following chemoimmunotherapy. The coding sequence of the DNA damage response gene, ataxia-telangiectasia-mutated (ATM), is contained in this deletion. The residual ATM allele is frequently mutated, suggesting a relationship between gene function and clinical response. To investigate this possibility, we sought to develop and validate an assay for the function of ATM protein in these patients. SMC1 (structural maintenance of chromosomes 1) and KAP1 (KRAB-associated protein 1) were found to be unique substrates of ATM kinase by immunoblot detection following ionizing radiation. Using a pool of eight fluorescence in situ hybridization-negative CLL samples as a standard, the phosphorylation of SMC1 and KAP1 from 46 del (11q22-23) samples was analyzed using normal mixture model-based clustering. This identified 13 samples (28%) that were deficient in ATM function. Targeted sequencing of the ATM gene of these samples, with reference to genomic DNA, revealed 12 somatic mutations and 15 germline mutations in these samples. No strong correlation was observed between ATM mutation and function. Therefore, mutation status may not be taken as an indicator of ATM function. Rather, a direct assay of the kinase activity should be used in the development of therapies.

  8. Rats with a missense mutation in Atm display neuroinflammation and neurodegeneration subsequent to accumulation of cytosolic DNA following unrepaired DNA damage.

    PubMed

    Quek, Hazel; Luff, John; Cheung, KaGeen; Kozlov, Sergei; Gatei, Magtouf; Lee, C Soon; Bellingham, Mark C; Noakes, Peter G; Lim, Yi Chieh; Barnett, Nigel L; Dingwall, Steven; Wolvetang, Ernst; Mashimo, Tomoji; Roberts, Tara L; Lavin, Martin F

    2017-04-01

    Mutations in the ataxia-telangiectasia (A-T)-mutated (ATM) gene give rise to the human genetic disorder A-T, characterized by immunodeficiency, cancer predisposition, and neurodegeneration. Whereas a series of animal models recapitulate much of the A-T phenotype, they fail to present with ataxia or neurodegeneration. We describe here the generation of an Atm missense mutant [amino acid change of leucine (L) to proline (P) at position 2262 (L2262P)] rat by intracytoplasmic injection (ICSI) of mutant sperm into oocytes. Atm-mutant rats (Atm(L2262P/L2262P) ) expressed low levels of ATM protein, suggesting a destabilizing effect of the mutation, and had a significantly reduced lifespan compared with Atm(+/+) Whereas these rats did not show cerebellar atrophy, they succumbed to hind-limb paralysis (45%), and the remainder developed tumors. Closer examination revealed the presence of both dsDNA and ssDNA in the cytoplasm of cells in the hippocampus, cerebellum, and spinal cord of Atm(L2262P/L2262P) rats. Significantly increased levels of IFN-β and IL-1β in all 3 tissues were indicative of DNA damage induction of the type 1 IFN response. This was further supported by NF-κB activation, as evidenced by p65 phosphorylation (P65) and translocation to the nucleus in the spinal cord and parahippocampus. Other evidence of neuroinflammation in the brain and spinal cord was the loss of motor neurons and the presence of increased activation of microglia. These data provide support for a proinflammatory phenotype that is manifested in the Atm mutant rat as hind-limb paralysis. This mutant represents a useful model to investigate the importance of neuroinflammation in A-T. © Society for Leukocyte Biology.

  9. Recently emerging signaling landscape of ataxia-telangiectasia mutated (ATM) kinase.

    PubMed

    Farooqi, Ammad Ahmad; Attar, Rukset; Arslan, Belkis Atasever; Romero, Mirna Azalea; ul Haq, Muhammad Fahim; Qadir, Muhammad Imran

    2014-01-01

    Research over the years has progressively and sequentially provided near complete resolution of regulators of the DNA repair pathways which are so important for cancer prevention. Ataxia-telangiectasia mutated kinase (ATM), a high-molecular-weight PI3K-family kinase has emerged as a master regulator of DNA damage signaling and extensive cross-talk between ATM and downstream proteins forms an interlaced signaling network. There is rapidly growing scientific evidence emphasizing newly emerging paradigms in ATM biology. In this review, we provide latest information regarding how oxidative stress induced activation of ATM can be utilized as a therapeutic target in different cancer cell lines and in xenografted mice. Moreover, crosstalk between autophagy and ATM is also discussed with focus on how autophagy inhibition induces apoptosis in cancer cells.

  10. The risk for developing cancer in Israeli ATM, BLM, and FANCC heterozygous mutation carriers.

    PubMed

    Laitman, Yael; Boker-Keinan, Lital; Berkenstadt, Michal; Liphsitz, Irena; Weissglas-Volkov, Daphna; Ries-Levavi, Liat; Sarouk, Ifat; Pras, Elon; Friedman, Eitan

    2016-03-01

    Cancer risks in heterozygous mutation carriers of the ATM, BLM, and FANCC genes are controversial. To shed light on this issue, cancer rates were evaluated by cross referencing asymptomatic Israeli heterozygous mutation carriers in the ATM, BLM, and FANCC genes with cancer diagnoses registered at the Israeli National Cancer Registry (INCR). Comparison of observed to expected Standardized Incidence Rates (SIR) was performed. Overall, 474 individuals participated in the study: 378 females; 25 Arab and 31 Jewish ATM carriers, 152 BLM carriers, and 170 FANCC carriers (all Ashkenazim). Age range at genotyping was 19-53 years (mean + SD 30.6 + 5 years). In addition, 96 males were included; 5, 34, and 57 ATM, BLM, and FANCC mutation carriers, respectively. Over 5-16 years from genotyping (4721 person/years), 15 new cancers were diagnosed in mutation carriers: 5 breast, 4 cervical, 3 melanomas, and one each bone sarcoma, pancreatic, and colorectal cancer. No single cancer diagnosis was more prevalent then expected in all groups combined or per gene analyzed. Specifically breast cancer SIR was 0.02-0.77. We conclude that Israeli ATM, BLM, and FANCC heterozygous mutation carriers are not at an increased risk for developing cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation.

    PubMed

    Lin, Zi-Li; Kim, Nam-Hyung

    2015-06-01

    Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes.

  12. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity.

    PubMed

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-07-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important.

  13. A-TWinnipeg: Pathogenesis of rare ATM missense mutation c.6200C>A with decreased protein expression and downstream signaling, early-onset dystonia, cancer, and life-threatening radiotoxicity

    PubMed Central

    Nakamura, Kotoka; Fike, Francesca; Haghayegh, Sara; Saunders-Pullman, Rachel; Dawson, Angelika J; Dörk, Thilo; Gatti, Richard A

    2014-01-01

    We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important. PMID:25077176

  14. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    PubMed

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  15. Almost 2% of Spanish breast cancer families are associated to germline pathogenic mutations in the ATM gene.

    PubMed

    Tavera-Tapia, A; Pérez-Cabornero, L; Macías, J A; Ceballos, M I; Roncador, G; de la Hoya, M; Barroso, A; Felipe-Ponce, V; Serrano-Blanch, R; Hinojo, C; Miramar-Gallart, M D; Urioste, M; Caldés, T; Santillan-Garzón, S; Benitez, J; Osorio, A

    2017-02-01

    There is still a considerable percentage of hereditary breast and ovarian cancer (HBOC) cases not explained by BRCA1 and BRCA2 genes. In this report, next-generation sequencing (NGS) techniques were applied to identify novel variants and/or genes involved in HBOC susceptibility. Using whole exome sequencing, we identified a novel germline mutation in the moderate-risk gene ATM (c.5441delT; p.Leu1814Trpfs*14) in a family negative for mutations in BRCA1/2 (BRCAX). A case-control association study was performed to establish its prevalence in Spanish population, in a series of 1477 BRCAX families and 589 controls further screened, and NGS panels were used for ATM mutational screening in a cohort of 392 HBOC Spanish BRCAX families and 350 patients affected with diseases not related to breast cancer. Although the interrogated mutation was not prevalent in case-control association study, a comprehensive mutational analysis of the ATM gene revealed 1.78% prevalence of mutations in the ATM gene in HBOC and 1.94% in breast cancer-only BRCAX families in Spanish population, where data about ATM mutations were very limited. ATM mutation prevalence in Spanish population highlights the importance of considering ATM pathogenic variants linked to breast cancer susceptibility.

  16. Inhibition of Ataxia Telangiectasia Mutated (ATM) Kinase Suppresses Herpes Simplex Virus Type 1 (HSV-1) Keratitis

    PubMed Central

    Alekseev, Oleg; Donovan, Kelly; Azizkhan-Clifford, Jane

    2014-01-01

    Purpose. Herpes keratitis (HK) remains the leading cause of cornea-derived blindness in the developed world, despite the availability of effective antiviral drugs. Treatment toxicity and the emergence of drug resistance highlight the need for additional therapeutic approaches. This study examined ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response, as a potential new target for the treatment of HK. Methods. Small molecule inhibitor of ATM (KU-55933) was used to treat herpes simplex virus type 1 (HSV-1) infection in three experimental models: (1) in vitro—cultured human corneal epithelial cells, hTCEpi, (2) ex vivo—organotypically explanted human and rabbit corneas, and (3) in vivo—corneal infection in young C57BL/6J mice. Infection productivity was assayed by plaque assay, real-time PCR, Western blot, and disease scoring. Results. Robust ATM activation was detected in HSV-1-infected human corneal epithelial cells. Inhibition of ATM greatly suppressed viral replication in cultured cells and in explanted human and rabbit corneas, and reduced the severity of stromal keratitis in mice. The antiviral effect of KU-55933 in combination with acyclovir was additive, and KU-55933 suppressed replication of a drug-resistant HSV-1 strain. KU-55933 caused minimal toxicity, as monitored by clonogenic survival assay and fluorescein staining. Conclusions. This study identifies ATM as a potential target for the treatment of HK. ATM inhibition by KU-55933 reduces epithelial infection and stromal disease severity without producing appreciable toxicity. These findings warrant further investigations into the DNA damage response as an area for therapeutic intervention in herpetic ocular diseases. PMID:24370835

  17. Inhibition of ataxia telangiectasia mutated (ATM) kinase suppresses herpes simplex virus type 1 (HSV-1) keratitis.

    PubMed

    Alekseev, Oleg; Donovan, Kelly; Azizkhan-Clifford, Jane

    2014-02-03

    Herpes keratitis (HK) remains the leading cause of cornea-derived blindness in the developed world, despite the availability of effective antiviral drugs. Treatment toxicity and the emergence of drug resistance highlight the need for additional therapeutic approaches. This study examined ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response, as a potential new target for the treatment of HK. Small molecule inhibitor of ATM (KU-55933) was used to treat herpes simplex virus type 1 (HSV-1) infection in three experimental models: (1) in vitro--cultured human corneal epithelial cells, hTCEpi, (2) ex vivo--organotypically explanted human and rabbit corneas, and (3) in vivo--corneal infection in young C57BL/6J mice. Infection productivity was assayed by plaque assay, real-time PCR, Western blot, and disease scoring. Robust ATM activation was detected in HSV-1-infected human corneal epithelial cells. Inhibition of ATM greatly suppressed viral replication in cultured cells and in explanted human and rabbit corneas, and reduced the severity of stromal keratitis in mice. The antiviral effect of KU-55933 in combination with acyclovir was additive, and KU-55933 suppressed replication of a drug-resistant HSV-1 strain. KU-55933 caused minimal toxicity, as monitored by clonogenic survival assay and fluorescein staining. This study identifies ATM as a potential target for the treatment of HK. ATM inhibition by KU-55933 reduces epithelial infection and stromal disease severity without producing appreciable toxicity. These findings warrant further investigations into the DNA damage response as an area for therapeutic intervention in herpetic ocular diseases.

  18. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells.

    PubMed

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-04-08

    During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    SciTech Connect

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-04-08

    Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  20. Ataxia-telangiectasia: Mutations in ATM cDNA detected by protein-truncation screening

    SciTech Connect

    Telatar, M.; Wang, Z.; Udar, N.

    1996-07-01

    We have examined the distal half of the ataxia-telangiectasia (A-T) gene transcript for truncation mutations in 48 A-T affecteds. We found 21 mutations; 4 of the mutations were seen in more than one individual. Genotyping of the individuals sharing mutations, by using nearby microsatellite markers, established that three of the four groups shared common haplotypes, indicating that these were probably founder effects, not public mutations. The one public mutation was found in two American families, one of Ashkenazi Jewish background and the other not. Most truncations deleted the PI3-kinase domain, although some exceptions to this were found in patients with typical A-T phenotypes. All patients not previously known to be consanguineous were found to be compound heterozygotes when mutations could be identified - that is, normal and abnormal protein segments were seen on SDS-PAGE gels. All 48 patients gave RT-PCR products, indicating the presence of relatively stable mRNAs despite their mutations. These results suggest that few public mutations or hot spots can be expected in the A-T gene and that epidemiological studies of A-T carrier status and associated health risks will have to be designed around populations with frequent founder-effect mutations, despite the obvious limitations of this approach. 20 refs., 4 figs., 1 tab.

  1. Concurrent Mutations in ATM and Genes Associated with Common γ Chain Signaling in Peripheral T Cell Lymphoma.

    PubMed

    Simpson, Haley M; Khan, Rashid Z; Song, Chang; Sharma, Deva; Sadashivaiah, Kavitha; Furusawa, Aki; Liu, Xinyue; Nagaraj, Sushma; Sengamalay, Naomi; Sadzewicz, Lisa; Tallon, Luke J; Chen, Qing C; Livak, Ferenc; Rapoport, Aaron P; Kimball, Amy; Banerjee, Arnob

    2015-01-01

    Peripheral T cell lymphoma (PTCL) is a heterogeneous malignancy with poor response to current therapeutic strategies and incompletely characterized genetics. We conducted whole exome sequencing of matched PTCL and non-malignant samples from 12 patients, spanning 8 subtypes, to identify potential oncogenic mutations in PTCL. Analysis of the mutations identified using computational algorithms, CHASM, PolyPhen2, PROVEAN, and MutationAssessor to predict the impact of these mutations on protein function and PTCL tumorigenesis, revealed 104 somatic mutations that were selected as high impact by all four algorithms. Our analysis identified recurrent somatic missense or nonsense mutations in 70 genes, 9 of which contained mutations predicted significant by all 4 algorithms: ATM, RUNX1T1, WDR17, NTRK3, TP53, TRMT12, CACNA2D1, INTS8, and KCNH8. We observed somatic mutations in ATM (ataxia telangiectasia-mutated) in 5 out of the 12 samples and mutations in the common gamma chain (γc) signaling pathway (JAK3, IL2RG, STAT5B) in 3 samples, all of which also harbored mutations in ATM. Our findings contribute insights into the genetics of PTCL and suggest a relationship between γc signaling and ATM in T cell malignancy.

  2. Lower ataxia telangiectasia mutated (ATM) mRNA expression is correlated with poor outcome of laryngeal and pharyngeal cancer patients.

    PubMed

    Lee, K-W; Tsai, Y-S; Chiang, F-Y; Huang, J-L; Ho, K-Y; Yang, Y-H; Kuo, W-R; Chen, M-K; Lin, C-S

    2011-05-01

    Ataxia telangiectasia mutated (ATM) kinase is a critical regulator in initiating DNA damage response and activating DNA repair. However, the correlation between ATM expression and the outcome of laryngopharyngeal cancer patients is unknown. We hypothesize that ATM expression is correlated with a worse outcome in laryngopharyngeal cancer patients. The ATM messenger RNA (mRNA) expression of 80 tumors of laryngeal and pharyngeal cancer was examined by real-time quantitative RT-PCR. Overall survival rates were measured using Kaplan-Meier estimates and the log-rank tests. The adjusted hazard rate ratios (HRRs) were computed by multivariate Cox regressions. Reduced ATM mRNA was found in 65 of 80 studied cases. Lower ATM expression [tumor/normal <0.3, HRR = 2.49; 95% confidence interval (CI) 1.27-4.88], younger age (<55 years, HRR = 2.71; 95% CI 1.16-6.32), and larger tumor (T(3)/T(4), HRR = 2.21; 95% CI 1.10-4.44) were independent risk factors for survival. Patients with lower ATM and younger age (HRR = 6.51; 95% CI 2.05-20.66) or with lower ATM and T(3)/T(4) tumor (HRR = 5.23; 95% CI 2.04-13.40) exhibited the poorest outcome. The expression of ATM mRNA, which is frequently downregulated in laryngeal and pharyngeal cancers, could be a valuable prognostic marker.

  3. Ataxia telangiectasia mutated (ATM)-mediated DNA damage response in oxidative stress-induced vascular endothelial cell senescence.

    PubMed

    Zhan, Hong; Suzuki, Toru; Aizawa, Kenichi; Miyagawa, Kiyoshi; Nagai, Ryozo

    2010-09-17

    Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

  4. Enhanced cytotoxicity of PARP inhibition in mantle cell lymphoma harbouring mutations in both ATM and p53

    PubMed Central

    Williamson, Chris T; Kubota, Eiji; Hamill, Jeffrey D; Klimowicz, Alexander; Ye, Ruiqiong; Muzik, Huong; Dean, Michelle; Tu, LiRen; Gilley, David; Magliocco, Anthony M; McKay, Bruce C; Bebb, D Gwyn; Lees-Miller, Susan P

    2012-01-01

    Poly-ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia-telangiectasia mutated (ATM)-deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM-deficient MCL cells, olaparib induced DNA-PK-dependent phosphorylation and stabilization of p53 as well as expression of p53-responsive cell cycle checkpoint regulators, and inhibition of DNA-PK reduced the toxicity of olaparib in ATM-deficient MCL cells. Thus, both DNA-PK and p53 regulate the response of ATM-deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53-deficient malignancies. PMID:22416035

  5. Tele-ultrasound using ATM over a T-1 satellite connection

    NASA Astrophysics Data System (ADS)

    Williamson, Morgan P.; Suitor, Charles T.; de Treville, Robert E.; Freckleton, Michael W.; Kinsey, Van; Goeringer, Fred; Lyche, David K.; Hunter, Bruce; Jennings, Neal E.; Shelton, Philip D.; Marcy, Jon; Poore, Tom; North, Jack

    1996-04-01

    In September 1995 the United States military conducted a demonstration project to provide live ultrasound video and diagnostic DICOM still images using GTE's asynchronous transfer mode (ATM) technologies over an Orion T-1 satellite link. Still images were frame-grabbed from a Diasonics ultrasound and sent to the ALI Wide Area Network system. A group of diagnostic images was then sent in DICOM 3.0 format over a virtual ethernet satellite link from Chantilly, Virginia to Dayton, Ohio. These images came across a DICOM gateway into the Medical Diagnostic Imaging Support (MDIS) System. Live video from the ultrasound was also routed through a CLI Radiance VTC over the satellite to a VTC in Ohio. The video bandwidth was progressively narrowed with two radiologists determining the minimal acceptable bandwidth for detecting test objects in a phantom. The radiologists accepted live video ultrasound at bandwidths as low as 384 kbps from the hands of an experienced ultrasonographer located hundreds of miles away. DICOM still images were sent uncompressed and were of acceptable image quality when viewed on the MDIS system. The technology demonstrated holds great promise for both deployed U.S. Military Forces and civil uses of remote radiology. Detailed network drawings and videotapes of the ultrasound examinations at the remote site are provided.

  6. A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints.

    PubMed

    Mu, Jung-Jung; Wang, Yi; Luo, Hao; Leng, Mei; Zhang, Jinglan; Yang, Tao; Besusso, Dario; Jung, Sung Yun; Qin, Jun

    2007-06-15

    ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

  7. Correlation of Ataxia-Telangiectasia-Mutated (ATM) gene loss with outcome in head and neck squamous cell carcinoma.

    PubMed

    Lim, Annette M; Young, Richard J; Collins, Marnie; Fox, Stephen B; McArthur, Grant A; Corry, June; Peters, Lester; Rischin, Danny; Solomon, Benjamin

    2012-08-01

    Ataxia-Telangiectasia-Mutated (ATM) gene loss has been associated with poor prognosis and treatment resistance in head and neck squamous cell carcinomas (HNSCC). We investigated the relationship between ATM loss detected by fluorescence in-situ hybridisation (FISH) with patient outcome, and its relationship with Human Papillomavirus (HPV)/p16(INK4A) status. Copy number of the ATM gene and chromosome 11 were determined by FISH and HPV status was determined using p16(INK4A) immunohistochemistry in 87 paraffin embedded tumour samples from patients with HNSCC treated with chemoradiation at a single institution. ATM loss was correlated with patient outcome as both a continuous and dichotomous variable. Of 73 evaluable patients, 44 (60.3%) demonstrated loss of the ATM gene. There was no correlation between ATM loss (defined as a mean ratio of ATM: chromosome 11<0.75) and overall survival (OS, p=0.67) or time to locoregional failure (TTLRF, p=0.72). Similarly, when evaluated as a continuous variable there was no significant relationship between ATM loss and patient outcome (OS, p=0.89; TTLRF, p=0.21). No significant relationship was found between p16(INK4A) status and ATM loss, for patient outcome. We found 35.6% (n=26) of patients demonstrated polysomy of chromosome 11 (defined as the presence of a mean >2.5 copies of chromosome 11) which was significantly associated with p16(INK4A) negative status (p=0.0004), but did not influence outcome. ATM loss is a frequent event in HNSCC, however it does not impact outcome after treatment with chemoradiation. Polysomy of chromosome 11 was significantly associated with p16(INK4A) negative status but also lacks prognostic significance. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

    PubMed

    Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

    2016-05-01

    Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. New Diagnosis of Atypical Ataxia-Telangiectasia in a 17-Year-Old Boy with T-Cell Acute Lymphoblastic Leukemia and a Novel ATM mutation

    PubMed Central

    Roohi, Jasmin; Crowe, Jennifer; Loredan, Denis; Anyane-Yeboa, Kwame; Mansukhani, Mahesh M.; Omesi, Lenore; Levine, Jennifer; Politi, Anya Revah; Zha, Shan

    2017-01-01

    Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM gene. Typically it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM (c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T. PMID:28123174

  10. New diagnosis of atypical ataxia-telangiectasia in a 17-year-old boy with T-cell acute lymphoblastic leukemia and a novel ATM mutation.

    PubMed

    Roohi, Jasmin; Crowe, Jennifer; Loredan, Denis; Anyane-Yeboa, Kwame; Mansukhani, Mahesh M; Omesi, Lenore; Levine, Jennifer; Revah Politi, Anya; Zha, Shan

    2017-04-01

    Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM gene. Typically, it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM (c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T.

  11. Cytoplasmic ATM in neurons modulates synaptic function.

    PubMed

    Li, Jiali; Han, Yu R; Plummer, Mark R; Herrup, Karl

    2009-12-29

    ATM is a PI 3-kinase involved in DNA double-strand break repair. ATM deficiency leads to ataxia-telangiectasia (A-T), a syndrome of cancer susceptibility, hypersensitivity to ionizing radiation, immune deficiency, and sterility [1, 2]-phenotypes that can straightforwardly be attributed to a defective response to DNA damage. Yet patients with A-T also suffer from ataxia, speech defects, and abnormal body movements [3-5]-neurological phenotypes whose origins remain largely unexplained. Compounding the discordance, Atm mutations in mouse interfere with DNA repair but have only mild neurological symptoms [6-9], suggesting that the link between DNA damage and the death of neurons can be broken [10-12]. We find that in neurons, ATM protein has a substantial cytoplasmic distribution. We show that in Atm(tm1Awb) mice, hippocampal long-term potentiation is significantly reduced, as is the rate of spontaneous vesicular dye release, suggesting a functional importance of cytoplasmic ATM. In the cytoplasm, ATM forms a complex with two synaptic vesicle proteins, VAMP2 and synapsin-I, both of which must be phosphorylated to bind ATM. Also, cytoplasmic ATM physically associates with the homologous PI 3-kinase, ATR. The neurological symptoms of ataxia-telangiectasia may thus result from defective nonnuclear functions of ATM not associated with DNA repair.

  12. The impact of an early truncating founder ATM mutation on immunoglobulins, specific antibodies and lymphocyte populations in ataxia-telangiectasia patients and their parents

    PubMed Central

    STRAY-PEDERSEN, A; JÓNSSON, T; HEIBERG, A; LINDMAN, C R; WIDING, E; AABERGE, I S; BORRESEN-DALE, A L; ABRAHAMSEN, T G

    2004-01-01

    Eleven Norwegian patients (aged 2–33 years, seven males and four females) with Ataxia-telangiectasia (A-T) and their parents were investigated. Five of the patients were homozygous for the same ATM mutation, 3245delATCinsTGAT, a Norwegian founder mutation. They had the lowest IgG2 levels; mean (95% confidence interval) 0·23 (0·05–0·41) g/l versus 0·91 (0·58–1·26) g/l in the other patients (P = 0·002). Among the 11 A-T patients, six had IgG2 deficiency, six had IgA deficiency (three in combination with IgG2 deficiency) and seven had low/undetectable IgE values. All patients had very low levels of antibodies to Streptococcus pneumoniae 0·9 (0·4–1·4) U/ml, while normal levels were found in their parents 11·1 (8·7–13·4) U/ml (P < 0·001). A positive linear relationship between pneumococcal antibodies and IgG2 (r = 0·85, P = 0·001) was found in the patients. Six of 11 had diphtheria antibodies and 7 of 11 tetanus antibodies after childhood vaccinations, while 4 of 7 Hemophilus influenzae type b (Hib) vaccinated patients had protective antibodies. Ten patients had low B cell (CD19+) counts, while six had low T cell (CD3+) counts. Of the T cell subpopulations, 11 had low CD4+ cell counts, six had reduced CD8+ cell counts, and four had an increased portion of double negative (CD3+/CD4-/CD8-) gamma delta T cells. Of the 22 parents (aged 23–64 years) 12 were heterozygous for the ATM founder mutation. Abnormalities in immunoglobulin levels and/or lymphocyte subpopulations were also observed in these carriers, with no correlation to a special ATM genotype. PMID:15196260

  13. Distinct in vitro sensitivity of p53-mutated and ATM-mutated chronic lymphocytic leukemia cells to ofatumumab and rituximab.

    PubMed

    Sebejova, Ludmila; Borsky, Marek; Jaskova, Zuzana; Potesil, David; Navrkalova, Veronika; Malcikova, Jitka; Sramek, Martin; Doubek, Michael; Loja, Tomas; Pospisilova, Sarka; Mayer, Jiri; Trbusek, Martin

    2014-10-01

    Abnormalities in ATM and TP53 genes represent important predictive factors in chronic lymphocytic leukemia (CLL); however, the efficacy of CD20 targeting immunotherapy is only poorly defined in the affected patients. Therefore, we tested the in vitro response to ofatumumab (OFA) and rituximab (RTX) in 75 CLL samples with clearly defined p53 or ATM inactivation. Using standard conditions allowing complement-dependent cytotoxicity, i.e., 10 μg/mL of antibodies and 20% active human serum, we observed clear differences among the tested genetic categories: ATM-mutated samples (n = 17) represented the most sensitive, wild-type samples (n = 31) intermediate, and TP53-mutated samples (n = 27) the most resistant group (ATM-mut vs. TP53-mut: P = 0.0005 for OFA and P = 0.01 for RTX). The response correlated with distinct levels of CD20 and critical complement inhibitors CD55 and CD59; CD20 level median was the highest in ATM-mutated and the lowest in TP53-mutated samples (difference between the groups P < 0.01), while the total level of complement inhibitors (CD55 plus CD59) was distributed in the opposite manner (P < 0.01). Negligible response to both OFA and RTX was noted in all cultures (n = 10) tested in the absence of active serum, which strongly indicated that complement-dependent cytotoxicity was a principal cell death mechanism. Our study shows that (1) common genetic defects in CLL cells significantly impact a primary response to anti-CD20 monoclonal antibodies and (2) ATM-mutated patients with currently poor prognosis may potentially benefit from immunotherapy targeting CD20. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  14. Dexamethasone partially rescues ataxia telangiectasia-mutated (ATM) deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity.

    PubMed

    Menotta, Michele; Biagiotti, Sara; Bianchi, Marzia; Chessa, Luciana; Magnani, Mauro

    2012-11-30

    Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of nucleotide residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.

  15. A new type of mutation causes a splicing defect in ATM.

    PubMed

    Pagani, Franco; Buratti, Emanuele; Stuani, Cristiana; Bendix, Regina; Dörk, Thilo; Baralle, Francisco E

    2002-04-01

    Disease-causing splicing mutations described in the literature primarily produce changes in splice sites and, to a lesser extent, variations in exon-regulatory sequences such as the enhancer elements. The gene ATM is mutated in individuals with ataxia-telangiectasia; we have identified the aberrant inclusion of a cryptic exon of 65 bp in one affected individual with a deletion of four nucleotides (GTAA) in intron 20. The deletion is located 12 bp downstream and 53 bp upstream from the 5' and 3' ends of the cryptic exon, respectively. Through analysis of the splicing defect using a hybrid minigene system, we identified a new intron-splicing processing element (ISPE) complementary to U1 snRNA, the RNA component of the U1 small nuclear ribonucleoprotein (snRNP). This element mediates accurate intron processing and interacts specifically with U1 snRNP particles. The 4-nt deletion completely abolished this interaction, causing activation of the cryptic exon. On the basis of this analysis, we describe a new type of U1 snRNP binding site in an intron that is essential for accurate intron removal. Deletion of this sequence is directly involved in the splicing processing defect.

  16. ATM mutation rather than BIRC3 deletion and/or mutation predicts reduced survival in 11q-deleted chronic lymphocytic leukemia: data from the UK LRF CLL4 trial.

    PubMed

    Rose-Zerilli, Matthew J J; Forster, Jade; Parker, Helen; Parker, Anton; Rodríguez, Ana E; Chaplin, Tracy; Gardiner, Anne; Steele, Andrew J; Collins, Andrew; Young, Bryan D; Skowronska, Anna; Catovsky, Daniel; Stankovic, Tatjana; Oscier, David G; Strefford, Jonathan C

    2014-04-01

    ATM mutation and BIRC3 deletion and/or mutation have independently been shown to have prognostic significance in chronic lymphocytic leukemia. However, the relative clinical importance of these abnormalities in patients with a deletion of 11q encompassing the ATM gene has not been established. We screened a cohort of 166 patients enriched for 11q-deletions for ATM mutations and BIRC3 deletion and mutation and determined the overall and progression-free survival among the 133 of these cases treated within the UK LRF CLL4 trial. SNP6.0 profiling demonstrated that BIRC3 deletion occurred in 83% of 11q-deleted cases and always co-existed with ATM deletion. For the first time we have demonstrated that 40% of BIRC3-deleted cases have concomitant deletion and mutation of ATM. While BIRC3 mutations were rare, they exclusively occurred with BIRC3 deletion and a wild-type residual ATM allele. In 11q-deleted cases, we confirmed that ATM mutation was associated with a reduced overall and progression-free survival comparable to that seen with TP53 abnormalities, whereas BIRC3 deletion and/or mutation had no impact on overall and progression-free survival. In conclusion, in 11q-deleted patients treated with first-line chemotherapy, ATM mutation rather than BIRC3 deletion and/or mutation identifies a subgroup with a poorer outcome.

  17. Nitric oxide induces ataxia telangiectasia mutated (ATM) protein-dependent γH2AX protein formation in pancreatic β cells.

    PubMed

    Oleson, Bryndon J; Broniowska, Katarzyna A; Schreiber, Katherine H; Tarakanova, Vera L; Corbett, John A

    2014-04-18

    In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

  18. ATM gene mutations result in both recessive and dominant expression phenotypes of genes and microRNAs.

    PubMed

    Smirnov, Denis A; Cheung, Vivian G

    2008-08-01

    The defining characteristic of recessive disorders is the absence of disease in heterozygous carriers of the mutant alleles. However, it has been recognized that recessive carriers may differ from noncarriers in some phenotypes. Here, we studied ataxia telangiectasia (AT), a classical recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. We compared the gene and microRNA expression phenotypes of noncarriers, AT carriers who have one copy of the ATM mutations, and AT patients with two copies of ATM mutations. We found that some phenotypes are more similar between noncarriers and AT carriers compared to AT patients, as expected for a recessive disorder. However, for some expression phenotypes, AT carriers are more similar to the patients than to the noncarriers. Analysis of one of these expression phenotypes, TNFSF4 level, allowed us to uncover a regulatory pathway where ATM regulates TNFSF4 expression through MIRN125B (also known as miR-125b or miR125b) [corrected] In AT carriers and AT patients, this pathway is disrupted. As a result, the level of MIRN125B is lower and the level of its target gene, TNFSF4, is higher than in noncarriers. A decreased level of MIRN125B is associated with breast cancer, and an elevated level of TNFSF4 is associated with atherosclerosis. Thus, our findings provide a mechanistic suggestion for the increased risk of breast cancer and heart disease in AT carriers. By integrating molecular and computational analyses of gene and microRNA expression, we show the complex consequences of a human gene mutation.

  19. Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism.

    PubMed

    Mandriota, Stefano J; Valentijn, Linda J; Lesne, Laurence; Betts, David R; Marino, Denis; Boudal-Khoshbeen, Mary; London, Wendy B; Rougemont, Anne-Laure; Attiyeh, Edward F; Maris, John M; Hogarty, Michael D; Koster, Jan; Molenaar, Jan J; Versteeg, Rogier; Ansari, Marc; Gumy-Pause, Fabienne

    2015-07-30

    Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma.

  20. Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism

    PubMed Central

    Mandriota, Stefano J.; Valentijn, Linda J.; Lesne, Laurence; Betts, David R.; Marino, Denis; Boudal-Khoshbeen, Mary; London, Wendy B.; Rougemont, Anne-Laure; Attiyeh, Edward F.; Maris, John M.; Hogarty, Michael D.; Koster, Jan; Molenaar, Jan J.; Versteeg, Rogier

    2015-01-01

    Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma. PMID:26053094

  1. ATM, radiation, and the risk of second primary breast cancer.

    PubMed

    Bernstein, Jonine L; Concannon, Patrick

    2017-07-27

    It was first suggested more than 40 years ago that heterozygous carriers for the human autosomal recessive disorder Ataxia-Telangiectasia (A-T) might also be at increased risk for cancer. Subsequent studies have identified the responsible gene, Ataxia-Telangiectasia Mutated (ATM), characterized genetic variation at this locus in A-T and a variety of different cancers, and described the functions of the ATM protein with regard to cellular DNA damage responses. However, an overall model of how ATM contributes to cancer risk, and in particular, the role of DNA damage in this process, remains lacking. This review considers these questions in the context of contralateral breast cancer (CBC). Heterozygous carriers of loss of function mutations in ATM that are A-T causing, are at increased risk of breast cancer. However, examination of a range of genetic variants, both rare and common, across multiple cancers, suggests that ATM may have additional effects on cancer risk that are allele-dependent. In the case of CBC, selected common alleles at ATM are associated with a reduced incidence of CBC, while other rare and predicted deleterious variants may act jointly with radiation exposure to increase risk. Further studies that characterize germline and somatic ATM mutations in breast cancer and relate the detected genetic changes to functional outcomes, particularly with regard to radiation responses, are needed to gain a complete picture of the complex relationship between ATM, radiation and breast cancer.

  2. Ataxia-telangiectasia mutated (ATM) participates in the regulation of ionizing radiation-induced cell death via MAPK14 in lung cancer H1299 cells.

    PubMed

    Liang, Nan; Zhong, Rui; Hou, Xue; Zhao, Gang; Ma, Shumei; Cheng, Guanghui; Liu, Xiaodong

    2015-10-01

    The role of Ataxia-telangiectasia mutated (ATM) in response to DNA damage has previously been studied, but its underlying mechanisms specific to ionizing radiation (IR) have remained to be elucidated. In this study, function of ATM on radiation-induced cell death in lung cancer H1299 cells was analysed. Human lung cancer cells, H1299, were used, and cell models with ATM(-/-) and MAPK14(-/-) were established by genetic engineering. Radiosensitivity was analysed using colony formation assays. Western blotting and co-immunoprecipitation were implemented to detect protein expression and interaction. MDC staining and GFP-LC3 relocalization were used to detect autophagy. Autophagy as well as phosphorylation of ATM was activated by ionizing radiation. Both the inhibitor of ATM, KU55933 and ATM silencing reduced phosphorylation of ATM and MAPKAPK2 expression. Both ATM(-/-) and MAPK14(-/-) cells displayed hypersensitivity. IR increased autophagy level by more than 129% in DMSO-treated cells, while only by 47% and 27% in KU55933-treated and ATM(-/-) cells respectively. MAPK14 knock-down alone gave rise to the basal autophagy level, but decreased notably after IR. KU55933 and ATM knock-down inhibited IR-induced autophagy by activating mTOR pathways. Both Beclin1-PI3KIII and Beclin1-MAPKAPK2 interactions as were remarkably affected by silencing either ATM or MAPK14. ATM promoted IR-induced autophagy via the MAPK14 pathway, mTOR pathway and Beclin1/PI3KIII complexes. MAPK14 contributed to radiosensitization of H1299 cells. © 2015 John Wiley & Sons Ltd.

  3. Strain background determines lymphoma incidence in Atm knockout mice.

    PubMed

    Genik, Paula C; Bielefeldt-Ohmann, Helle; Liu, Xianan; Story, Michael D; Ding, Lianghao; Bush, Jamie M; Fallgren, Christina M; Weil, Michael M

    2014-02-01

    About 10% to 30% of patients with ataxia-telangiectasia (A-T) develop leukemias or lymphomas. There is considerable interpatient variation in the age of onset and leukemia/lymphoma type. The incomplete penetrance and variable age of onset may be attributable to several factors. These include competing mortality from other A-T-associated pathologies, particularly neurodegeneration and interstitial lung disease, allele-specific effects of ataxia-telangiectasia mutated (ATM) gene mutations. There is also limited evidence from clinical observations and studies using Atm knockout mice that modifier genes may account for some variation in leukemia/lymphoma susceptibility. We have introgressed the Atm(tm1Awb) knockout allele (Atm(-)) onto several inbred murine strains and observed differences in thymic lymphoma incidence and latency between Atm(-/-) mice on the different strain backgrounds and between their F1 hybrids. The lymphomas that arose in these mice had a pattern of sequence gains and losses that were similar to those previously described by others. These results provide further evidence for the existence of modifier genes controlling lymphomagenesis in individuals carrying defective copies of Atm, at least in mice, the characterized Atm(-) congenic strain set provides a resource with which to identify these genes. In addition, we found that fewer than expected Atm(-/-) pups were weaned on two strain backgrounds and that there was no correlation between body weight of young Atm-/- mice and lymphoma incidence or latency. Copyright © 2014 Neoplasia Press, Inc. All rights reserved.

  4. A new series of small molecular weight compounds induce read through of all three types of nonsense mutations in the ATM gene.

    PubMed

    Du, Liutao; Jung, Michael E; Damoiseaux, Robert; Completo, Gladys; Fike, Francesca; Ku, Jin-Mo; Nahas, Shareef; Piao, Cijing; Hu, Hailiang; Gatti, Richard A

    2013-09-01

    Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs.

  5. A New Series of Small Molecular Weight Compounds Induce Read Through of All Three Types of Nonsense Mutations in the ATM Gene

    PubMed Central

    Du, Liutao; Jung, Michael E; Damoiseaux, Robert; Completo, Gladys; Fike, Francesca; Ku, Jin-Mo; Nahas, Shareef; Piao, Cijing; Hu, Hailiang; Gatti, Richard A

    2013-01-01

    Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs. PMID:23774824

  6. Variable in vivo embryoprotective role for ataxia-telangiectasia-mutated against constitutive and phenytoin-enhanced oxidative stress in atm knockout mice.

    PubMed

    Bhuller, Yadvinder; Jeng, Winnie; Wells, Peter G

    2006-09-01

    Knockout mice lacking the ataxia-telangiectasia-mutated (Atm) protein exhibit impaired detection and repair of DNA damage and increased embryopathies from ionizing radiation in vivo, and vehicle or phenytoin in embryo culture. Here we determined if Atm-deficient mice are more susceptible in vivo to phenytoin embryopathies. Wild-type (+/+) or heterozygous (+/-) Atm knockout dams were mated with +/- males, pregnant dams were treated with phenytoin (65 mg/kg ip) or its vehicle, and resorptions and fetuses were genotyped and characterized. This strain proved resistant to phenytoin-initiated cleft palates but not to other spontaneous and phenytoin-enhanced embryopathies. With vehicle-treated +/- dams, fetal body weight was lower in homozygous Atm-null (-/-) fetuses compared to +/- and +/+ littermates (p < 0.05). Phenytoin enhanced this Atm-dependent embryopathic pattern (p < 0.05). It also enhanced DNA oxidation in -/- Atm-deficient embryos compared to its +/- Atm-deficient (p < 0.001) and +/+ Atm-normal (p < 0.001), phenytoin-exposed littermates and to its -/- vehicle controls (p < 0.01). Postpartum lethality was greater in both +/- and -/- Atm-deficient fetuses compared to +/+ littermates, independent of treatment (0.05 < p < 0.1). By maternal genotype, +/- Atm-deficient dams had fewer implantations than +/+ dams, independent of treatment, and phenytoin decreased litter size (p < 0.05). Conversely, phenytoin-exposed +/+ fetuses were more likely than -/- littermates to die in utero (p < 0.05), and in +/+ dams fetal resorptions and postpartum lethality were variably higher and enhanced by phenytoin (p < 0.05). Despite variable actions in vivo, the embryoprotective effects of Atm suggest a role for reactive oxygen species and oxidative DNA damage in some spontaneous and phenytoin-enhanced embryopathies.

  7. Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*

    PubMed Central

    Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl

    2015-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis). PMID:25561503

  8. ATM mutations and E-cadherin expression define sensitivity to EGFR-targeted therapy in colorectal cancer.

    PubMed

    Geißler, Anna-Lena; Geißler, Miriam; Kottmann, Daniel; Lutz, Lisa; Fichter, Christiane D; Fritsch, Ralph; Weddeling, Britta; Makowiec, Frank; Werner, Martin; Lassmann, Silke

    2017-02-09

    EGFR-targeted therapy is a key treatment approach in patients with RAS wildtype metastatic colorectal cancers (CRC). Still, also RAS wildtype CRC may be resistant to EGFR-targeted therapy, with few predictive markers available for improved stratification of patients. Here, we investigated response of 7 CRC cell lines (Caco-2, DLD1, HCT116, HT29, LS174T, RKO, SW480) to Cetuximab and correlated this to NGS-based mutation profiles, EGFR promoter methylation and EGFR expression status as well as to E-cadherin expression. Moreover, tissue specimens of primary and/or recurrent tumors as well as liver and/or lung metastases of 25 CRC patients having received Cetuximab and/or Panitumumab were examined for the same molecular markers. In vitro and in situ analyses showed that EGFR promoter methylation and EGFR expression as well as the MSI and or CIMP-type status did not guide treatment responses. In fact, EGFR-targeted treatment responses were also observed in RAS exon 2 p.G13 mutated CRC cell lines or CRC cases and were further linked to PIK3CA exon 9 mutations. In contrast, non-response to EGFR-targeted treatment was associated with ATM mutations and low E-cadherin expression. Moreover, down-regulation of E-cadherin by siRNA in otherwise Cetuximab responding E-cadherin positive cells abrogated their response. Hence, we here identify ATM and E-cadherin expression as potential novel supportive predictive markers for EGFR-targeted therapy.

  9. Concordance of ATM (ataxia telangiectasia mutated) immunohistochemistry between biopsy or metastatic tumor samples and primary tumors in gastric cancer patients.

    PubMed

    Kim, Hee Sung; Kim, Min A; Hodgson, Darren; Harbron, Christopher; Wellings, Robert; O'Connor, Mark J; Womack, Christopher; Yin, Xiaolu; Bang, Yung-Jue; Im, Seok-Ah; Lee, Byung Lan; Kim, Woo Ho

    2013-01-01

    ATM (ataxia telangiectasia mutated) is one of several DNA repair proteins that are suggested to sensitize tumor cells to the poly(ADP-ribose) polymerase inhibitor olaparib when deficient. The aim of this study was to assess the spatiotemporal concordance of ATM immunohistochemistry (IHC) in gastric cancer in order to determine if measurements made at the level of various sample types and times could be inferred as having the potential to be relevant to treatment decisions made at the patient level. Two independent cohorts composed of 591 gastric cancer patients divided into a gastrectomy cohort (n = 450) and a metastasis cohort (n = 141) were used in this study. A total of 2,705 ATM IHC samples were examined, including 450 whole tissue, 3 sets of 450 tissue microarray (TMA), 301 biopsy, 222 metastatic tumor and 2 additional whole tissue samples of 50 cases from the gastrectomy cohort, and 141 pairs of primary and metastatic tumors from the metastasis cohort. The prevalence of ATM negativity was 13.1% in biopsies, 13.9, 15.1, and 16.0% in TMAs and 15.9% in whole tissue samples of the gastrectomy cohort, and 21.4% in primary tumor and 21.5% in metastatic tumor samples of the metastasis cohort. coefficients were 0.341 for biopsy, 0.572 as the average of 3 TMAs and 0.415 for the largely synchronous metastatic tumors of the gastrectomy cohort, and 0.153 for the largely asynchronous metastatic tumors of the metastasis cohort. Using whole tissue sections from tumor resections or primary tumor, respectively, as the reference standards, specificity and sensitivity were 91.6 and 41.0% for biopsy, 93.9 and 61.9% as the average of 3 TMAs, and 86.6 and 58.8% for metastatic tumors of the gastrectomy cohort and 81.7 and 33.3% for metastatic tumors of the metastasis cohort, respectively. Although we have demonstrated that the IHC assay for ATM was robust and reproducible in gastric tumor samples, we have also found that measurements were subject to significant discordance across

  10. Strain Background Determines Lymphoma Incidence in Atm Knockout Mice12

    PubMed Central

    Genik, Paula C; Bielefeldt-Ohmann, Helle; Liu, Xianan; Story, Michael D; Ding, Lianghao; Bush, Jamie M; Fallgren, Christina M; Weil, Michael M

    2014-01-01

    About 10% to 30% of patients with ataxia-telangiectasia (A-T) develop leukemias or lymphomas. There is considerable interpatient variation in the age of onset and leukemia/lymphoma type. The incomplete penetrance and variable age of onset may be attributable to several factors. These include competing mortality from other A-T-associated pathologies, particularly neurodegeneration and interstitial lung disease, and allele-specific effects of ataxia-telangiectasia mutated (ATM) gene mutations. There is also limited evidence from clinical observations and studies using Atm knockout mice that modifier genes may account for some variation in leukemia/lymphoma susceptibility. We have introgressed the Atmtm1Awb knockout allele (Atm-) onto several inbred murine strains and observed differences in thymic lymphoma incidence and latency between Atm-/- mice on the different strain backgrounds and between their F1 hybrids. The lymphomas that arose in these mice had a pattern of sequence gains and losses that were similar to those previously described by others. These results provide further evidence for the existence of modifier genes controlling lymphomagenesis in individuals carrying defective copies of Atm, at least in mice, and the characterized Atm- congenic strain set provides a resource with which to identify these genes. In addition, we found that fewer than expected Atm-/- pups were weaned on two strain backgrounds and that there was no correlation between body weight of young Atm-/- mice and lymphoma incidence or latency. PMID:24709420

  11. Loss of ATM kinase activity leads to embryonic lethality in mice

    PubMed Central

    Pellegrini, Manuela; Lee, Baeck-Seung; Guo, Zhi; Filsuf, Darius; Belkina, Natalya V.; You, Zhongsheng; Paull, Tanya T.; Sleckman, Barry P.; Feigenbaum, Lionel

    2012-01-01

    Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis. PMID:22869595

  12. Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia

    PubMed Central

    Nadeu, Ferran; Delgado, Julio; Royo, Cristina; Baumann, Tycho; Stankovic, Tatjana; Pinyol, Magda; Jares, Pedro; Navarro, Alba; Martín-García, David; Beà, Sílvia; Salaverria, Itziar; Oldreive, Ceri; Aymerich, Marta; Suárez-Cisneros, Helena; Rozman, Maria; Villamor, Neus; Colomer, Dolors; López-Guillermo, Armando; González, Marcos; Alcoceba, Miguel; Terol, Maria José; Colado, Enrique; Puente, Xose S.; López-Otín, Carlos; Enjuanes, Anna

    2016-01-01

    Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients. PMID:26837699

  13. Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia.

    PubMed

    Nadeu, Ferran; Delgado, Julio; Royo, Cristina; Baumann, Tycho; Stankovic, Tatjana; Pinyol, Magda; Jares, Pedro; Navarro, Alba; Martín-García, David; Beà, Sílvia; Salaverria, Itziar; Oldreive, Ceri; Aymerich, Marta; Suárez-Cisneros, Helena; Rozman, Maria; Villamor, Neus; Colomer, Dolors; López-Guillermo, Armando; González, Marcos; Alcoceba, Miguel; Terol, Maria José; Colado, Enrique; Puente, Xose S; López-Otín, Carlos; Enjuanes, Anna; Campo, Elías

    2016-04-28

    Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients. © 2016 by The American Society of Hematology.

  14. Isolation and characterization of Xenopus ATM (X-ATM): expression, localization, and complex formation during oogenesis and early development.

    PubMed

    Robertson, K; Hensey, C; Gautier, J

    1999-11-25

    ATM, the gene product mutated in Ataxia Telangiectasia (A-T) encodes a 350-kDa protein involved in the regulation of several cellular responses to DNA breaks. We used a degenerate PCR-based strategy to isolate a partial clone of X-ATM, the Xenopus homologue of human ATM. Sequence analysis and confirmed that the clone was most closely related to human ATM. Xenopus ATM protein (X-ATM) is 85% identical to human ATM within the kinase domain and 71% identical over the carboxyl-terminal half of the protein. Polyclonal antibodies raised against recombinant X-ATM are highly specific for the ATM protein and recognize a single polypeptide of 370-kDa in oocytes, embryos, egg extracts and a Xenopus cell line. We found that X-ATM was expressed maternally in eggs and as early as stage II pre-vitellogenic oocytes, and the protein and mRNA were present at relatively constant levels throughout development. Subcellular fractionation showed that the protein was nuclear in both the female and male germlines. The level of X-ATM protein did not change throughout the meiotic divisions or the synchronous mitotic cycles of cleavage stage embryos. In addition, we did not observe any change in the level or mobility of X-ATM protein following gamma-irradiation of embryos. Finally, we also demonstrated that X-ATM was present in a high molecular weight complex of approximately 500 kDa containing the X-ATM protein and other, as yet unidentified component(s).

  15. Bone marrow transplantation improves the outcome of Atm-deficient mice through the migration of ATM-competent cells.

    PubMed

    Pietzner, J; Baer, P C; Duecker, R P; Merscher, M B; Satzger-Prodinger, C; Bechmann, I; Wietelmann, A; Del Turco, D; Doering, C; Kuci, S; Bader, P; Schirmer, S; Zielen, S; Schubert, R

    2013-02-01

    Ataxia telangiectasia (A-T) is a highly pleiotropic disorder. Patients suffer from progressive neurodegeneration, severe bronchial complications, immunodeficiency, hypersensitivity to radiotherapy and elevated risk of malignancies. Leukemia and lymphoma, along with lung failure, are the main causes of morbidity and mortality in A-T patients. At present, no effective therapy for A-T exists. One promising therapeutic approach is bone marrow transplantation (BMT) that is already used as a curative therapy for other genomic instability syndromes. We used an established clinically relevant non-myeloablative host-conditioning regimen and transplanted green fluorescent protein (GFP)-expressing ataxia telangiectasia mutated (ATM)-competent bone marrow-derived cells (BMDCs) into Atm-deficient mice. GFP expression allowed tracking of the potential migration of the cells into the tissues of recipient animals. Donor BMDCs migrated into the bone marrow, blood, thymus, spleen and lung tissue of Atm-deficient mice showing an ATM-competent phenotype. BMT inhibited thymic lymphomas, normalized T-lymphocyte populations, improved weight gain and rearing activity of Atm-deficient mice. In contrast, no GFP(+) cells were found in the cerebellum or cerebrum, and we detected decreased size index in MRI imaging of the cerebellum in 8-month-old transplanted Atm-deficient mice in comparison to wild-type mice. The repopulation with ATM-competent BMDCs is associated with a prolonged lifespan and significantly improved the phenotype of Atm-deficient mice.

  16. ATM protein and p53-serine 15 phosphorylation in ataxia-telangiectasia (AT) patients and at heterozygotes

    PubMed Central

    Delia, D; Mizutani, S; Panigone, S; Tagliabue, E; Fontanella, E; Asada, M; Yamada, T; Taya, Y; Prudente, S; Saviozzi, S; Frati, L; Pierotti, M A; Chessa, L

    2000-01-01

    ATM (ataxia-telangiectasia mutated) gene plays a central role in the DNA-damage response pathway. We characterized the ATM protein expression in immortalized cells from AT and AT-variant patients, and heterozygotes and correlated it with two ATM-dependent radiation responses, G1 checkpoint arrest and p53-Ser 15 phosphorylation. On Western blots, the full-length ATM protein was detected in eight of 18 AT cases, albeit at 1–32% of the normal levels, whereas a truncated ATM protein was detected in a single case, despite the prevalence among cases of truncation mutations. Of two ataxia without telangiectasia [A-(T)] cases, one expressed 20% and the other ~70% of the normal ATM levels. Noteworthy, among ten asymptomatic heterozygous carriers for AT, normal amounts of ATM protein were found in one and reduced by 40–50% in the remaining cases. The radiation-induced phosphorylation of p53 protein at serine 15, largely mediated by ATM kinase, was defective in AT, A(-T) and in 2/4 heterozygous carriers, while the G1 cell cycle checkpoint was disrupted in all AT and A(-T) cases, and in 3/10 AT heterozygotes. Altogether, our study shows that AT and A(-T) cases bearing truncation mutations of the ATM gene can produce modest amounts of full-length (and only rarely truncated) ATM protein. However, this limited expression of ATM protein provides no benefit regarding the ATM-dependent responses related to G1 arrest and p53-ser15 phosphorylation. Our study additionally shows that the majority of AT heterozygotes express almost halved levels of ATM protein, sufficient in most cases to normally regulate the ATM-dependent DNA damage-response pathway. © 2000 Cancer Research Campaign PMID:10864201

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

    PubMed Central

    Reiman, A; Srinivasan, V; Barone, G; Last, J I; Wootton, L L; Davies, E G; Verhagen, M M; Willemsen, M A; Weemaes, C M; Byrd, P J; Izatt, L; Easton, D F; Thompson, D J; Taylor, A M

    2011-01-01

    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. PMID:21792198

  18. Recessive mutations in the cancer gene Ataxia Telangiectasia Mutated (ATM), at a locus previously associated with metformin response, cause dysglycaemia and insulin resistance.

    PubMed

    Connelly, P J; Smith, N; Chadwick, R; Exley, A R; Shneerson, J M; Pearson, E R

    2016-03-01

    To investigate glucose and insulin metabolism in participants with ataxia telangiectasia in the absence of a diagnosis of diabetes. A standard oral glucose tolerance test was performed in participants with ataxia telangiectasia (n = 10) and in a control cohort (n = 10). Serial glucose and insulin measurements were taken to permit cohort comparisons of glucose-insulin homeostasis and indices of insulin secretion and sensitivity. During the oral glucose tolerance test, the 2-h glucose (6.75 vs 4.93 mmol/l; P = 0.029), insulin concentrations (285.6 vs 148.5 pmol/l; P = 0.043), incremental area under the curve for glucose (314 vs 161 mmol/l/min; P = 0.036) and incremental area under the curve for insulin (37,720 vs 18,080 pmol/l/min; P = 0.03) were higher in participants with ataxia telangiectasia than in the controls. There were no significant differences between groups in fasting glucose, insulin concentrations or insulinogenic index measurement (0.94 vs 0.95; P = 0.95). The Matsuda index, reflecting whole-body insulin sensitivity, was lower in participants with ataxia telangiectasia (5.96 vs 11.03; P = 0.019) than in control subjects. Mutations in Ataxia Telangiectasia Mutated (ATM) that cause ataxia telangiectasia are associated with elevated glycaemia and low insulin sensitivity in participants without diabetes. This indicates a role of ATM in glucose and insulin metabolic pathways. © 2016 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.

  19. Autophosphorylation and ATM Activation

    PubMed Central

    Kozlov, Sergei V.; Graham, Mark E.; Jakob, Burkhard; Tobias, Frank; Kijas, Amanda W.; Tanuji, Marcel; Chen, Philip; Robinson, Phillip J.; Taucher-Scholz, Gisela; Suzuki, Keiji; So, Sairai; Chen, David; Lavin, Martin F.

    2011-01-01

    The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser367, Ser1893, and Ser1981) and acetylation on Lys3016. We now describe the identification of a new ATM phosphorylation site, Thr(P)1885 and an additional autophosphorylation site, Ser(P)2996, that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser367 and Ser2996, for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)1981, ATM phosphorylated at Ser367 and Ser2996 localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser367 and Ser2996 was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo. PMID:21149446

  20. Absence of Wip1 partially rescues Atm deficiency phenotypes in mice

    PubMed Central

    Darlington, Yolanda; Nguyen, Thuy-Ai; Moon, Sung-Hwan; Herron, Alan; Rao, Pulivarthi; Zhu, Chengming; Lu, Xiongbin; Donehower, Lawrence A.

    2011-01-01

    Wildtype p53-Induced Phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may play a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared to Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared to their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared to Atm null mice. Finally, doubly null mice were partially rescued from infertility defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular. PMID:21765465

  1. DMAP1 is an essential regulator of ATM activity and function.

    PubMed

    Penicud, K; Behrens, A

    2014-01-23

    The hereditary autosomal recessive disease ataxia telangiectasia (A-T) is caused by mutation in the DNA damage kinase ATM. ATM's main function is to orchestrate DNA repair, thereby maintaining genomic stability. ATM activity is increased in response to several stimuli, including ionising radiation (IR) and hypotonic stress. DNMT1-associated protein 1 (DMAP1) is a member of the TIP60-p400 histone acetyl transferase (HAT) complex, which acetylates histone H4 at lysine 16 (H4K16) to affect chromatin relaxation and modulate ATM activation. Here we demonstrate that DMAP1 is required for both modes of ATM activation. Knockdown of DMAP1 impaired IR-induced ATM activation and consequently resulted in radiosensitivity and impaired the G2/M checkpoint. Moreover, DMAP1 was also required for efficient ATM signalling in response to hypotonic stress. Overexpression of DMAP1 increased IR-induced ATM substrate phosphorylation, suggesting that DMAP1 function is rate limiting for ATM signalling. DMAP1 associated with TIP60-dependent HAT activity, and depletion of DMAP1 reduced H4K16 acetylation in response to DNA damage. Treatment with histone deacetylase inhibitors rescued IR-induced ATM signalling in Dmap1-depleted cells. These results suggest that DMAP1 is a critical regulator of ATM activity and function.

  2. Absence of Wip1 partially rescues Atm deficiency phenotypes in mice.

    PubMed

    Darlington, Y; Nguyen, T-A; Moon, S-H; Herron, A; Rao, P; Zhu, C; Lu, X; Donehower, L A

    2012-03-01

    Wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may have a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared with Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared with their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared with Atm null mice. Finally, doubly null mice were partially rescued from gametogenesis defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular.

  3. The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.

    PubMed

    Kemp, Michael G; Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

  4. ATM and the epigenetics of the neuronal genome

    PubMed Central

    Herrup, Karl

    2013-01-01

    Ataxia-telangiectasia (A-T) is a neurodegenerative syndrome caused by the mutation of the ATM gene. The ATM protein is a PI3kinase family member best known for its role in the DNA damage response. While repair of DNA damage is a critical function that every CNS neuron must perform, a growing body of evidence indicates that the full range of ATM functions includes some that are unrelated to DNA damage yet are essential to neuronal survival and normal function. For example, ATM participates in the regulation of synaptic vesicle trafficking and is essential for the maintenance of normal LTP. In addition ATM helps to ensure the cytoplasmic localization of HDAC4 and thus maintains the histone ‘code’ of the neuronal genome by suppressing genome-wide histone deacetylation, which alters the message and protein levels of many genes that are important for neuronal survival and function. The growing list of ATM functions that go beyond its role in the DNA damage response offers a new perspective on why individuals with A-T express such a wide range of neurological symptoms, and suggests that not all A-T symptoms need to be understood in the context of the DNA repair process. PMID:23707635

  5. ATM and the epigenetics of the neuronal genome.

    PubMed

    Herrup, Karl

    2013-10-01

    Ataxia-telangiectasia (A-T) is a neurodegenerative syndrome caused by the mutation of the ATM gene. The ATM protein is a PI3kinase family member best known for its role in the DNA damage response. While repair of DNA damage is a critical function that every CNS neuron must perform, a growing body of evidence indicates that the full range of ATM functions includes some that are unrelated to DNA damage yet are essential to neuronal survival and normal function. For example, ATM participates in the regulation of synaptic vesicle trafficking and is essential for the maintenance of normal LTP. In addition ATM helps to ensure the cytoplasmic localization of HDAC4 and thus maintains the histone 'code' of the neuronal genome by suppressing genome-wide histone deacetylation, which alters the message and protein levels of many genes that are important for neuronal survival and function. The growing list of ATM functions that go beyond its role in the DNA damage response offers a new perspective on why individuals with A-T express such a wide range of neurological symptoms, and suggests that not all A-T symptoms need to be understood in the context of the DNA repair process. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  6. Germline Mutations in ATM and BRCA1/2 Distinguish Risk for Lethal and Indolent Prostate Cancer and are Associated with Early Age at Death.

    PubMed

    Na, Rong; Zheng, S Lilly; Han, Misop; Yu, Hongjie; Jiang, Deke; Shah, Sameep; Ewing, Charles M; Zhang, Liti; Novakovic, Kristian; Petkewicz, Jacqueline; Gulukota, Kamalakar; Helseth, Donald L; Quinn, Margo; Humphries, Elizabeth; Wiley, Kathleen E; Isaacs, Sarah D; Wu, Yishuo; Liu, Xu; Zhang, Ning; Wang, Chi-Hsiung; Khandekar, Janardan; Hulick, Peter J; Shevrin, Daniel H; Cooney, Kathleen A; Shen, Zhoujun; Partin, Alan W; Carter, H Ballentine; Carducci, Michael A; Eisenberger, Mario A; Denmeade, Sam R; McGuire, Michael; Walsh, Patrick C; Helfand, Brian T; Brendler, Charles B; Ding, Qiang; Xu, Jianfeng; Isaacs, William B

    2017-05-01

    Germline mutations in BRCA1/2 and ATM have been associated with prostate cancer (PCa) risk. To directly assess whether germline mutations in these three genes distinguish lethal from indolent PCa and whether they confer any effect on age at death. A retrospective case-case study of 313 patients who died of PCa and 486 patients with low-risk localized PCa of European, African, and Chinese descent. Germline DNA of each of the 799 patients was sequenced for these three genes. Mutation carrier rates and their effect on lethal PCa were analyzed using the Fisher's exact test and Cox regression analysis, respectively. The combined BRCA1/2 and ATM mutation carrier rate was significantly higher in lethal PCa patients (6.07%) than localized PCa patients (1.44%), p=0.0007. The rate also differed significantly among lethal PCa patients as a function of age at death (10.00%, 9.08%, 8.33%, 4.94%, and 2.97% in patients who died ≤ 60 yr, 61-65 yr, 66-70 yr, 71-75 yr, and over 75 yr, respectively, p=0.046) and time to death after diagnosis (12.26%, 4.76%, and 0.98% in patients who died ≤ 5 yr, 6-10 yr, and>10 yr after a PCa diagnosis, respectively, p=0.0006). Survival analysis in the entire cohort revealed mutation carriers remained an independent predictor of lethal PCa after adjusting for race and age, prostate-specific antigen, and Gleason score at the time of diagnosis (hazard ratio=2.13, 95% confidence interval: 1.24-3.66, p=0.004). A limitation of this study is that other DNA repair genes were not analyzed. Mutation status of BRCA1/2 and ATM distinguishes risk for lethal and indolent PCa and is associated with earlier age at death and shorter survival time. Prostate cancer patients with inherited mutations in BRCA1/2 and ATM are more likely to die of prostate cancer and do so at an earlier age. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  7. A new role for ATM: regulating mitochondrial function and mitophagy.

    PubMed

    Valentin-Vega, Yasmine A; Kastan, Michael B

    2012-05-01

    The various pathologies in ataxia telangiectasia (A-T) patients including T-cell lymphomagenesis have been attributed to defects in the DNA damage response pathway because ATM, the gene mutated in this disease, is a key mediator of this process. Analysis of Atm-deficient thymocytes in mice reveals that the absence of this gene results in altered mitochondrial homeostasis, a phenomenon that appears to result from abnormal mitophagy engagement. Interestingly, allelic loss of the autophagic gene Becn1 delays tumorigenesis in Atm-null mice presumably by reversing the mitochondrial abnormalities and not by improving the DNA damage response (DDR) pathway. Thus, ATM plays a critical role in modulating mitochondrial homeostasis perhaps by regulating mitophagy.

  8. Disruption of Xpg increases spontaneous mutation frequency, particularly A:T to C:G transversion.

    PubMed

    Shiomi, N; Hayashi, E; Sasanuma, S; Mita, K; Shiomi, T

    2001-12-19

    Cells isolated from Xpg (the mouse counterpart of XPG)-disrupted mice underwent premature senescence and showed early onset of immortalization, suggesting that Xpg might be involved in genetic stability. Recent studies showed that human XPG, in addition to its function in the nucleotide excision repair (NER), was involved in the repair of oxidative base damages such as thymine glycol (Tg) and 8-oxo-guanine (8-oxoG), and this may explain the genetic instability observed in Xpg-deficient cells. To clarify this point, we determined spontaneous mutation frequencies and the type of spontaneous base substitution mutations in cells obtained from normal and Xpg-deficient mice using the supF shuttle vector (pNY200) for mutation assay. The spontaneous mutation frequency of the supF gene in pNY200 propagated in the Xpg-deficient cells was about three times higher than that in normal cells, indicating the importance of Xpg in reducing the frequency of spontaneous mutations. The frequency of spontaneous base substitution mutations at A:T sites, particularly that of the A:T to C:G transversion, increased markedly in the Xpg-deficient cells.

  9. Morbidity and mortality from ataxia-telangiectasia are associated with ATM genotype.

    PubMed

    Micol, Romain; Ben Slama, Lilia; Suarez, Felipe; Le Mignot, Loïc; Beauté, Julien; Mahlaoui, Nizar; Dubois d'Enghien, Catherine; Laugé, Anthony; Hall, Janet; Couturier, Jérôme; Vallée, Louis; Delobel, Bruno; Rivier, François; Nguyen, Karine; Billette de Villemeur, Thierry; Stephan, Jean-Louis; Bordigoni, Pierre; Bertrand, Yves; Aladjidi, Nathalie; Pedespan, Jean-Michel; Thomas, Caroline; Pellier, Isabelle; Koenig, Michel; Hermine, Olivier; Picard, Capucine; Moshous, Despina; Neven, Bénédicte; Lanternier, Fanny; Blanche, Stéphane; Tardieu, Marc; Debré, Marianne; Fischer, Alain; Stoppa-Lyonnet, Dominique

    2011-08-01

    Ataxia-telangiectasia (A-T) is a rare genetic disease caused by germline biallelic mutations in the ataxia-telangiectasia mutated gene (ATM) that result in partial or complete loss of ATM expression or activity. The course of the disease is characterized by neurologic manifestations, infections, and cancers. We studied A-T progression and investigated whether manifestations were associated with the ATM genotype. We performed a retrospective cohort study in France of 240 patients with A-T born from 1954 to 2005 and analyzed ATM mutations in 184 patients, along with neurologic manifestations, infections, and cancers. Among patients with A-T, the Kaplan-Meier 20-year survival rate was 53.4%; the prognosis for these patients has not changed since 1954. Life expectancy was lower among patients with mutations in ATM that caused total loss of expression or function of the gene product (null mutations) compared with that seen in patients with hypomorphic mutations because of earlier onset of cancer (mainly hematologic malignancies). Cancer (hazard ratio, 2.7; 95% CI, 1.6-4.5) and respiratory tract infections (hazard ratio, 2.3; 95% CI, 1.4-3.8) were independently associated with mortality. Cancer (hazard ratio, 5.8; 95% CI, 2.9-11.6) was a major risk factor for mortality among patients with null mutations, whereas respiratory tract infections (hazard ratio, 4.1; 95% CI, 1.8-9.1) were the leading cause of death among patients with hypomorphic mutations. Morbidity and mortality among patients with A-T are associated with ATM genotype. This information could improve our prognostic ability and lead to adapted therapeutic strategies. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  10. Neurons in Vulnerable Regions of the Alzheimer’s Disease Brain Display Reduced ATM Signaling123

    PubMed Central

    Shen, Xuting; Chen, Jianmin; Li, Jiali; Kofler, Julia

    2016-01-01

    Abstract Ataxia telangiectasia (A-T) is a multisystemic disease caused by mutations in the ATM (A-T mutated) gene. It strikes before 5 years of age and leads to dysfunctions in many tissues, including the CNS, where it leads to neurodegeneration, primarily in cerebellum. Alzheimer’s disease (AD), by contrast, is a largely sporadic neurodegenerative disorder that rarely strikes before the 7th decade of life with primary neuronal losses in hippocampus, frontal cortex, and certain subcortical nuclei. Despite these differences, we present data supporting the hypothesis that a failure of ATM signaling is involved in the neuronal death in individuals with AD. In both, partially ATM-deficient mice and AD mouse models, neurons show evidence for a loss of ATM. In human AD, three independent indices of reduced ATM function—nuclear translocation of histone deacetylase 4, trimethylation of histone H3, and the presence of cell cycle activity—appear coordinately in neurons in regions where degeneration is prevalent. These same neurons also show reduced ATM protein levels. And though they represent only a fraction of the total neurons in each affected region, their numbers significantly correlate with disease stage. This previously unknown role for the ATM kinase in AD pathogenesis suggests that the failure of ATM function may be an important contributor to the death of neurons in AD individuals. PMID:27022623

  11. Neurons in Vulnerable Regions of the Alzheimer's Disease Brain Display Reduced ATM Signaling.

    PubMed

    Shen, Xuting; Chen, Jianmin; Li, Jiali; Kofler, Julia; Herrup, Karl

    2016-01-01

    Ataxia telangiectasia (A-T) is a multisystemic disease caused by mutations in the ATM (A-T mutated) gene. It strikes before 5 years of age and leads to dysfunctions in many tissues, including the CNS, where it leads to neurodegeneration, primarily in cerebellum. Alzheimer's disease (AD), by contrast, is a largely sporadic neurodegenerative disorder that rarely strikes before the 7th decade of life with primary neuronal losses in hippocampus, frontal cortex, and certain subcortical nuclei. Despite these differences, we present data supporting the hypothesis that a failure of ATM signaling is involved in the neuronal death in individuals with AD. In both, partially ATM-deficient mice and AD mouse models, neurons show evidence for a loss of ATM. In human AD, three independent indices of reduced ATM function-nuclear translocation of histone deacetylase 4, trimethylation of histone H3, and the presence of cell cycle activity-appear coordinately in neurons in regions where degeneration is prevalent. These same neurons also show reduced ATM protein levels. And though they represent only a fraction of the total neurons in each affected region, their numbers significantly correlate with disease stage. This previously unknown role for the ATM kinase in AD pathogenesis suggests that the failure of ATM function may be an important contributor to the death of neurons in AD individuals.

  12. ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila.

    PubMed

    Petersen, Andrew J; Rimkus, Stacey A; Wassarman, David A

    2012-03-13

    To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia-telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non-cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimer's in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.

  13. Etoposide Induces ATM-Dependent Mitochondrial Biogenesis through AMPK Activation

    PubMed Central

    Lyu, Yi Lisa; Liu, Leroy F.; Qi, Haiyan

    2008-01-01

    biogenesis may play a role in DNA damage response and ROS regulation, and that defect in ATM-dependent mitochondrial biogenesis could contribute to the manifestations of A-T disease. PMID:18431490

  14. ATM mediates repression of DNA end-degradation in an ATP-dependent manner.

    PubMed

    Rahal, Elias A; Henricksen, Leigh A; Li, Yuling; Turchi, John J; Pawelczak, Katherine S; Dixon, Kathleen

    2008-03-01

    Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.

  15. Oxidative DNA damage is involved in ochratoxin A-induced G2 arrest through ataxia telangiectasia-mutated (ATM) pathways in human gastric epithelium GES-1 cells in vitro.

    PubMed

    Cui, Jinfeng; Liu, Jing; Wu, Sha; Wang, Yuan; Shen, Haitao; Xing, Lingxiao; Wang, Junling; Yan, Xia; Zhang, Xianghong

    2013-10-01

    Ochratoxin A (OTA), one of the most abundant mycotoxin food contaminants, is classified as "possibly carcinogenic to humans." Our previous study showed that OTA could induce a G2 arrest in immortalized human gastric epithelium cells (GES-1). To explore the putative roles of oxidative DNA damage and the ataxia telangiectasia-mutated (ATM) pathways on the OTA-induced G2 arrest, the current study systematically evaluated the roles of reactive oxygen species (ROS) production, DNA damage, and ATM-dependent pathway activation on the OTA-induced G2 phase arrest in GES-1 cells. The results showed that OTA exposure elevated intracellular ROS production, which directly induced DNA damage and increased the levels of 8-OHdG and DNA double-strand breaks (DSBs). In addition, it was found that OTA treatment induced the phosphorylation of the ATM protein, as well as its downstream molecules Chk2 and p53, in response to DNA DSBs. Inhibition of ATM by the pharmacological inhibitor caffeine or siRNA effectively prevented the activation of ATM-dependent pathways and rescued the G2 arrest elicited by OTA. Finally, pretreatment with the antioxidant N-acetyl-L-cysteine (NAC) reduced the OTA-induced DNA DSBs, ATM phosphorylation, and G2 arrest. In conclusion, the results of this study suggested that OTA-induced oxidative DNA damage triggered the ATM-dependent pathways, which ultimately elicited a G2 arrest in GES-1 cells.

  16. Expression pattern of ataxia telangiectasia mutated (ATM), p53, Akt, and glycogen synthase kinase-3β in the striatum of rats treated with 3-nitropropionic acid.

    PubMed

    Duran-Vilaregut, Joaquim; Manich, Gemma; Del Valle, Jaume; Camins, Antoni; Pallàs, Mercè; Vilaplana, Jordi; Pelegrí, Carme

    2012-09-01

    3-Nitropropionic acid (3-NPA) is a mitochondrial toxin used in the laboratory to replicate neurodegenerative conditions that are accompanied by degeneration of the caudate-putamen. 3-NPA induces depletion in ATP production, reactive oxygen species production, and secondary excitotoxicity mediated by activation of N-methyl-D-aspartate receptors that culminates in the triggering of cell death mechanisms, including apoptosis. We here examined by immunohistochemical methods whether cellular expression of phospho(Ser1981) -ataxia telangiectasia mutated (ATM), phospho(Ser15) -p53, phospho(Ser473) -Akt, and phospho(Ser9) -glycogen synthase kinase-3β (GSK3β), which are key signal molecules that play a critical role in regulating cellular processes related to cell survival and demise, were involved in the striatal neurodegeneration in the brains of rats treated with 3-NPA. Our results indicate that the toxin induced the activation of ATM and p53 only in astrocytes, and a role for these proteins in neuronal degeneration was ruled out. On the other hand, striatal neurons lost the active form of Akt as soon as they began to appear pyknotic, indicating impairment of the PI3K/Akt/GSK3 pathway in their degenerative process. The inactive form of GSK3β was detected extensively, mainly in the rim of the striatal lesions around degenerating neurons, which could be attributed to a cell death or cell survival response.

  17. A Novel Role for ATM in Regulating Proteasome-Mediated Protein Degradation through Suppression of the ISG15 Conjugation Pathway

    PubMed Central

    Wood, Laurence M.; Sankar, Surendran; Reed, Ryan E.; Haas, Arthur L.; Liu, Leroy F.; McKinnon, Peter; Desai, Shyamal D.

    2011-01-01

    Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients. PMID:21298066

  18. Molecular Imaging of the ATM Kinase Activity

    SciTech Connect

    Williams, Terence M.; Nyati, Shyam; Ross, Brian D.; Rehemtulla, Alnawaz

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  19. Aberrant TCRδ rearrangement underlies the T-cell lymphocytopenia and t(12;14) translocation associated with ATM deficiency.

    PubMed

    Jiang, Wenxia; Lee, Brian J; Li, Chen; Dubois, Richard L; Gostissa, Monica; Alt, Frederick W; Zha, Shan

    2015-04-23

    Ataxia telangiectasia mutated (ATM) is a protein kinase and a master regulator of DNA-damage responses. Germline ATM inactivation causes ataxia-telangiectasia (A-T) syndrome with severe lymphocytopenia and greatly increased risk for T-cell lymphomas/leukemia. Both A-T and T-cell prolymphoblastic leukemia patients with somatic mutations of ATM frequently carry inv(14;14) between the T-cell receptor α/δ (TCRα/δ) and immunoglobulin H loci, but the molecular origin of this translocation remains elusive. ATM(-/-) mice recapitulate lymphocytopenia of A-T patients and routinely succumb to thymic lymphomas with t(12;14) translocation, syntenic to inv(14;14) in humans. Here we report that deletion of the TCRδ enhancer (Eδ), which initiates TCRδ rearrangement, significantly improves αβ T cell output and effectively prevents t(12;14) translocations in ATM(-/-) mice. These findings identify the genomic instability associated with V(D)J recombination at the TCRδ locus as the molecular origin of both lymphocytopenia and the signature t(12;14) translocations associated with ATM deficiency. © 2015 by The American Society of Hematology.

  20. ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

    PubMed Central

    Fang, Ling; Choudhary, Sanjeev; Zhao, Yingxin; Edeh, Chukwudi B; Yang, Chunying; Boldogh, Istvan; Brasier, Allan R.

    2014-01-01

    Ataxia-telangiectasia mutated (ATM), a member of the phosphatidylinositol 3 kinase-like kinase family, is a master regulator of the double strand DNA break-repair pathway after genotoxic stress. Here, we found ATM serves as an essential regulator of TNF-induced NF-kB pathway. We observed that TNF exposure of cells rapidly induced DNA double strand breaks and activates ATM. TNF-induced ROS promote nuclear IKKγ association with ubiquitin and its complex formation with ATM for nuclear export. Activated cytoplasmic ATM is involved in the selective recruitment of the E3-ubiquitin ligase β-TrCP to phospho-IκBα proteosomal degradation. Importantly, ATM binds and activates the catalytic subunit of protein kinase A (PKAc), ribosmal S6 kinase that controls RelA Ser 276 phosphorylation. In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased. We further observed decreased binding and recruitment of the transcriptional elongation complex containing cyclin dependent kinase-9 (CDK9; a kinase necessary for triggering transcriptional elongation) to promoters of NF-κB-dependent immediate-early cytokine genes, in ATM knockdown cells. We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation. Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation. PMID:24957606

  1. ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor

    PubMed Central

    Lavin, Martin F.; Kozlov, Sergei; Gatei, Magtouf; Kijas, Amanda W.

    2015-01-01

    The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes. PMID:26512707

  2. ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor.

    PubMed

    Lavin, Martin F; Kozlov, Sergei; Gatei, Magtouf; Kijas, Amanda W

    2015-10-23

    The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

  3. Assessment of targeted and non-targeted responses in cells deficient in ATM function following exposure to low and high dose X-rays.

    PubMed

    Kiuru, Anne; Kämäräinen, Meerit; Heinävaara, Sirpa; Pylkäs, Katri; Chapman, Kim; Koivistoinen, Armi; Parviainen, Teuvo; Winqvist, Robert; Kadhim, Munira; Launonen, Virpi; Lindholm, Carita

    2014-01-01

    Radiation sensitivity at low and high dose exposure to X-rays was investigated by means of chromosomal aberration (CA) analysis in heterozygous ATM mutation carrier and A-T patient (biallelic ATM mutation) lymphoblastoid cell lines (LCLs). Targeted and non-targeted responses to acutely delivered irradiation were examined by applying a co-culture system that enables study of both directly irradiated cells and medium-mediated bystander effects in the same experimental setting. No indication of radiation hypersensitivity was observed at doses of 0.01 Gy or 0.1 Gy for the ATM mutation carrier LCL. The A-T patient cells also did not show low-dose response. There was significant increase in unstable CA yields for both ATM mutation carrier and A-T LCLs at 1 and 2 Gy, the A-T cells displaying more distinct dose dependency. Both chromosome and chromatid type aberrations were induced at an increased rate in the irradiated A-T cells, whereas for ATM carrier cells, only unstable chromosomal aberrations were increased above the level observed in the wild type cell line. No bystander effect could be demonstrated in any of the cell lines or doses applied. Characteristics typical for the A-T cell line were detected, i.e., high baseline frequency of CA that increased with dose. In addition, dose-dependent loss of cell viability was observed. In conclusion, CA analysis did not demonstrate low-dose (≤100 mGy) radiosensitivity in ATM mutation carrier cells or A-T patient cells. However, both cell lines showed increased radiosensitivity at high dose exposure.

  4. Assessment of Targeted and Non-Targeted Responses in Cells Deficient in ATM Function following Exposure to Low and High Dose X-Rays

    PubMed Central

    Heinävaara, Sirpa; Pylkäs, Katri; Chapman, Kim; Koivistoinen, Armi; Parviainen, Teuvo; Winqvist, Robert; Kadhim, Munira; Launonen, Virpi; Lindholm, Carita

    2014-01-01

    Radiation sensitivity at low and high dose exposure to X-rays was investigated by means of chromosomal aberration (CA) analysis in heterozygous ATM mutation carrier and A-T patient (biallelic ATM mutation) lymphoblastoid cell lines (LCLs). Targeted and non-targeted responses to acutely delivered irradiation were examined by applying a co-culture system that enables study of both directly irradiated cells and medium-mediated bystander effects in the same experimental setting. No indication of radiation hypersensitivity was observed at doses of 0.01 Gy or 0.1 Gy for the ATM mutation carrier LCL. The A-T patient cells also did not show low-dose response. There was significant increase in unstable CA yields for both ATM mutation carrier and A-T LCLs at 1 and 2 Gy, the A-T cells displaying more distinct dose dependency. Both chromosome and chromatid type aberrations were induced at an increased rate in the irradiated A-T cells, whereas for ATM carrier cells, only unstable chromosomal aberrations were increased above the level observed in the wild type cell line. No bystander effect could be demonstrated in any of the cell lines or doses applied. Characteristics typical for the A-T cell line were detected, i.e., high baseline frequency of CA that increased with dose. In addition, dose-dependent loss of cell viability was observed. In conclusion, CA analysis did not demonstrate low-dose (≤100 mGy) radiosensitivity in ATM mutation carrier cells or A-T patient cells. However, both cell lines showed increased radiosensitivity at high dose exposure. PMID:24681528

  5. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells

    PubMed Central

    Coufal, Nicole G.; Garcia-Perez, Josè Luis; Peng, Grace E.; Marchetto, Maria C. N.; Muotri, Alysson R.; Mu, Yangling; Carson, Christian T.; Macia, Angela; Moran, John V.; Gage, Fred H.

    2011-01-01

    Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition. PMID:22159035

  6. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells.

    PubMed

    Coufal, Nicole G; Garcia-Perez, Josè Luis; Peng, Grace E; Marchetto, Maria C N; Muotri, Alysson R; Mu, Yangling; Carson, Christian T; Macia, Angela; Moran, John V; Gage, Fred H

    2011-12-20

    Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition.

  7. Significance of ATM Gene Polymorphisms in Chronic Myeloid Leukemia - a Case Control Study from India.

    PubMed

    Gorre, Manjula; Mohandas, Prajitha Edathara; Kagita, Sailaja; Cingeetham, Anuradha; Vuree, Sugunakar; Jarjapu, Sarika; Nanchari, Santhoshirani; Meka, Phanni Bhushann; Annamaneni, Sandhya; Dunna, Nageswara Rao; Digumarti, Raghunadharao; Satti, Vishnupriya

    2016-01-01

    Development of chronic myeloid leukemia (CML) involves formation of double strand breaks (DSBs) which are initially sensed by the ataxia telangiectasia mutated (ATM) signal kinase to induce a DNA damage response (DDR). Mutations or single nucleotide polymorphisms in ATM gene are known to influence the signaling capacity resulting in susceptibility to certain genetic diseases such as cancers. In the present study, we have analyzed -5144A>T (rs228589) and C4138T (rs3092856) polymorphisms of theATM gene through polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 925 subjects (476 CML cases and 449 controls). The A allele of -5144A>T polymorphism and T allele of C4138T polymorphism which were known to be influencing ATM signaling capacity are significantly associated with enhanced risk for CML independently and also in combination (evident from the haplotype and diplotype analyses). Significant elevation in the frequencies of both the risk alleles among high risk groups under European Treatment and Outcome Study (EUTOS) score suggests the possible role of these polymorphisms in predicting the prognosis of CML patients. This study provides the first evidence of association of functional ATM gene polymorphisms with the increased risk of CML development as well as progression.

  8. Ataxia-Telangiectasia, Mutated (ATM)/Nuclear Factor κ light chain enhancer of activated B cells (NFκB) signaling controls basal and DNA damage-induced transglutaminase 2 expression.

    PubMed

    Ai, Lingbao; Skehan, Ryan R; Saydi, John; Lin, Tong; Brown, Kevin D

    2012-05-25

    Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that cross-links proteins and its overexpression, linked to a drug resistant phenotype, is commonly observed in cancer cells. Further, up-regulation of TG2 expression occurs during response to various forms of cell stress; however, the molecular mechanisms that drive inducible expression of the TG2 gene (TGM2) require elucidation. Here we show that genotoxic stress induces TG2 expression through the Ataxia-Telangiectasia, Mutated (ATM)/Nuclear Factor κ light chain enhancer of activated B cells (NFκB) signaling pathway. We further document that NFκB is both necessary and sufficient to drive constitutive TG2 expression in cultured cell lines. Additionally, shRNA-mediated knockdown or pharmacological inhibition of the ATM kinase results in reduced constitutive TG2 expression and NFκB transcriptional activity. We document that the NFκB subunit p65 (RelA) interacts with two independent consensus NFκB binding sites within the TGM2 promoter, that mutation of either site or pharmacological inhibition of NFκB reduces TGM2 promoter activity, and genotoxic stress drives heightened association of p65 with the TGM2 promoter. Finally, we observed that knockdown of either p65 or ATM in MDA-MB-468 breast cancer cells expressing recombinant TG2 partially reduces resistance to doxorubicin, indicating that the drug resistance linked to overexpression of TG2 functions, in part, through p65 and ATM. This work establishes a novel ATM-dependent signaling loop where TG2 and NFκB activate each other resulting in sustained activation of NFκB and acquisition of a drug-resistant phenotype.

  9. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    SciTech Connect

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung; Choi, Seong-Jun; Shim, Hosup

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  10. Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice

    PubMed Central

    Borghesani, Paul R.; Alt, Frederick W.; Bottaro, Andrea; Davidson, Laurie; Aksoy, Saime; Rathbun, Gary A.; Roberts, Thomas M.; Swat, Wojciech; Segal, Rosalind A.; Gu, Yansong

    2000-01-01

    Motor incoordination, immune deficiencies, and an increased risk of cancer are the characteristic features of the hereditary disease ataxia–telangiectasia (A-T), which is caused by mutations in the ATM gene. Through gene targeting, we have generated a line of Atm mutant mice, Atmy/y mice. In contrast to other Atm mutant mice, Atmy/y mice show a lower incidence of thymic lymphoma and survive beyond a few months of age. Atmy/y mice exhibit deficits in motor learning indicative of cerebellar dysfunction. Even though we found no gross cerebellar degeneration in older Atmy/y animals, ectopic and abnormally differentiated Purkinje cells were apparent in mutant mice of all ages. These findings establish that some neuropathological abnormalities seen in A-T patients also are present in Atm mutant mice. In addition, we report a previously unrecognized effect of Atm deficiency on development or maintenance of CD4+8+ thymocytes. We discuss these findings in the context of the hypothesis that abnormal development of Purkinje cells and lymphocytes contributes to the pathogenesis of A-T. PMID:10716718

  11. Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression.

    PubMed

    Kang, Jian; Ferguson, David; Song, Hoseok; Bassing, Craig; Eckersdorff, Mark; Alt, Frederick W; Xu, Yang

    2005-01-01

    Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.

  12. Structure of the human dimeric ATM kinase

    PubMed Central

    Lau, Wilson C. Y.; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S. Y.

    2016-01-01

    ABSTRACT DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  13. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer.

    PubMed

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung; Choi, Seong-Jun; Shim, Hosup

    2014-10-03

    Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  14. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    PubMed

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  15. ATM technology and beyond

    NASA Technical Reports Server (NTRS)

    Cheung, Nim K.

    1993-01-01

    Networks based on Asynchronous Transfer Mode (ATM) are expected to provide cost-effective and ubiquitous infrastructure to support broadband and multimedia services. In this paper, we give an overview of the ATM standards and its associated physical layer transport technologies. We use the experimental HIPPI-ATM-SONET (HAS) interface in the Nectar Gigabit Testbed to illustrate how one can use the SONET/ATM public network to provide transport for bursty gigabit applications.

  16. Pharmacologic manipulation of the ataxia-telangiectasia mutated gene product as an intervention in age-related disease.

    PubMed

    Shackelford, Rodney E

    2005-01-01

    Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by progressive ataxia, elevated cancer incidence, and premature aging. A-T cells, Atm-deficient mice, and individuals with A-T show increased oxidant sensitivity, genomic instability, altered IGF-1 and p53 signaling, and rapid telomere shortening compared to normal controls. The gene mutated in A-T, ATM, regulates DNA repair, IGF-1 and p53 signaling, age pigment removal, antioxidant capacity, and telomere maintenance - pathways involved in and often attenuated with aging. Interestingly, flavonoids with chemopreventative effects, such as quercetin, genistein, and epigallocatechin gallate activate ATM. Since ATM activates pathways which increase genomic stability, oxidant resistance, and/or telomere stability, and since many diseases of old age (i.e., cancer, cardiovascular and neurodegenerative disease), result from attenuation of these pathways, pharmacologic manipulation of ATM activity via flavonoid intake may prove useful in slowing the appearance of age-associated disease.

  17. ATM CMG/EPEA

    NASA Technical Reports Server (NTRS)

    Abramowitz, R.; Kovek, J.; Teimer, W.; Haddad, S. P.

    1975-01-01

    The Apollo Telescope mount double gimballed control moment gyro ATM CMG is described. Photographs of the CMG and its subassemblies are presented along with a functional block diagram of the CMG subsystem. Analog processing electronics for ATM vehicle pointing control and ATM experiment package pointing control are also described.

  18. Investigation of the Functional Link between ATM and NBS1 in the DNA Damage Response in the Mouse Cerebellum*

    PubMed Central

    Dar, Inbal; Yosha, Galit; Elfassy, Ronen; Galron, Ronit; Wang, Zhao-Qi; Shiloh, Yosef; Barzilai, Ari

    2011-01-01

    Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are related genomic instability syndromes characterized by neurological deficits. The NBS1 protein that is defective in NBS is a component of the Mre11/RAD50/NBS1 (MRN) complex, which plays a major role in the early phase of the complex cellular response to double strand breaks (DSBs) in the DNA. Among others, Mre11/RAD50/NBS1 is required for timely activation of the protein kinase ATM (A-T, mutated), which is missing or inactivated in patients with A-T. Understanding the molecular pathology of A-T, primarily its cardinal symptom, cerebellar degeneration, requires investigation of the DSB response in cerebellar neurons, particularly Purkinje cells, which are the first to be lost in A-T patients. Cerebellar cultures derived from mice with different mutations in DNA damage response genes is a useful experimental system to study malfunctioning of the damage response in the nervous system. To clarify the interrelations between murine Nbs1 and Atm, we generated a mouse strain with specific disruption of the Nbs1 gene in the central nervous system on the background of general Atm deficiency (Nbs1-CNS-Δ//Atm−/−). This genotype exacerbated several features of both conditions and led to a markedly reduced life span, dramatic decline in the number of cerebellar granule neurons with considerable cerebellar disorganization, abolishment of the white matter, severe reduction in glial cell proliferation, and delayed DSB repair in cerebellar tissue. Combined loss of Nbs1 and Atm in the CNS significantly abrogated the DSB response compared with the single mutation genotypes. Importantly, the data indicate that Atm has cellular roles not regulated by Nbs1 in the murine cerebellum. PMID:21300797

  19. ATMIN defines an NBS1-independent pathway of ATM signalling

    PubMed Central

    Kanu, Nnennaya; Behrens, Axel

    2007-01-01

    The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM colocalised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling. PMID:17525732

  20. 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.

    PubMed

    Campbell, Andrew; Krupp, Brittany; Bushman, Jared; Noble, Mark; Pröschel, Christoph; Mayer-Pröschel, Margot

    2015-11-15

    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. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Pathogenesis of ataxia-telangiectasia: the next generation of ATM functions

    PubMed Central

    Ambrose, Mark

    2013-01-01

    In 1988, the gene responsible for the autosomal recessive disease ataxia- telangiectasia (A-T) was localized to 11q22.3-23.1. It was eventually cloned in 1995. Many independent laboratories have since demonstrated that in replicating cells, ataxia telangiectasia mutated (ATM) is predominantly a nuclear protein that is involved in the early recognition and response to double-stranded DNA breaks. ATM is a high-molecular-weight PI3K-family kinase. ATM also plays many important cytoplasmic roles where it phosphorylates hundreds of protein substrates that activate and coordinate cell-signaling pathways involved in cell-cycle checkpoints, nuclear localization, gene transcription and expression, the response to oxidative stress, apoptosis, nonsense-mediated decay, and others. Appreciating these roles helps to provide new insights into the diverse clinical phenotypes exhibited by A-T patients—children and adults alike—which include neurodegeneration, high cancer risk, adverse reactions to radiation and chemotherapy, pulmonary failure, immunodeficiency, glucose transporter aberrations, insulin-resistant diabetogenic responses, and distinct chromosomal and chromatin changes. An exciting recent development is the ATM-dependent pathology encountered in mitochondria, leading to inefficient respiration and energy metabolism and the excessive generation of free radicals that themselves create life-threatening DNA lesions that must be repaired within minutes to minimize individual cell losses. PMID:23440242

  2. Pathogenesis of ataxia-telangiectasia: the next generation of ATM functions.

    PubMed

    Ambrose, Mark; Gatti, Richard A

    2013-05-16

    In 1988, the gene responsible for the autosomal recessive disease ataxia- telangiectasia (A-T) was localized to 11q22.3-23.1. It was eventually cloned in 1995. Many independent laboratories have since demonstrated that in replicating cells, ataxia telangiectasia mutated (ATM) is predominantly a nuclear protein that is involved in the early recognition and response to double-stranded DNA breaks. ATM is a high-molecular-weight PI3K-family kinase. ATM also plays many important cytoplasmic roles where it phosphorylates hundreds of protein substrates that activate and coordinate cell-signaling pathways involved in cell-cycle checkpoints, nuclear localization, gene transcription and expression, the response to oxidative stress, apoptosis, nonsense-mediated decay, and others. Appreciating these roles helps to provide new insights into the diverse clinical phenotypes exhibited by A-T patients-children and adults alike-which include neurodegeneration, high cancer risk, adverse reactions to radiation and chemotherapy, pulmonary failure, immunodeficiency, glucose transporter aberrations, insulin-resistant diabetogenic responses, and distinct chromosomal and chromatin changes. An exciting recent development is the ATM-dependent pathology encountered in mitochondria, leading to inefficient respiration and energy metabolism and the excessive generation of free radicals that themselves create life-threatening DNA lesions that must be repaired within minutes to minimize individual cell losses.

  3. ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion.

    PubMed

    Chen, Wei-Ta; Ebelt, Nancy D; Stracker, Travis H; Xhemalce, Blerta; Van Den Berg, Carla L; Miller, Kyle M

    2015-06-01

    Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression.

  4. Founder Effect of a c.828+3A>T Splice Site Mutation in Peripherin 2 (PRPH2) Causing Autosomal Dominant Retinal Dystrophies.

    PubMed

    Shankar, Suma P; Birch, David G; Ruiz, Richard S; Hughbanks-Wheaton, Dianna K; Sullivan, Lori S; Bowne, Sara J; Stone, Edwin M; Daiger, Stephen P

    2015-05-01

    Screening for splice site mutation c.828+3A>T in the peripherin 2 (PRPH2) gene should be a high priority in families with highly variable retinal dystrophies. The correction of missplicing is a potential therapeutic target. To determine the prevalence, genetic origin, and molecular mechanism of a donor c.828+3A>T mutation in the PRPH2 (peripherin 2, retinal degeneration slow) gene in individuals with retinal dystrophies. Case-control study that took place at the University of Texas Health Science Center, the University of Iowa, and the Retina Foundation of the Southwest, from January 1, 1987, to August 1, 2014, including affected individuals from 200 families with a diagnosis of autosomal dominant retinitis pigmentosa, 35 families with unspecified macular dystrophies, and 116 families with pattern dystrophy. Participants were screened for the c.828+3A>T mutation by restriction-enzyme digest, single-strand conformational polymorphism screening, or bidirectional sequencing. Haplotypes of polymorphic markers flanking the PRPH2 locus and sequence variants within the gene were determined by denaturing gel electrophoresis or automated capillary-based cycle sequencing. The effect of the splice site mutation on the PRPH2 transcript was analyzed using NetGene2, a splice prediction program and by the reverse transcription polymerase chain reaction of illegitimate transcripts from peripheral white blood cells. Results of testing for splice site mutation, haplotypes, and alternate transcripts. The PRPH2 mutation was found in 97 individuals of 19 independently ascertained families with a clinical diagnosis of retinitis pigmentosa, macular dystrophy, and/or pattern dystrophy. All affected individuals also shared a rare haplotype of approximately 644 kilobase pairs containing the c.828+3A>T mutation, which extends from the short tandem repeat polymorphism D6S282 to c.1013G>A (rs434102, a single-nucleotide polymorphism) in exon 3 of PRPH2, suggesting this mutation is from a common

  5. WWOX guards genome stability by activating ATM.

    PubMed

    Hazan, Idit; Abu-Odeh, Mohammad; Hofmann, Thomas G; Aqeilan, Rami I

    2015-01-01

    Common fragile sites (CFSs) tend to break upon replication stress and have been suggested to be "hot spots" for genomic instability. Recent evidence, however, implies that in the wake of DNA damage, WW domain-containing oxidoreductase (WWOX, the gene product of the FRA16D fragile site), associates with ataxia telangiectasia-mutated (ATM) and regulates its activation to maintain genomic integrity.

  6. ATM activation in hypoxia - causes and consequences.

    PubMed

    Olcina, Monica M; Grand, Roger Ja; Hammond, Ester M

    2014-01-01

    The DNA damage response is a complex signaling cascade that is triggered by cellular stress. This response is essential for the maintenance of genomic integrity and is considered to act as a barrier to the early stages of tumorigenesis. The integral role of ataxia telangiectasia mutated (ATM) kinase in the response to DNA damaging agents is well characterized; however, ATM can also be activated by non-DNA damaging agents. In fact, much has been learnt recently about the mechanism of ATM activation in response to physiologic stresses such as hypoxia that do not induce DNA damage. Regions of low oxygen concentrations that occur in solid tumors are associated with a poor prognostic outcome irrespective of treatment modality. Severe levels of hypoxia induce replication stress and trigger the activation of DNA damage response pathways including ataxia telangiectasia and Rad3-related (ATR)- and ATM-mediated signaling. In this review, we discuss hypoxia-driven ATM signaling and the possible contribution of ATM activation in this context to tumorigenesis.

  7. Aven-dependent activation of ATM following DNA damage

    PubMed Central

    Guo, Jessie Yanxiang; Yamada, Ayumi; Kajino, Taisuke; Wu, Judy Qiju; Tang, Wanli; Freel, Christopher D.; Feng, Junjie; Chau, B. Nelson; Wang, Michael Zhuo; Margolis, Seth; Yoo, Hae Yong; Wang, Xiao-Fan; Dunphy, William G.; Irusta, Pablo M.; Hardwick, J. Marie; Kornbluth, Sally

    2009-01-01

    Summary Background In response to DNA damage, cells either undergo cell cycle arrest or apoptosis, depending on the extent of damage and the cell’s capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors such as Chk2 and p53 to inhibit cell cycle progression. ATM is recruited to double stranded DNA breaks by a complex of sensor proteins including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. Results In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpression in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knock-down of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM following DNA damage is enhanced by ATM-mediated Aven phosphorylation. Conclusions These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA damage signal. PMID:18571408

  8. Recurrence of the 'deep-intronic' FGG IVS6-320A>T mutation causing quantitative fibrinogen deficiency in the Italian population of Veneto.

    PubMed

    Platè, Manuela; Duga, Stefano; Castaman, Giancarlo; Rodeghiero, Francesco; Asselta, Rosanna

    2009-07-01

    Quantitative fibrinogen deficiency is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to mutations in one of the three fibrinogen genes: FGA, FGB, and FGG, coding for A alpha, B beta, and gamma chain, respectively. Although the partial defect (hypofibrinogenemia) is due to mutations occurring in the heterozygous state, homozygosity or compound heterozygosity for the same genetic defects give rise to the more severe afibrinogenemia. Mutations responsible for these conditions are scattered throughout the three fibrinogen genes, with only few sites representing relative mutational hot spots. In this study, we report the identification of the FGG IVS6-320A>T mutation in an Italian hypofibrinogenemic patient from Veneto (a region of North-Eastern Italy). This 'deep-intronic' mutation, which would go unnoticed by using conventional mutational screening strategies was previously reported in an afibrinogenemic family from Vicenza (a province of Veneto). The geographic clustering of patients carrying the FGG IVS6-320A>T mutation and the results of haplotype analysis suggest the existence of a common founder. This information will be useful to direct future genetic screenings in patients coming from the same geographic area.

  9. Pathological neoangiogenesis depends on oxidative stress regulation by ATM.

    PubMed

    Okuno, Yuji; Nakamura-Ishizu, Ayako; Otsu, Kinya; Suda, Toshio; Kubota, Yoshiaki

    2012-08-01

    The ataxia telangiectasia mutated (ATM) kinase, a master regulator of the DNA damage response (DDR), acts as a barrier to cellular senescence and tumorigenesis. Aside from DDR signaling, ATM also functions in oxidative defense. Here we show that Atm in mice is activated specifically in immature vessels in response to the accumulation of reactive oxygen species (ROS). Global or endothelial-specific Atm deficiency in mice blocked pathological neoangiogenesis in the retina. This block resulted from increased amounts of ROS and excessive activation of the mitogen activated kinase p38α rather than from defects in the canonical DDR pathway. Atm deficiency also lowered tumor angiogenesis and enhanced the antiangiogenic action of vascular endothelial growth factor (Vegf) blockade. These data suggest that pathological neoangiogenesis requires ATM-mediated oxidative defense and that agents that promote excessive ROS generation may have beneficial effects in the treatment of neovascular disease.

  10. Susceptibility of ATM-deficient pancreatic cancer cells to radiation.

    PubMed

    Ayars, Michael; Eshleman, James; Goggins, Michael

    2017-05-19

    Ataxia telangiectasia mutated (ATM) is inactivated in a significant minority of pancreatic ductal adenocarcinomas and may be predictor of treatment response. We determined if ATM deficiency renders pancreatic cancer cells more sensitive to fractionated radiation or commonly used chemotherapeutics. ATM expression was knocked down in three pancreatic cancer cell lines using ATM-targeting shRNA. Isogenic cell lines were tested for sensitivity to several chemotherapeutic agents and radiation. DNA repair kinetics were analyzed in irradiated cells using the comet assay. We find that while rendering pancreatic cancer cells ATM-deficient did not significantly change their sensitivity to several chemotherapeutics, it did render them exquisitely sensitized to radiation. Pancreatic cancer ATM status may help predict response to radiotherapy.

  11. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors

    PubMed Central

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-01-01

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (AtmKD/-) is more oncogenic than loss of ATM (Atm-/-) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate AtmKD/-, but not Atm-proficientor Atm-/- leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy. DOI: http://dx.doi.org/10.7554/eLife.14709.001 PMID:27304073

  12. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

    PubMed

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-06-15

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

  13. Detecting ATM-dependent chromatin modification in DNA damage response.

    PubMed

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lopa; Hunt, Clayton; Pandita, Tej K

    2015-01-01

    Loss of function or mutation of the ataxia-telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage.

  14. Detecting ATM-Dependent Chromatin Modification in DNA Damage Response

    PubMed Central

    Udayakumar, Durga; Horikoshi, Nobuo; Mishra, Lope; Hunt, Clayton; Pandita, Tej K.

    2015-01-01

    Loss of function or mutation of the ataxia–telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage. PMID:25827888

  15. 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

    PubMed Central

    Campbell, Andrew; Krupp, Brittany; Bushman, Jared; Noble, Mark; Pröschel, Christoph; Mayer-Pröschel, Margot

    2015-01-01

    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 (Atmtm1Mmpl/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. PMID:26310626

  16. Linking ATM Promoter Methylation to Cell Cycle Protein Expression in Brain Tumor Patients: Cellular Molecular Triangle Correlation in ATM Territory.

    PubMed

    Mehdipour, P; Karami, F; Javan, Firouzeh; Mehrazin, M

    2015-08-01

    Ataxia telangiectasia mutated (ATM) is a key gene in DNA double-strand break (DSB), and therefore, most of its disabling genetic alterations play an important initiative role in many types of cancer. However, the exact role of ATM gene and its epigenetic alterations, especially promoter methylation in different grades of brain tumors, remains elusive. The current study was conducted to query possible correlations among methylation statue of ATM gene, ATM/ retinoblastoma (RB) protein expression, D1853N ATM polymorphism, telomere length (TL), and clinicopathological characteristics of various types of brain tumors. Isolated DNA from 30 fresh tissues was extracted from different types of brain tumors and two brain tissues from deceased normal healthy individuals. DNAs were treated with bisulfate sodium using DNA modification kit (Qiagen). Methylation-specific polymerase chain reaction (MSP-PCR) was implicated to determine the methylation status of treated DNA templates confirmed by promoter sequencing. Besides, the ATM and RB protein levels were determined by immunofluorescence (IF) assay using monoclonal mouse antihuman against ATM, P53, and RB proteins. To achieve an interactive correlation, the methylation data were statistically analyzed by considering TL and D1853N ATM polymorphism. More than 73% of the brain tumors were methylated in ATM gene promoter. There was strong correlation between ATM promoter methylation and its protein expression (p < 0.001). As a triangle, meaningful correlation was also found between methylated ATM promoter and ATM protein expression with D1853N ATM polymorphism (p = 0.01). ATM protein expression was not in line with RB protein expression while it was found to be significantly correlated with ATM promoter methylation (p = 0.01). There was significant correlation between TL neither with ATM promoter methylation nor with ATM protein expression nor with D1853N polymorphism. However, TL has shown strong correlation with patient's age and

  17. Prognostic Significance of Nuclear Phospho-ATM Expression in Melanoma

    PubMed Central

    Bhandaru, Madhuri; Martinka, Magdalena; McElwee, Kevin J.; Rotte, Anand

    2015-01-01

    UV radiation induced genomic instability is one of the leading causes for melanoma. Phosphorylation of Ataxia Telangiectasia Mutated (ATM) is one of the initial events that follow DNA damage. Phospho-ATM (p-ATM) plays a key role in the activation of DNA repair and several oncogenic pathways as well as in the maintenance of genomic integrity. The present study was therefore performed to understand the significance of p-ATM in melanoma progression and to correlate it with patient prognosis. Tissue microarray and immunohistochemical analysis were employed to study the expression of p-ATM in melanoma patients. A total of 366 melanoma patients (230 primary melanoma and 136 metastatic melanoma) were used for the study. Chi-square test, Kaplan-Meier, univariate and multivariate Cox regression analysis were used to elucidate the prognostic significance of p-ATM expression. Results revealed that both loss of, and gain in, p-ATM expression were associated with progression of melanoma from normal nevi to metastatic melanoma. Patients whose samples showed negative or strong p-ATM staining had significantly worse 5-year survival compared to patients who had weak to moderate expression. Loss of p-ATM expression was associated with relatively better 5-year survival, but the corresponding 10-year survival curve almost overlapped with that of strong p-ATM expression. p-ATM expression was found to be an independent prognostic factor for 5-year but not for 10-year patient survival. In conclusion our findings show that loss of p-ATM expression and gain-in p-ATM expression are indicators of worse melanoma patient survival. PMID:26275218

  18. Prognostic Significance of Nuclear Phospho-ATM Expression in Melanoma.

    PubMed

    Bhandaru, Madhuri; Martinka, Magdalena; McElwee, Kevin J; Rotte, Anand

    2015-01-01

    UV radiation induced genomic instability is one of the leading causes for melanoma. Phosphorylation of Ataxia Telangiectasia Mutated (ATM) is one of the initial events that follow DNA damage. Phospho-ATM (p-ATM) plays a key role in the activation of DNA repair and several oncogenic pathways as well as in the maintenance of genomic integrity. The present study was therefore performed to understand the significance of p-ATM in melanoma progression and to correlate it with patient prognosis. Tissue microarray and immunohistochemical analysis were employed to study the expression of p-ATM in melanoma patients. A total of 366 melanoma patients (230 primary melanoma and 136 metastatic melanoma) were used for the study. Chi-square test, Kaplan-Meier, univariate and multivariate Cox regression analysis were used to elucidate the prognostic significance of p-ATM expression. Results revealed that both loss of, and gain in, p-ATM expression were associated with progression of melanoma from normal nevi to metastatic melanoma. Patients whose samples showed negative or strong p-ATM staining had significantly worse 5-year survival compared to patients who had weak to moderate expression. Loss of p-ATM expression was associated with relatively better 5-year survival, but the corresponding 10-year survival curve almost overlapped with that of strong p-ATM expression. p-ATM expression was found to be an independent prognostic factor for 5-year but not for 10-year patient survival. In conclusion our findings show that loss of p-ATM expression and gain-in p-ATM expression are indicators of worse melanoma patient survival.

  19. Electrophysiological and trafficking defects of the SCN5A T353I mutation in Brugada syndrome are rescued by alpha-allocryptopine.

    PubMed

    Zhang, Jiancheng; Chen, Yu; Yang, Jie; Xu, Bin; Wen, Yi; Xiang, Guojian; Wei, Guoliang; Zhu, Chao; Xing, Yanwei; Li, Yang

    2015-01-05

    Brugada syndrome (BrS), which causes arrhythmias that lead to sudden cardiac death, is linked to loss-of-function mutations that affect sodium channels. Here, we investigate the rescue effect of alpha-allocryptopine (All) from Chinese herbal medicine in a T353I mutation of SCN5A, which combines trafficking abnormalities with Brugada syndrome. SCN5A-T353I expressed in HEK293 cells showed a small peak current (I(peak)) of only 59.6% of WT and an observably sustained current (I(sus)). We found that All strongly enhanced the I(peak) of the T353I channel by enhancing the plasma membrane (PM) expression of Nav1.5 and rescued defective trafficking after co-incubation with HEK293 cells that carry mutation channel 24 h. It is also beneficial to increase the I(peak) of the T353I mutation by All by prolonging the closed-state inactivation (CSI) process and shortening the recovery from inactivation of the T353I mutation. Interestingly, the I(sus) of T353I was significantly inhibited by All, which reduces the occurrence of LQT syndrome 3 (LQT3). We provide evidence that All can rescue the trafficking deficiencies and restore the cellular electrophysiological characteristics of SCN5A-T353I. This feature of All may benefit patients with the BrS-associated Nav1.5 channel and might have other potential therapeutic effects.

  20. Immunodeficiency in ataxia telangiectasia is correlated strongly with the presence of two null mutations in the ataxia telangiectasia mutated gene

    PubMed Central

    Staples, E R; McDermott, E M; Reiman, A; Byrd, P J; Ritchie, S; Taylor, A M R; Davies, E G

    2008-01-01

    Immunodeficiency affects over half of all patients with ataxia telangiectasia (A-T) and when present can contribute significantly to morbidity and mortality. A retrospective review of clinical history, immunological findings, ataxia telangiectasia mutated (ATM) enzyme activity and ATM mutation type was conducted on 80 consecutive patients attending the National Clinic for Ataxia Telangiectasia, Nottingham, UK between 1994 and 2006. The aim was to characterize the immunodeficiency in A-T and determine its relationship to the ATM mutations present. Sixty-one patients had mutations resulting in complete loss of ATM kinase activity (group A) and 19 patients had leaky splice or missense mutations resulting in residual kinase activity (group B). There was a significantly higher proportion of patients with recurrent sinopulmonary infections in group A compared with group B (31 of 61 versus four of 19 P = 0·03) and a greater need for prophylactic antibiotics (30 of 61 versus one of 19 P = 0·001). Comparing group A with group B patients, 25 of 46 had undetectable/low immunoglobulin A (IgA) levels compared with none of 19; T cell lymphopenia was found in 28 of 56 compared with one of 18 and B cell lymphopenia in 35 of 55 compared with four of 18 patients (P = 0·00004, 0·001 and 0·003 respectively). Low IgG2 subclass levels and low levels of antibodies to pneumococcal polysaccharide were more common in group A than group B (16 of 27 versus one of 11 P = 0·01; 34/43 versus six of 17 P = 0·002) patients. Ig replacement therapy was required in 10 (12·5%) of the whole cohort, all in group A. In conclusion, A-T patients with no ATM kinase activity had a markedly more severe immunological phenotype than those expressing low levels of ATM activity. PMID:18505428

  1. ATM kinase activity modulates ITCH E3-ubiquitin ligase activity

    PubMed Central

    Santini, Simonetta; Stagni, Venturina; Giambruno, Roberto; Fianco, Giulia; Di Benedetto, Anna; Mottolese, Marcella; Pellegrini, Manuela; Barilà, Daniela

    2014-01-01

    Ataxia Telangiectasia Mutated (ATM) kinase, a central regulator of the DNA damage response regulates the activity of several E3-ubiquitin ligases and the ubiquitination-proteasome system is a consistent target of ATM. ITCH is an E3-ubiquitin ligase that modulates the ubiquitination of several targets, therefore participating to the regulation of several cellular responses, among which the DNA damage response, TNFα, Notch and Hedgehog signalling and T cell development. Here we uncover ATM as a novel positive modulator of ITCH E3-ubiquitin ligase activity. A single residue on ITCH protein, S161, which is part of an ATM SQ consensus motif, is required for ATM-dependent activation of ITCH. ATM activity enhances ITCH enzymatic activity, which in turn drives the ubiquitination and degradation of c-FLIP-L and c-Jun, previously identified as ITCH substrates. Importantly, Atm deficient mice show resistance to hepatocyte cell death, similarly to Itch deficient animals, providing in vivo genetic evidence for this circuit. Our data identify ITCH as a novel component of the ATM-dependent signaling pathway and suggest that the impairment of the correct functionality of ITCH caused by Atm deficiency may contribute to the complex clinical features linked to Ataxia Telangiectasia. PMID:23435430

  2. Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

    PubMed

    Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

    2015-08-01

    High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity.

  3. Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction

    PubMed Central

    Resseguie, Emily A.; Staversky, Rhonda J.; Brookes, Paul S.; O’Reilly, Michael A.

    2015-01-01

    High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12 h and basal respiration by 48 h. ROS were significantly increased at 24 h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. PMID:25967673

  4. Mode of ATM-dependent suppression of chromosome translocation

    SciTech Connect

    Yamauchi, Motohiro; Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer We addressed how ATM suppresses frequency of chromosome translocation. Black-Right-Pointing-Pointer We found ATM/p53-dependent G1 checkpoint suppresses translocation frequency. Black-Right-Pointing-Pointer We found ATM and DNA-PKcs function in a common pathway to suppress translocation. -- Abstract: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

  5. Pleiotropic Effects of the Trichloroethylene-Associated P81S VHL Mutation on Metabolism, Apoptosis, and ATM-Mediated DNA Damage Response

    PubMed Central

    2013-01-01

    Background The risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations. Methods VHL deficient (Vhl -/-) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided. Results The P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation–induced apoptosis. Conclusions The TCE-associated P81S VHL mutation can initiate a unique adaptive response required

  6. Effect of TERT and ATM on gene expression profiles in human fibroblasts.

    PubMed

    Baross, Agnes; Schertzer, Mike; Zuyderduyn, Scott D; Jones, Steven J M; Marra, Marco A; Lansdorp, Peter M

    2004-04-01

    Telomeres protect chromosomes from degradation, end-to-end fusion, and illegitimate recombination. Loss of telomeres may lead to cell death or senescence or may cause genomic instability, leading to tumor formation. Expression of human telomerase reverse transcriptase (TERT) in human fibroblast cells elongates their telomeres and extends their lifespan. Ataxia telangiectasia mutated (ATM) deficiency in A-T human fibroblasts results in accelerated telomere shortening, abnormal cell-cycle response to DNA damage, and early senescence. Gene expression profiling was performed by serial analysis of gene expression (SAGE) on BJ normal human skin fibroblasts, A-T cells, and BJ and A-T cells transduced with TERT cDNA and expressing telomerase activity. In the four SAGE libraries, 36,921 unique SAGE tags were detected. Pairwise comparisons between the libraries showed differential expression levels of 1%-8% of the tags. Transcripts affected by both TERT and ATM were identified according to expression patterns, making them good candidates for further studies of pathways affected by both TERT and ATM. These include MT2A, P4HB, LGALS1, CFL1, LDHA, S100A10, EIF3S8, RANBP9, and SEC63. These genes are involved in apoptosis or processes related to cell growth, and most have been found to be deregulated in cancer. Our results have provided further insight into the roles of TERT and ATM by identifying genes likely to be involved in their function. Supplementary material for this article can be found on the Genes, Chromosomes and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html. Copyright 2004 Wiley-Liss, Inc.

  7. Founder Effect of a c.828+3A>T Splice Site Mutation in Peripherin 2 (PRPH2) Causing Autosomal Dominant Retinal Dystrophies

    PubMed Central

    Shankar, Suma P.; Birch, David G.; Ruiz, Richard S.; Hughbanks-Wheaton, Dianna K.; Sullivan, Lori S.; Bowne, Sara J.; Stone, Edwin M.; Daiger, Stephen P.

    2015-01-01

    Importance Screening for splice site mutation c.828+3A>T in the peripherin 2 (PRPH2) gene should be a high priority in families with highly variable retinal dystrophies. The correction of missplicing is a potential therapeutic target. Objective To determine the prevalence, genetic origin, and molecular mechanism of a donor c.828+3A>T mutation in the PRPH2 (peripherin 2, retinal degeneration slow) gene in individuals with retinal dystrophies. Design, Setting, and Participants Case-control study that took place at the University of Texas Health Science Center, the University of Iowa, and the Retina Foundation of the Southwest, from January 1, 1987, to August 1, 2014, including affected individuals from 200 families with a diagnosis of autosomal dominant retinitis pigmentosa, 35 families with unspecified macular dystrophies, and 116 families with pattern dystrophy. Participants were screened for the c.828+3A>T mutation by restriction-enzyme digest, single-strand conformational polymorphism screening, or bidirectional sequencing. Haplotypes of polymorphic markers flanking the PRPH2 locus and sequence variants within the gene were determined by denaturing gel electrophoresis or automated capillary-based cycle sequencing. The effect of the splice site mutation on the PRPH2 transcript was analyzed using NetGene2, a splice prediction program and by the reverse transcription polymerase chain reaction of illegitimate transcripts from peripheral white blood cells. Main Outcomes and Measures Results of testing for splice site mutation, haplotypes, and alternate transcripts. Results The PRPH2 mutation was found in 97 individuals of 19 independently ascertained families with a clinical diagnosis of retinitis pigmentosa, macular dystrophy, and/or pattern dystrophy. All affected individuals also shared a rare haplotype of approximately 644 kilobase pairs containing the c.828+3A>T mutation, which extends from the short tandem repeat polymorphism D6S282 to c.1013G>A (rs434102, a

  8. A new ataxia-telangiectasia mutation in an 11-year-old female.

    PubMed

    Mortaz, Esmaeil; Marashian, Sayed Mehran; Ghaffaripour, Hosseinali; Varahram, Mohammad; Mehrian, Payam; Dorudinia, Atosa; Garssen, Johan; Adcock, Ian M; Taylor, Malcolm; Mahdaviani, Seyed Alireza

    2017-07-01

    Ataxia-telangiectasia (A-T), a rare inherited disorder, usually affects the nervous and immune systems, and occasionally other organs. A-T is associated mainly with mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a protein kinase that has a major role in the cellular response to DNA damage. We report here a novel ATM mutation (c.3244_3245insG; p.His1082fs) in an 11-year old female. This subject presented with typical features, with the addition of chest manifestations including mediastinal lymphadenopathy and diffuse bilateral micronodular infiltration of the lungs, along with a high EBV titer. The subject died as a result of rapid B-cell lymphoma progression before chemotherapy could be initiated. This case highlights the need for the rapid diagnosis of A-T mutations and the detection of associated life-threatening outcomes such as cancers.

  9. ATM facilitates mouse gammaherpesvirus reactivation from myeloid cells during chronic infection.

    PubMed

    Kulinski, Joseph M; Darrah, Eric J; Broniowska, Katarzyna A; Mboko, Wadzanai P; Mounce, Bryan C; Malherbe, Laurent P; Corbett, John A; Gauld, Stephen B; Tarakanova, Vera L

    2015-09-01

    Gammaherpesviruses are cancer-associated pathogens that establish life-long infection in most adults. Insufficiency of Ataxia-Telangiectasia mutated (ATM) kinase leads to a poor control of chronic gammaherpesvirus infection via an unknown mechanism that likely involves a suboptimal antiviral response. In contrast to the phenotype in the intact host, ATM facilitates gammaherpesvirus reactivation and replication in vitro. We hypothesized that ATM mediates both pro- and antiviral activities to regulate chronic gammaherpesvirus infection in an immunocompetent host. To test the proposed proviral activity of ATM in vivo, we generated mice with ATM deficiency limited to myeloid cells. Myeloid-specific ATM deficiency attenuated gammaherpesvirus infection during the establishment of viral latency. The results of our study uncover a proviral role of ATM in the context of gammaherpesvirus infection in vivo and support a model where ATM combines pro- and antiviral functions to facilitate both gammaherpesvirus-specific T cell immune response and viral reactivation in vivo.

  10. Induction of A.T to G.C mutations by erroneous repair of depurinated DNA following estrogen treatment of the mammary gland of ACI rats.

    PubMed

    Mailander, Paula C; Meza, Jane L; Higginbotham, Sheila; Chakravarti, Dhrubajyoti

    2006-11-01

    Evidence suggests that the genotoxic mechanism of estrogens (estrone/estradiol) in breast cancer involves their oxidation to 3,4-quinones and reaction with DNA to form depurinating N3Ade and N7Gua adducts. We examined whether estrogen genotoxicity is mutagenic in the mammary gland of the female ACI rat, a model for estrogen-dependent breast cancer. Mutagenesis was studied by PCR amplification of the H-ras1 gene (exons 1-2), cloning in pUC18, transforming Escherichia coli, and sequencing the inserts in plasmids from individual colonies. Mammary glands of both estrogen-responsive (ACI and DA) and resistant (Sprague-Dawley) rats contained pre-existing mutations at frequencies of (39.8-58.8)x10(-5), the majority (62.5-100%) of which were A.T to G.C transitions. Estradiol-3,4-quinone (200 nmol) treatment of ACI rats caused rapid (6h to 1 day) mutagenesis (frequency (83.3-156.1)x10(-5); A.T to G.C 70-73.3%). The estrogen-induced A.T to G.C mutations were detected as G.T heteroduplexes, as would be expected if N3Ade depurinations caused Gua misincorporations by erroneous repair. These heteroduplexes were identified by the T.G-DNA glycosylase (TDG) assay. TDG converts G.T heteroduplexes to G.abasic sites, rendering DNA templates refractory to PCR amplification. Consequently, A.T to G.C mutations present as G.T heteroduplexes in the DNA are eliminated from the spectra. TDG treatment of mammary DNA from estradiol-3,4-quinone-treated ACI rats brought A.T to G.C mutations down to pre-existing frequencies. Our results demonstrate that treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A.T to G.C mutations in the DNA of the mammary gland of ACI rats.

  11. WWOX guards genome stability by activating ATM

    PubMed Central

    Hazan, Idit; Abu-Odeh, Mohammad; Hofmann, Thomas G; Aqeilan, Rami I

    2015-01-01

    Common fragile sites (CFSs) tend to break upon replication stress and have been suggested to be “hot spots” for genomic instability. Recent evidence, however, implies that in the wake of DNA damage, WW domain-containing oxidoreductase (WWOX, the gene product of the FRA16D fragile site), associates with ataxia telangiectasia-mutated (ATM) and regulates its activation to maintain genomic integrity. PMID:27308504

  12. Autosomal Dominant Retinal Dystrophies Caused by a Founder Splice Site Mutation, c.828+3A>T, in PRPH2 and Protein Haplotypes in trans as Modifiers

    PubMed Central

    Shankar, Suma P.; Hughbanks-Wheaton, Dianna K.; Birch, David G.; Sullivan, Lori S.; Conneely, Karen N.; Bowne, Sara J.; Stone, Edwin M.; Daiger, Stephen P.

    2016-01-01

    Purpose We determined the phenotypic variation, disease progression, and potential modifiers of autosomal dominant retinal dystrophies caused by a splice site founder mutation, c.828+3A>T, in the PRPH2 gene. Methods A total of 62 individuals (19 families) harboring the PRPH2 c.828+3A>T mutation, had phenotype analysis by fundus appearance, electrophysiology, and visual fields. The PRPH2 haplotypes in trans were sequenced for potential modifying variants and generalized estimating equations (GEE) used for statistical analysis. Results Several distinct phenotypes caused by the PRPH2 c.828+3A>T mutation were observed and fell into two clinical categories: Group I (N = 44) with mild pattern dystrophies (PD) and Group II (N = 18) with more severe cone-rod dystrophy (CRD), retinitis pigmentosa (RP), and central areolar chorioretinal dystrophy (CACD). The PRPH2 Gln304-Lys310-Asp338 protein haplotype in trans was found in Group I only (29.6% vs. 0%), whereas the Glu304-Lys310-Gly338 haplotype was predominant in Group II (94.4% vs. 70.4%). Generalized estimating equations analysis for PD versus the CRD/CACD/RP phenotypes in individuals over 43 years alone with the PRPH2 haplotypes in trans and age as predictors, adjusted for correlation within families, confirmed a significant effect of haplotype on severity (P = 0.03) with an estimated odds ratio of 7.16 (95% confidence interval [CI] = [2.8, 18.4]). Conclusions The PRPH2 c.828+3A>T mutation results in multiple distinct phenotypes likely modified by protein haplotypes in trans; the odds of having the CACD/RP-like phenotype (versus the PD phenotype) are 7.16 times greater with a Glu304-Lys310-Gly338 haplotype in trans. Further functional studies of the modifying haplotypes in trans and PRPH2 splice variants may offer therapeutic targets. PMID:26842753

  13. DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)*

    PubMed Central

    Zhou, Yi; Paull, Tanya T.

    2013-01-01

    The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice. PMID:24220101

  14. ATMS Step By Step.

    ERIC Educational Resources Information Center

    National Library of Australia, Canberra.

    This manual is designed to provide an introduction and basic guide to the use of IBM's Advanced Text Management System (ATMS), the text processing system to be used for the creation of Australian data bases within AUSINET. Instructions are provided for using the system to enter, store, retrieve, and modify data, which may then be displayed at the…

  15. Variants in the ATM gene associated with a reduced risk of contralateral breast cancer.

    PubMed

    Concannon, Patrick; Haile, Robert W; Børresen-Dale, Anne-Lise; Rosenstein, Barry S; Gatti, Richard A; Teraoka, Sharon N; Diep, T Anh; Jansen, Laila; Atencio, David P; Langholz, Bryan; Capanu, Marinela; Liang, Xiaolin; Begg, Colin B; Thomas, Duncan C; Bernstein, Leslie; Olsen, Jørgen H; Malone, Kathleen E; Lynch, Charles F; Anton-Culver, Hoda; Bernstein, Jonine L

    2008-08-15

    Between 5% and 10% of women who survive a first primary breast cancer will subsequently develop a second primary cancer in the contralateral breast. The Women's Environment, Cancer, and Radiation Epidemiology Study was designed to identify genetic and environmental determinants of contralateral breast cancer (CBC). In this study, 708 women with asynchronous CBC served as cases and 1,397 women with unilateral breast cancer served as controls. ATM, a serine-threonine kinase, controls the cellular response to DNA double-strand breaks, and has been implicated in breast cancer risk. Complete mutation screening of the ATM gene in all 2,105 study participants identified 240 distinct sequence variants; only 15 were observed in >1% of subjects. Among the rare variants, deleterious alleles resulting in loss of ATM function were associated with a nonsignificant increase in risk of CBC. In contrast, carriers of common variants had a statistically significant reduction in risk of CBC. Four of these 15 variants were individually associated with a significantly decreased risk of second primary breast cancer [c.1899-55T>G, rate ratio (RR), 0.5; 95% confidence interval (CI), 0.3-0.8; c.3161C>G, RR, 0.5; 95% CI, 0.3-0.9; c.5558A>T, RR, 0.2; 95% CI, 0.1-0.6; c.6348-54T>C RR, 0.2; 95% CI, 0.1-0.8]. These data suggest that some alleles of ATM may exert an antineoplastic effect, perhaps by altering the activity of ATM as an initiator of DNA damage responses or a regulator of p53.

  16. Suppressor Mutations within the Core Binding Factor (CBF/AML1) Binding Site of a T-Cell Lymphomagenic Retrovirus

    PubMed Central

    Martiney, Marita J.; Levy, Laura S.; Lenz, Jack

    1999-01-01

    The transcriptional enhancer of the lymphomagenic mouse retrovirus SL3 contains a binding site for the transcription factor core binding factor (CBF; also called AML1, PEBP2, and SEF1). The SL3 CBF binding site is called the core. It differs from the core of the weakly lymphomagenic mouse retrovirus Akv by one nucleotide (the sequences are TGTGGTTAA and TGTGGTCAA, respectively). A mutant virus called SAA that was identical to SL3 except that its core was mutated to the Akv sequence was only moderately attenuated for lymphomagenicity. In most SAA-infected mice, tumor proviruses contained either reversions of the original mutation or one of two novel core sequences. In 20% of the SAA-infected mice, tumor proviruses retained the original SAA/Akv core mutation but acquired one of two additional mutations (underlined), TGCGGTCAA or TGTGGTCTA, that generated core elements called So and T*, respectively. We tested whether the novel base changes in the So and T* cores were suppressor mutations. SL3 mutants that contained So or T* cores in place of the wild-type sequence were generated. These viruses induced T-cell lymphomas in mice more quickly than SAA. Therefore, the mutations in the So and T* cores are indeed second-site suppressor mutations. The suppressor mutations increased CBF binding in vitro and transcriptional activity of the viral long terminal repeats (LTRs) in T lymphocytes to levels comparable to those of SL3. Thus, CBF binding was increased by any of three different nucleotide changes within the sequence of the SAA core. Increased CBF binding resulted in increased LTR transcriptional activity in T cells and in increased viral lymphomagenicity. PMID:9971797

  17. Gadd45a deletion aggravates hematopoietic stem cell dysfunction in ATM-deficient mice.

    PubMed

    Chen, Yulin; Yang, Runan; Guo, Peng; Ju, Zhenyu

    2014-01-01

    Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM(-/-)) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM(-/-) HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM(-/-) mice. Instead, ATM and Gadd45a double knockout (ATM(-/-) Gadd45a(-/-)) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM(-/-) HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM(-/-) Gadd45a(-/-) HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM(-/-) Gadd45a(-/-) mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM(-/-) mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM(-/-) HSCs.

  18. Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.

    PubMed

    Moding, Everett J; Lee, Chang-Lung; Castle, Katherine D; Oh, Patrick; Mao, Lan; Zha, Shan; Min, Hooney D; Ma, Yan; Das, Shiva; Kirsch, David G

    2014-08-01

    Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

  19. Acute Megakaryoblastic Leukemia with Myelodysplasia-related Changes Associated with ATM Gene Deletion.

    PubMed

    Ureshino, Hiroshi; Tanabe, Momoka; Kurogi, Kazuya; Miyahara, Masaharu; Kimura, Shinya

    2016-01-01

    Ataxia telangiectasia mutated (ATM) is a tumor suppressor gene, and its somatic inactivation plays a role in the pathogenesis of lymphoid malignancies. However, the role of ATM in patients with myeloid malignancies is still unknown. We herein report a case of acute megakaryoblastic leukemia (AMKL) with ATM gene deletion. An 84-year-old Japanese woman presenting with a pale face and pancytopenia was admitted to our institution and diagnosed to have AMKL with ATM gene deletion. She was treated with intravenous azacitidine. The azacitidine treatment was effective for approximately 1 year. Somatic inactivation of the ATM gene may therefore be involved in the pathogenesis of AMKL.

  20. [Research advance of retinal pathological angiogenesis related to ATM protein kinase].

    PubMed

    Lu, Li; Zheng, Zhi; Li, Chunxia

    2015-05-01

    Ataxia-telangiectasia mutated (ATM) gene could cause ataxia telangiectasia which is an autosomal recessive disease. The ATM protein kinase encoded by the ATM gene mainly distributed in nucleus as a master regulator of the DNA damage response and apoptosis via cell signaling pathways. The ATM kinase plays a key role in the pathogenesis of cellular senescence and tumor genesis. Recently, some studies have indicated that ATM protein kinase is involved in pathological neovascularization, suggesting that it could be a novel potential therapeutic target in diseases associated with pathological angiogenesis.

  1. Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation.

    PubMed

    Stavnezer, Janet; Linehan, Erin K; Thompson, Mikayla R; Habboub, Ghaith; Ucher, Anna J; Kadungure, Tatenda; Tsuchimoto, Daisuke; Nakabeppu, Yusaku; Schrader, Carol E

    2014-06-24

    Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase β. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.

  2. Combinations of Affinity-Enhancing Mutations in a T Cell Receptor Reveal Highly Nonadditive Effects within and between CDRs and Chains

    PubMed Central

    Pierce, Brian G.; Haidar, Jaafar N.; Yu, Yong; Weng, Zhiping

    2010-01-01

    Understanding the energetic and structural response to multiple mutations in a protein-protein interface is a key aspect of rational protein design. Here we investigate the cooperativity of combinations of point mutations of a T cell receptor (TCR) that binds in vivo to the HLA-A2 MHC and a viral peptide. The mutations were obtained from two sources: a structure-based design study on the TCR α chain (9 mutations) and an in vitro selection study on the TCR β chain (4 mutations). In addition to combining the highest affinity variants from each chain, we tested other combinations of mutations within and among the chains, for a total of 23 TCR mutants that we measured for binding kinetics to the peptide/MHC. A wide range of binding affinities was observed, from 2-fold to 1000-fold binding improvement over wild-type, with significant nonadditive effects observed within and between TCR chains. This included an amino acid dependent cooperative interaction between CDR1 and CDR3 residues that are separated by over 9 Å in the wild-type complex. When analyzing the kinetics of the mutations, we found that the association rates were primarily responsible for the cooperativity, while the dissociation rates were responsible for the anticooperativity (less-than-additive energetics). Based on structural modeling of anticooperative mutants we determined that side chain clash between proximal mutants likely led to nonadditive binding energies. These results highlight the complex nature of TCR association and binding and will be informative in future design efforts that combine multiple mutant residues. PMID:20681514

  3. ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib.

    PubMed

    Wang, Chen; Jette, Nicholas; Moussienko, Daniel; Bebb, D Gwyn; Lees-Miller, Susan P

    2017-04-01

    The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.

  4. A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA.

    PubMed

    Bloom, Kristie; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-01

    Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.

  5. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    PubMed

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. ATM-deficiency increases genomic instability and metastatic potential in a mouse model of pancreatic cancer.

    PubMed

    Drosos, Yiannis; Escobar, David; Chiang, Ming-Yi; Roys, Kathryn; Valentine, Virginia; Valentine, Marc B; Rehg, Jerold E; Sahai, Vaibhav; Begley, Lesa A; Ye, Jianming; Paul, Leena; McKinnon, Peter J; Sosa-Pineda, Beatriz

    2017-09-11

    Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (Kras(G12D)). We show that partial or total ATM deficiency cooperates with Kras(G12D) to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16(Ink4a) and p19(Arf). However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.

  7. Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington's disease.

    PubMed

    Lu, Xiao-Hong; Mattis, Virginia B; Wang, Nan; Al-Ramahi, Ismael; van den Berg, Nick; Fratantoni, Silvina A; Waldvogel, Henry; Greiner, Erin; Osmand, Alex; Elzein, Karla; Xiao, Jingbo; Dijkstra, Sipke; de Pril, Remko; Vinters, Harry V; Faull, Richard; Signer, Ethan; Kwak, Seung; Marugan, Juan J; Botas, Juan; Fischer, David F; Svendsen, Clive N; Munoz-Sanjuan, Ignacio; Yang, X William

    2014-12-24

    Age-related neurodegenerative disorders including Alzheimer's disease and Huntington's disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer's disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD. Copyright © 2014, American Association for the Advancement of Science.

  8. ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

    PubMed Central

    Andrisse, Stanley; Patel, Gaytri D.; Chen, Joseph E.; Webber, Andrea M.; Spears, Larry D.; Koehler, Rikki M.; Robinson-Hill, Rona M.; Ching, James K.; Jeong, Imju; Fisher, Jonathan S.

    2013-01-01

    Objective The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1. Research Design and Methods L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity. Results In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL. Conclusions These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction. PMID:23776597

  9. A novel mutation c.1048A>T at codon 350(Lys>Stop) in PROC gene causing neonatal purpura fulminans.

    PubMed

    Jain, Rakhi; T, Leenath; Chandran, Jolly; Jayandharan, Giridhara R; Palle, Arpana; Moses, Prabhakar D

    2013-12-01

    Purpura fulminans in the neonatal period due to severe congenital protein C deficiency (protein C activity <1 IU/dl) is a rare autosomal recessive disorder. If untreated, it is fatal. Early identification of such patients may be lifesaving. Acquired deficiency of protein C caused by increased consumption as overt disseminated intravascular coagulation (DIC) and severe infection creates a diagnostic dilemma. Mutation analysis plays a critical role in confirming the diagnosis of the disease and offering prenatal diagnosis. In this report, we describe a newborn who presented with purpura fulminans and DIC, molecular analysis showed a novel c.1048A>T transversion in a homozygous state at codon 350 (Lys>Stop) of protein C (PROC) gene. Prenatal diagnosis in subsequent pregnancy was done which revealed the affected fetus had the same mutation in homozygous form.

  10. Phosphorylation of p300 by ATM controls the stability of NBS1

    SciTech Connect

    Jang, Eun Ryoung; Choi, Jae Duk; Jeong, Gajin; Lee, Jong-Soo

    2010-07-09

    Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon {gamma}-irradiation in ATM{sup +} cells but not ATM{sup -} cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

  11. Phosphorylation of p300 by ATM controls the stability of NBS1.

    PubMed

    Jang, Eun Ryoung; Choi, Jae Duk; Jeong, Gajin; Lee, Jong-Soo

    2010-07-09

    Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon gamma-irradiation in ATM(+) cells but not ATM(-) cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

  12. Histone deacetylase regulation of ATM-mediated DNA damage signaling.

    PubMed

    Thurn, K Ted; Thomas, Scott; Raha, Paromita; Qureshi, Ian; Munster, Pamela N

    2013-10-01

    Ataxia-telangiectasia mutated (ATM) is a major regulator of the DNA damage response. ATM promotes the activation of BRCA1, CHK2, and p53 leading to the induction of response genes such as CDKN1A (p21), GADD45A, and RRM2B that promote cell-cycle arrest and DNA repair. The upregulation of these response genes may contribute to resistance of cancer cells to genotoxic therapies. Here, we show that histone deacetylases (HDAC) play a major role in mitigating the response of the ATM pathway to DNA damage. HDAC inhibition decreased ATM activation and expression, and attenuated the activation of p53 in vitro and in vivo. Select depletion of HDAC1 and HDAC2 was sufficient to modulate ATM activation, reduce GADD45A and RRM2B induction, and increase sensitivity to DNA strand breaks. The regulation of ATM by HDAC enzymes therefore suggests a vital role for HDAC1 and HDAC2 in the DNA damage response, and the potential use of the ATM pathway as a pharmacodynamic marker for combination therapies involving HDAC inhibitors. ©2013 AACR.

  13. Structure of the intact ATM/Tel1 kinase.

    PubMed

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-27

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  14. Structure of the intact ATM/Tel1 kinase

    NASA Astrophysics Data System (ADS)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  15. Structure of the intact ATM/Tel1 kinase

    PubMed Central

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-01-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents. PMID:27229179

  16. Variable phenotypic expressivity in a Swiss family with autosomal dominant retinitis pigmentosa due to a T494M mutation in the PRPF3 gene

    PubMed Central

    Gaillard, Marie-Claire; Tiab, L.; Schorderet, Daniel F.; Munier, Francis L.

    2010-01-01

    Purpose To characterize the clinical, psychophysical, and electrophysiological phenotypes in a five-generation Swiss family with dominantly inherited retinitis pigmentosa caused by a T494M mutation in the Precursor mRNA-Processing factor 3 (PRPF3) gene, and to relate the phenotype to the underlying genetic mutation. Methods Eleven affected patients were ascertained for phenotypic and genotypic characterization. Ophthalmologic evaluations included color vision testing, Goldmann perimetry, and digital fundus photography. Some patients had autofluorescence imaging, Optical Coherence Tomography, and ISCEV-standard full-field electroretinography. All affected patients had genetic testing. Results The age of onset of night blindness and the severity of the progression of the disease varied between members of the family. Some patients reported early onset of night blindness at age three, with subsequent severe deterioration of visual acuity, which was 0.4 in the best eye after their fifties. The second group of patients had a later onset of night blindness, in the mid-twenties, with a milder disease progression and a visual acuity of 0.8 at age 70. Fundus autofluorescence imaging and electrophysiological and visual field abnormalities also showed some degree of varying phenotypes. The autofluorescence imaging showed a large high-density ring bilaterally. Myopia (range: −0.75 to −8) was found in 10/11 affected subjects. Fundus findings showed areas of atrophy along the arcades. A T494M change was found in exon 11 of the PRPF3 gene. The change segregates with the disease in the family. Conclusions A mutation in the PRPF3 gene is rare compared to other genes causing autosomal dominant retinitis pigmentosa (ADRP). Although a T494M change has been reported, the family in our study is the first with variable expressivity. Mutations in the PRPF3 gene can cause a variable ADRP phenotype, unlike in the previously described Danish, English, and Japanese families. Our report

  17. ATM kinase: Much more than a DNA damage responsive protein.

    PubMed

    Guleria, Ayushi; Chandna, Sudhir

    2016-03-01

    ATM, mutation of which causes Ataxia telangiectasia, has emerged as a cardinal multifunctional protein kinase during past two decades as evidenced by various studies from around the globe. Further to its well established and predominant role in DNA damage response, ATM has also been understood to help in maintaining overall functional integrity of cells; since its mutation, inactivation or deficiency results in a variety of pathological manifestations besides DNA damage. These include oxidative stress, metabolic syndrome, mitochondrial dysfunction as well as neurodegeneration. Recently, high throughput screening using proteomics, metabolomics and transcriptomic studies revealed several proteins which might be acting as substrates of ATM. Studies that can help in identifying effective regulatory controls within the ATM-mediated pathways/mechanisms can help in developing better therapeutics. In fact, more in-depth understanding of ATM-dependent cellular signals could also help in the treatment of variety of other disease conditions since these pathways seem to control many critical cellular functions. In this review, we have attempted to put together a detailed yet lucid picture of the present-day understanding of ATM's role in various pathophysiological conditions involving DNA damage and beyond. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. ATM controls meiotic double-strand-break formation.

    PubMed

    Lange, Julian; Pan, Jing; Cole, Francesca; Thelen, Michael P; Jasin, Maria; Keeney, Scott

    2011-10-16

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

  19. Antisense Oligonucleotides Modulating Activation of a Nonsense-Mediated RNA Decay Switch Exon in the ATM Gene

    PubMed Central

    Kralovicova, Jana; Moreno, Pedro M.D.; Cross, Nicholas C.P.; Pêgo, Ana Paula

    2016-01-01

    ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs. PMID:27658045

  20. Mode of ATM-dependent suppression of chromosome translocation.

    PubMed

    Yamauchi, Motohiro; Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi

    2011-12-09

    It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Loss of ATM accelerates pancreatic cancer formation and epithelial-mesenchymal transition.

    PubMed

    Russell, Ronan; Perkhofer, Lukas; Liebau, Stefan; Lin, Qiong; Lechel, André; Feld, Fenja M; Hessmann, Elisabeth; Gaedcke, Jochen; Güthle, Melanie; Zenke, Martin; Hartmann, Daniel; von Figura, Guido; Weissinger, Stephanie E; Rudolph, Karl-Lenhard; Möller, Peter; Lennerz, Jochen K; Seufferlein, Thomas; Wagner, Martin; Kleger, Alexander

    2015-07-29

    Pancreatic ductal adenocarcinoma (PDAC) is associated with accumulation of particular oncogenic mutations and recent genetic sequencing studies have identified ataxia telangiectasia-mutated (ATM) mutations in PDAC cohorts. Here we report that conditional deletion of ATM in a mouse model of PDAC induces a greater number of proliferative precursor lesions coupled with a pronounced fibrotic reaction. ATM-targeted mice display altered TGFβ-superfamily signalling and enhanced epithelial-to-mesenchymal transition (EMT) coupled with shortened survival. Notably, our mouse model recapitulates many features of more aggressive human PDAC subtypes. Particularly, we report that low expression of ATM predicts EMT, a gene signature specific for Bmp4 signalling and poor prognosis in human PDAC. Our data suggest an intimate link between ATM expression and pancreatic cancer progression in mice and men.

  2. Loss of ATM accelerates pancreatic cancer formation and epithelial–mesenchymal transition

    PubMed Central

    Russell, Ronan; Perkhofer, Lukas; Liebau, Stefan; Lin, Qiong; Lechel, André; Feld, Fenja M; Hessmann, Elisabeth; Gaedcke, Jochen; Güthle, Melanie; Zenke, Martin; Hartmann, Daniel; von Figura, Guido; Weissinger, Stephanie E; Rudolph, Karl-Lenhard; Möller, Peter; Lennerz, Jochen K; Seufferlein, Thomas; Wagner, Martin; Kleger, Alexander

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is associated with accumulation of particular oncogenic mutations and recent genetic sequencing studies have identified ataxia telangiectasia-mutated (ATM) mutations in PDAC cohorts. Here we report that conditional deletion of ATM in a mouse model of PDAC induces a greater number of proliferative precursor lesions coupled with a pronounced fibrotic reaction. ATM-targeted mice display altered TGFβ-superfamily signalling and enhanced epithelial-to-mesenchymal transition (EMT) coupled with shortened survival. Notably, our mouse model recapitulates many features of more aggressive human PDAC subtypes. Particularly, we report that low expression of ATM predicts EMT, a gene signature specific for Bmp4 signalling and poor prognosis in human PDAC. Our data suggest an intimate link between ATM expression and pancreatic cancer progression in mice and men. PMID:26220524

  3. ATM modulates transcription in response to histone deacetylase inhibition as part of its DNA damage response.

    PubMed

    Jang, Eun Ryoung; Choi, Jae Duk; Park, Mi Ae; Jeong, Gajin; Cho, Hyeseong; Lee, Jong-Soo

    2010-03-31

    Chromatin structure has a crucial role in a diversity of physiological processes, including development, differentiation and stress responses, via regulation of transcription, DNA replication and DNA damage repair. Histone deacetylase (HDAC) inhibitors regulate chromatin structure and activate the DNA damage checkpoint pathway involving Ataxia-telangiectasia mutated (ATM). Herein, we investigated the impact of histone acetylation/deacetylation modification on the ATM-mediated transcriptional modulation to provide a better understanding of the transcriptional function of ATM. The prototype HDAC inhibitor trichostain A (TSA) reprograms expression of the myeloid cell leukemia-1 (MCL1) and Gadd45 genes via the ATM-mediated signal pathway. Transcription of MCL1 and Gadd45alpha is enhanced following TSA treatment in ATM(+) cells, but not in isogenic ATM(-) or kinase-dead ATM expressing cells, in the ATM-activated E2F1 or BRCA1- dependent manner, respectively. These findings suggest that ATM and its kinase activity are essential for the TSA-induced regulation of gene expression. In summary, ATM controls the transcriptional upregulation of MCL1 and Gadd45 through the activation of the ATM-mediated signal pathway in response to HDAC inhibition. These findings are important in helping to design combinatory treatment schedules for anticancer radio- or chemo-therapy with HDAC inhibitors.

  4. Coexistence of a T118M PMP22 missense mutation and chromosome 17 (17p11.2-p12) deletion.

    PubMed

    Jerath, Nivedita U; Kamholz, John; Grider, Tiffany; Harper, Amy; Swenson, Andrea; Shy, Michael E

    2015-11-01

    We describe a 6-year-old girl with a T118M PMP22 mutation and heterozygous deletion of PMP22 on chromosome 17 (17p11.2-p12) resulting in a severe sensorimotor polyneuropathy. This study is a case report in which the relevant mutations are described. Foot pain, cavovarus feet, tibialis anterior atrophy, absent reflexes, and inability to walk were found when the patient was age 6 years. Nerve conduction studies showed evidence of a sensorimotor polyneuropathy and compressive mononeuropathies of bilateral median nerves at the wrist and ulnar nerves at the elbow. Genetic testing revealed deletion of a PMP22 allele and T118M PMP22 mutation in the remaining allele. The severe sensorimotor polyneuropathy and hereditary neuropathy with liability to pressure palsies (HNPP) in this patient was likely a consequence of both decreased expression of PMP22 causing features consistent with HNPP and unopposed expression of the T118M mutant form of PMP22 that is relatively benign in the heterozygous state. The T118M mutant form of PMP22 can be disease-modifying in the appropriate circumstances. © 2015 Wiley Periodicals, Inc.

  5. Satellite Communications for ATM

    NASA Technical Reports Server (NTRS)

    Shamma, Mohammed A.

    2003-01-01

    This presentation is an overview on Satellite Communication for the Aeronautical Telecommunication Management (ATM) research. Satellite Communications are being considered by the FAA and NASA as a possible alternative to the present and future ground systems supporting Air Traffic Communications. The international Civil Aviation Organization (ICAO) have in place Standards and Recommended Practices (SARPS) for the Aeronautical Mobile Satellite Services (AMSS) which is mainly derived from the pre-existing Inmarsat service that has been in service since the 1980s. The Working Group A of the Aeronautical Mobile Communication Panel of ICAO has also been investigating SARPS for what is called the Next Generation Satellite Service (NGSS) which conforms less to the Inmarsat based architecture and explores wider options in terms of satellite architectures. Several designs are being proposed by Firms such as Boeing, ESA, NASA that are geared toward full or secondary usage of satellite communications for ATM. Satellite communications for ATM can serve several purposes ranging from primary usage where ground services would play a minimal backup role, to an integrated solution where it will be used to cover services, or areas that are less likely to be supported by the proposed and existing ground infrastructure. Such Integrated roles can include usage of satellite communications for oceanic and remote land areas for example. It also can include relieving the capacity of the ground network by providing broadcast based services of Traffic Information Services messages (TIS-B), or Flight Information Services (FIS-B) which can take a significant portion of the ground system capacity. Additionally, satellite communication can play a backup role to support any needs for ground replacement, or additional needed capacity even after the new digital systems are in place. The additional bandwidth that can be provided via satellite communications can also open the door for many new

  6. Connecting Remote Clusters with ATM

    SciTech Connect

    Hu, T.C.; Wyckoff, P.S.

    1998-10-01

    Sandia's entry into utilizing clusters of networked workstations is called Computational Plant or CPlant for short. The design of CPlant uses Ethernet to boot the individual nodes, Myrinet to communicate within a node cluster, and ATM to connect between remote clusters. This SAND document covers the work done to enable the use of ATM on the CPlant nodes in the Fall of 1997.

  7. Mutations in a tRNA import signal define distinct receptors at the two membranes of Leishmania mitochondria.

    PubMed

    Bhattacharyya, S N; Mukherjee, S; Adhya, S

    2000-10-01

    Nucleus-encoded tRNAs are selectively imported into the mitochondrion of Leishmania, a kinetoplastid protozoan. An oligoribonucleotide constituting the D stem-loop import signal of tRNA(Tyr)(GUA) was efficiently transported into the mitochondrial matrix in organello as well as in vivo. Transfer through the inner membrane could be uncoupled from that through the outer membrane and was resistant to antibody against the outer membrane receptor TAB. A number of mutations in the import signal had differential effects on outer and inner membrane transfer. Some mutants which efficiently traversed the outer membrane were unable to enter the matrix. Conversely, restoration of the loop-closing GC pair in reverse resulted in reversion of transfer through the inner, but not the outer, membrane, and binding of the RNA to the inner membrane was restored. These experiments indicate the presence at the two membranes of receptors with distinct specificities which mediate stepwise transfer into the mitochondrial matrix. The combination of oligonucleotide mutagenesis and biochemical fractionation may provide a general tool for the identification of tRNA transport factors.

  8. AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair.

    PubMed

    Lai, Yanhao; Jiang, Zhongliang; Zhou, Jing; Osemota, Emmanuel; Liu, Yuan

    2016-07-01

    Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3'-5' exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3'-5' exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. C/G to A/T transversions represent the main type of mutation induced by gamma-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution.

    PubMed

    Braun, J E; Wanamarta, A H; van den Akker, E; Lafleur, M V; Retèl, J

    1993-10-01

    To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays. Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH). The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene. A very specific mutation spectrum was obtained with respect to the type of mutations. Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion. The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots. When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.

  10. ATM CMG bearing failure analysis

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The cause or causes for the failure of ATM CMG S/N 5 (Skylab 1) and the anomalies associated with ATM CMG S/N 6 (Skylab 2) were investigated. Skylab telemetry data were reviewed and presented in the form of parameter distributions. The theory that the problems were caused by marginal bearing lubrication was studied along with the effects of orbital conditions on lubricants. Bearing tests were performed to investigate the effect of lubricant or lack of lubricant in the ATM CMG bearings and the dispersion and migration of the lubricant. The vacuum and weightless conditions of space were simulated in the bearing tests. Analysis of the results of the tests conducted points to inadequate lubrication as the predominant factor causing the failure of ATM CMG S/N 5 (Skylab 1) and the anomalies associated with ATM CMG S/N 6 (Skylab 2).

  11. Chemical screening identifies ATM as a target for alleviating senescence.

    PubMed

    Kang, Hyun Tae; Park, Joon Tae; Choi, Kobong; Kim, Yongsub; Choi, Hyo Jei Claudia; Jung, Chul Won; Lee, Young-Sam; Park, Sang Chul

    2017-06-01

    Senescence, defined as irreversible cell-cycle arrest, is the main driving force of aging and age-related diseases. Here, we performed high-throughput screening to identify compounds that alleviate senescence and identified the ataxia telangiectasia mutated (ATM) inhibitor KU-60019 as an effective agent. To elucidate the mechanism underlying ATM's role in senescence, we performed a yeast two-hybrid screen and found that ATM interacted with the vacuolar ATPase V1 subunits ATP6V1E1 and ATP6V1G1. Specifically, ATM decreased E-G dimerization through direct phosphorylation of ATP6V1G1. Attenuation of ATM activity restored the dimerization, thus consequently facilitating assembly of the V1 and V0 domains with concomitant reacidification of the lysosome. In turn, this reacidification induced the functional recovery of the lysosome/autophagy system and was coupled with mitochondrial functional recovery and metabolic reprogramming. Together, our data reveal a new mechanism through which senescence is controlled by the lysosomal-mitochondrial axis, whose function is modulated by the fine-tuning of ATM activity.

  12. Quantitative and Dynamic Imaging of ATM Kinase Activity.

    PubMed

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  13. Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC

    PubMed Central

    Petersen, Lars F.; Klimowicz, Alexander C.; Otsuka, Shannon; Elegbede, Anifat A.; Petrillo, Stephanie K.; Williamson, Tyler; Williamson, Chris T.; Konno, Mie; Lees-Miller, Susan P.; Hao, Desiree; Morris, Don; Magliocco, Anthony M.; Bebb, D. Gwyn

    2017-01-01

    Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p < 0.01). This effect was pronounced in stage II/III patients, even after adjusting for other clinical co-variates (p < 0.001). Additionally, we provide evidence that ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients. PMID:28418844

  14. Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC.

    PubMed

    Petersen, Lars F; Klimowicz, Alexander C; Otsuka, Shannon; Elegbede, Anifat A; Petrillo, Stephanie K; Williamson, Tyler; Williamson, Chris T; Konno, Mie; Lees-Miller, Susan P; Hao, Desiree; Morris, Don; Magliocco, Anthony M; Bebb, D Gwyn

    2017-06-13

    Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p < 0.01). This effect was pronounced in stage II/III patients, even after adjusting for other clinical co-variates (p < 0.001). Additionally, we provide evidence that ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients.

  15. Global Analysis of ATM Polymorphism Reveals Significant Functional Constraint

    PubMed Central

    Thorstenson, Yvonne R.; Shen, Peidong; Tusher, Virginia G.; Wayne, Tierney L.; Davis, Ronald W.; Chu, Gilbert; Oefner, Peter J.

    2001-01-01

    ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%–100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure. PMID:11443540

  16. ATM-deficiency sensitizes Mantle Cell Lymphoma cells to PARP-1 inhibitors

    PubMed Central

    Williamson, Chris T.; Muzik, Huong; Turhan, Ali G.; Zamò, Alberto; O’Connor, Mark J.; Bebb, D. Gwyn; Lees-Miller, Susan P.

    2013-01-01

    Poly-ADP ribose polymerase-1 (PARP-1) inhibition is toxic to cells with mutations in the breast and ovarian cancer susceptibility genes BRCA1 or BRCA2, a concept, termed synthetic lethality. However, whether this approach is applicable to other human cancers with defects in other DNA repair genes has yet to be determined. The Ataxia-Telangiectasia Mutated (ATM) gene is altered in a number of human cancers including Mantle Cell Lymphoma (MCL). Here, we characterize a panel of MCL cell lines for ATM status and function and investigate the potential for synthetic lethality in MCL in the presence of small molecule inhibitors of PARP-1. We show that Granta-519 and UPN2 cells have low levels of ATM protein, are defective in DNA damage-induced ATM-dependent signaling, are radiation sensitive and have cell cycle checkpoint defects: all characteristics of defective ATM function. Significantly, Granta-519 and UPN2 cells were more sensitive to PARP-1 inhibition, than were the ATM-proficient MCL cell lines examined. Furthermore, the PARP-1 inhibitor olaparib (previously known as AZD2281/KU-0059436) significantly decreased tumour growth and increased overall survival in mice bearing subcutaneous xenografts of ATM-deficient Granta-519 cells, while producing only a modest effect on overall survival of mice bearing xenografts of the ATM-proficient cell line, Z138. Thus, PARP inhibitors have therapeutic potential in the treatment of MCL and the concept of synthetic lethality extends to human cancers with ATM alterations. PMID:20124459

  17. Ten new ATM alterations in Polish patients with ataxia-telangiectasia

    PubMed Central

    Podralska, Marta Joanna; Stembalska, Agnieszka; Ślęzak, Ryszard; Lewandowicz-Uszyńska, Aleksandra; Pietrucha, Barbara; Kołtan, Sylwia; Wigowska-Sowińska, Jadwiga; Pilch, Jacek; Mosor, Maria; Ziółkowska-Suchanek, Iwona; Dzikiewicz-Krawczyk, Agnieszka; Słomski, Ryszard

    2014-01-01

    Inherited biallelic mutations of the ATM gene are responsible for the development of ataxia telangiectasia (AT). The objective of the present study was to conduct molecular analysis of the ATM gene in a cohort of 24 Polish patients with ataxia-telangiectasia with aim being to provide an updated mutational spectrum in Polish AT patients. As a result of molecular analysis, the status of recurrent mutation was confirmed and ten new ATM variants were detected. Application of MLPA analysis allowed the detection of large genomic deletion. Previously, this type of mutation had never been seen in our population. Finally, in silico analysis was carried out for newly detected ATM alterations. In addition, functional analysis was performed to evaluate the effects of intronic variants: c.3402+30_3402+32delATC. PMID:25614872

  18. Mobile phone signal exposure triggers a hormesis-like effect in Atm(+/+) and Atm(-/-) mouse embryonic fibroblasts.

    PubMed

    Sun, Chuan; Wei, Xiaoxia; Fei, Yue; Su, Liling; Zhao, Xinyuan; Chen, Guangdi; Xu, Zhengping

    2016-11-18

    Radiofrequency electromagnetic fields (RF-EMFs) have been classified by the International Agency for Research on Cancer as possible carcinogens to humans; however, this conclusion is based on limited epidemiological findings and lacks solid support from experimental studies. In particular, there are no consistent data regarding the genotoxicity of RF-EMFs. Ataxia telangiectasia mutated (ATM) is recognised as a chief guardian of genomic stability. To address the debate on whether RF-EMFs are genotoxic, we compared the effects of 1,800 MHz RF-EMF exposure on genomic DNA in mouse embryonic fibroblasts (MEFs) with proficient (Atm(+/+)) or deficient (Atm(-/-)) ATM. In Atm(+/+) MEFs, RF-EMF exposure for 1 h at an average special absorption rate of 4.0 W/kg induced significant DNA single-strand breaks (SSBs) and activated the SSB repair mechanism. This effect reduced the DNA damage to less than that of the background level after 36 hours of exposure. In the Atm(-/-) MEFs, the same RF-EMF exposure for 12 h induced both SSBs and double-strand breaks and activated the two repair processes, which also reduced the DNA damage to less than the control level after prolonged exposure. The observed phenomenon is similar to the hormesis of a toxic substance at a low dose. To the best of our knowledge, this study is the first to report a hormesis-like effect of an RF-EMF.

  19. Tug of War between Survival and Death: Exploring ATM Function in Cancer

    PubMed Central

    Stagni, Venturina; Oropallo, Veronica; Fianco, Giulia; Antonelli, Martina; Cinà, Irene; Barilà, Daniela

    2014-01-01

    Ataxia-telangiectasia mutated (ATM) kinase is a one of the main guardian of genome stability and plays a central role in the DNA damage response (DDR). The deregulation of these pathways is strongly linked to cancer initiation and progression as well as to the development of therapeutic approaches. These observations, along with reports that identify ATM loss of function as an event that may promote tumor initiation and progression, point to ATM as a bona fide tumor suppressor. The identification of ATM as a positive modulator of several signalling networks that sustain tumorigenesis, including oxidative stress, hypoxia, receptor tyrosine kinase and AKT serine-threonine kinase activation, raise the question of whether ATM function in cancer may be more complex. This review aims to give a complete overview on the work of several labs that links ATM to the control of the balance between cell survival, proliferation and death in cancer. PMID:24681585

  20. Coexistence of a T118M PMP22 missense mutation and chromosome 17 (17p11.2-p12) deletion

    PubMed Central

    Jerath, Nivedita U.; Kamholz, John; Grider, Tiffany; Harper, Amy; Swenson, Andrea; Shy, Michael E.

    2015-01-01

    We describe a 6-year-old girl with a T118M PMP22 mutation and heterozygous deletion of PMP22 on chromosome 17 (17p11.2-p12) resulting in a severe sensorimotor polyneuropathy. Methods Case Report Results Foot pain, cavovarus feet, tibialis anterior atrophy, absent reflexes, and inability to walk were found at age 6. Nerve conduction studies showed evidence of a sensorimotor polyneuropathy and compressive mononeuropathies of bilateral median nerves at the wrist and ulnar nerves at the elbow. Genetic testing revealed a deletion of a PMP22 allele and T118M PMP22 mutation in the remaining allele. Conclusions The severe presentation of sensory motor polyneuropathy and HNPP in this patient is likely a consequence of both decreased expression of PMP22 causing features consistent with HNPP, and unopposed expression of the T118M mutant form of PMP22 that is relatively benign in the heterozygous state. The T118M mutant form of PMP22 can be disease-modifying in the appropriate circumstances. PMID:26012543

  1. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    SciTech Connect

    Morita, Akinori; Tanimoto, Keiji; Murakami, Tomoki; Morinaga, Takeshi; Hosoi, Yoshio

    2014-01-24

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  2. Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

    PubMed

    Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

    2016-11-01

    The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P < .0001), and the primary sites of metastatic carcinomas (P < .0001) compared with normal mammary glands. No significant differences in ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

  3. ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion

    PubMed Central

    Chen, Wei-Ta; Ebelt, Nancy D; Stracker, Travis H; Xhemalce, Blerta; Van Den Berg, Carla L; Miller, Kyle M

    2015-01-01

    Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression. DOI: http://dx.doi.org/10.7554/eLife.07270.001 PMID:26030852

  4. ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

    PubMed

    Barlow, C; Ribaut-Barassin, C; Zwingman, T A; Pope, A J; Brown, K D; Owens, J W; Larson, D; Harrington, E A; Haeberle, A M; Mariani, J; Eckhaus, M; Herrup, K; Bailly, Y; Wynshaw-Boris, A

    2000-01-18

    We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

  5. The Role of the DNA Damage Response Kinase Ataxia Telangiectasia Mutated in Neuroprotection

    PubMed Central

    Marinoglou, Konstantina

    2012-01-01

    It has been estimated that a human cell is confronted with 1 million DNA lesions every day, one fifth of which may originate from the activity of Reactive Oxygen Species (ROS) alone [1,2]. Terminally differentiated neurons are highly active cells with, if any, very restricted regeneration potential [3]. In addition, genome integrity and maintenance during neuronal development is crucial for the organism. Therefore, highly accurate and robust mechanisms for DNA repair are vital for neuronal cells. This requirement is emphasized by the long list of human diseases with neurodegenerative phenotypes, which are either caused by or associated with impaired function of proteins involved in the cellular response to genotoxic stress [4-8]. Ataxia Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage Response (DDR), is a node that links DDR, neuronal development, and neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to Ataxia-Telangiectasia (A-T) disease [11,13], which is characterized by severe cerebellar neurodegeneration, indicating an important protective function of ATM in the nervous system [14]. Despite the large number of studies on the molecular cause of A-T, the neuroprotective role of ATM is not well established and is contradictory to its general proapoptotic function. This review discusses the putative functions of ATM in neuronal cells and how they might contribute to neuroprotection. PMID:23239948

  6. Induction of p53 renders ATM-deficient mice refractory to hepatocarcinogenesis.

    PubMed

    Teoh, Narci; Pyakurel, Pawan; Dan, Yock Young; Swisshelm, Karen; Hou, Jing; Mitchell, Claudia; Fausto, Nelson; Gu, Yansong; Farrell, Geoffrey

    2010-03-01

    p53 Mutations are very common in human hepatocellular carcinoma, and induction of hepatic p53 expression causes lysis of implanted hepatoblastoma cells in a chimeric mouse. Ataxia Telangiectasia Mutated (ATM) kinase senses DNA strand breaks and induces p53. Our aims were to establish whether ATM deficiency alters the carcinogenic response of hepatocytes to diethylnitrosamine (DEN). Male ATM-deficient (ATM(-/-)), heterozygote (ATM(+/-)), and wild-type (WT) mice were injected with DEN at age 15 days, and animals were killed up to 12 months to assess p53, cell cycle, apoptosis, and liver tumor development. Whereas >80% of WT and ATM(+/-) mice developed hepatocellular carcinoma (HCC), at 9-12 months, ATM(-/-) mice remained refractory to DEN-induced HCC up to 15 months. At 6 and 9 months, and compared with WT mice, p53 and p19(ARF) expression were greatly enhanced in ATM(-/-) liver associated with up-regulation of ATR and Chk1; cleaved caspase-3 immunohistochemistry and caspase-3 activity were also significantly increased. Whereas livers of DEN-treated ATM(-/-) mice showed markers of senescence (beta-galactosidase, Cxcl-1), up-regulation of telomerase occurred concurrently. The possibility that such balanced senescence could result in immortalization was demonstrated in hepatocytes prepared at 9 months from DEN-treated ATM(-/-) liver. Hepatocarcinogenesis is abrogated in ATM-deficient mice in association with induction of ATR, Chk1, p53, and p19(ARF). Resultant cell cycle arrest and apoptosis of DNA-damaged cells are possible mechanisms that underlie this unique "refractoriness" to malignant transformation in DEN-initiated ATM(-/-) hepatocytes. The findings also show that prolonged up-regulation of p53 associated with some features of senescence does not inevitably cause organ failure. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. ATM-Dependent Hyper-Radiosensitivity in Mammalian Cells Irradiated by Heavy Ions

    SciTech Connect

    Xue Lian; Yu Dong Furusawa, Yoshiya; Cao Jianping; Okayasu, Ryuichi; Fan Saijun

    2009-09-01

    Purpose: Low-dose hyper-radiosensitivity (HRS) and the later appearing radioresistance (termed induced radioresistance [IRR]) was mainly studied in low linear energy transfer (LET) radiation with survival observation. The aim of this study was to find out whether equivalent hypersensitivity occurred in high LET radiation, and the roles of ataxia telangiectasia mutated (ATM) kinase. Methods and Materials: Survival and mutation were measured by clonogenic assay and HPRT mutation assay. ATM Ser1981 activation was detected by Western blotting and immunofluorescent staining. Pretreatment of specific ATM inhibitor (10 {mu}M KU55933) and activator (20 {mu}g/mL chloroquine) before carbon radiation were adopted to explore the involvement of ATM. The roles of ATM were also investigated in its G2/M checkpoint function with histone H3 phosphorylation analysis and flow cytometric assay, and DNA double strand break (DSB) repair function measured using {gamma}-H2AX foci assay. Results: HRS/IRR was observed with survival and mutation in normal human skin fibroblast cells by carbon ions, while impaired in cells with intrinsic ATM deficiency or normal cells modified with specific ATM activator or inhibitor before irradiation. The dose-response pattern of ATM kinase activation was concordant with the transition from HRS to IRR. The ATM-dependent 'early' G2 checkpoint arrest and DNA DSB repair efficiency could explain the difference between HRS and IRR. Conclusions: These data demonstrate that the HRS/IRR by carbon ion radiation is an ATM-dependent phenomenon in the cellular response to DNA damage.

  8. Sandia ATM SONET Interface Logic

    SciTech Connect

    Kitta, Joseph P.

    1994-07-21

    SASIL is used to program the EPLD's (Erasable Programmable Logic Devices) and PAL's (Programmable Array Logic) that make up a large percentage of the Sandia ATM SONET Interface (OC3 version) for the INTEL Paragon.

  9. ATM may be a protective factor in endometrial carcinogenesis with the progesterone pathway.

    PubMed

    Shan, Weiwei; Wang, Chao; Zhang, Zhenbo; Luo, Xuezhen; Ning, Chengcheng; Yu, Yinhua; Feng, Youji; Gu, Chao; Chen, Xiaojun

    2015-03-01

    The purpose of the study was to explore the role and mechanism of ataxia-telangiectasia mutated (ATM) protein in endometrial carcinogenesis. A reverse-phase protein array (RPPA) was used to analyze the expression of ATM signal pathway proteins in Ishikawa and progesterone-insensitive Ishikawa. ATM expression was detected in endometrium specimens by immunohistochemistry, including 8 cases with proliferative endometrium, 6 cases with secretory endometrium, 10 cases with simple hyperplasia (SH), 13 cases of complex hyperplasia (CH), 11 cases of endometrial atypical hyperplasia (EAH), and 83 cases with type I endometrial cancer. The relationship between ATM expression and other clinicopathological indicators was also examined in type I endometrial cancer patients. The mechanisms of ATM were explored in vitro with the endometrial cell lines Ishikawa and RL95-2. A cell counting kit-8 (CCK-8) test and Western blot analysis were performed to test proliferation and protein expression. Statistical analysis was performed with SPSS19.0. The significance level was set at 0.05. ATM was increased with medroxyprogesterone acetate (MPA) stimulation in Ishikawa in RPPA. ATM expression gradually decreased in endometrial hyperplasic lesions compared with the normal proliferative and secretory endometrium and was the lowest in type I endometrial cancer. ATM expression was negatively correlated with pathological grades in type I endometrial cancer. In vitro, ATM silencing retarded proliferation inhibition in Ishikawa and RL95-2 treated with MPA. ATM silencing could down-regulate the MPA-stimulated signal proteins, including Chk2, P53, and caspase-3 in vitro. MPA might exert its role through activating the ATM-associated pathway, ATM-Chk2-P53-caspase-3 (active), preserving normal endometrium and protecting it from malignancies. ATM might be a promising indicator for endometrial hyperplasia and cancer.

  10. Using ATM over SATCOM links

    NASA Technical Reports Server (NTRS)

    Comparetto, Gary M.

    1995-01-01

    The Asynchronous Transfer Mode (ATM) protocol is studied from the standpoint of determining what limitations, if any, exist in using it over satellite links. It is concluded that, while there is nothing intrinsic about ATM that would generally preclude its use over satellite links, there are, however, several intrinsic characteristics of satellite links, as well as some satellite system configuration-specific issues, that must be taken into account.

  11. Security Services Discovery by ATM Endsystems

    SciTech Connect

    Sholander, Peter; Tarman, Thomas

    1999-07-15

    This contribution proposes strawman techniques for Security Service Discovery by ATM endsystems in ATM networks. Candidate techniques include ILMI extensions, ANS extensions and new ATM anycast addresses. Another option is a new protocol based on an IETF service discovery protocol, such as Service Location Protocol (SLP). Finally, this contribution provides strawman requirements for Security-Based Routing in ATM networks.

  12. Somatic inactivation of ATM in hematopoietic cells predisposes mice to cyclin D3 dependent T cell acute lymphoblastic leukemia.

    PubMed

    Ehrlich, Lori A; Yang-Iott, Katherine; DeMicco, Amy; Bassing, Craig H

    2015-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of immature T cells that exhibits heterogeneity of oncogenic lesions, providing an obstacle for development of more effective and less toxic therapies. Inherited deficiency of ATM, a regulator of the cellular DNA damage response, predisposes young humans and mice to T-ALLs with clonal chromosome translocations. While acquired ATM mutation or deletion occurs in pediatric T-ALLs, the role of somatic ATM alterations in T-ALL pathogenesis remains unknown. We demonstrate here that somatic Atm inactivation in haematopoietic cells starting as these cells differentiate in utero predisposes mice to T-ALL at similar young ages and harboring analogous translocations as germline Atm-deficient mice. However, some T-ALLs from haematopoietic cell specific deletion of Atm were of more mature thymocytes, revealing that the developmental timing and celluar origin of Atm inactivation influences the phenotype of ATM-deficient T-ALLs. Although it has been hypothesized that ATM suppresses cancer by preventing deletion and inactivation of TP53, we find that Atm inhibits T-ALL independent of Tp53 deletion. Finally, we demonstrate that the Cyclin D3 protein that drives immature T cell proliferation is essential for transformation of Atm-deficient thymocytes. Our study establishes a pre-clinical model for pediatric T-ALLs with acquired ATM inactivation and identifies the cell cycle machinery as a therapeutic target for this aggressive childhood T-ALL subtype.

  13. Somatic inactivation of ATM in hematopoietic cells predisposes mice to cyclin D3 dependent T cell acute lymphoblastic leukemia

    PubMed Central

    Ehrlich, Lori A; Yang-Iott, Katherine; DeMicco, Amy; Bassing, Craig H

    2015-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of immature T cells that exhibits heterogeneity of oncogenic lesions, providing an obstacle for development of more effective and less toxic therapies. Inherited deficiency of ATM, a regulator of the cellular DNA damage response, predisposes young humans and mice to T-ALLs with clonal chromosome translocations. While acquired ATM mutation or deletion occurs in pediatric T-ALLs, the role of somatic ATM alterations in T-ALL pathogenesis remains unknown. We demonstrate here that somatic Atm inactivation in haematopoietic cells starting as these cells differentiate in utero predisposes mice to T-ALL at similar young ages and harboring analogous translocations as germline Atm-deficient mice. However, some T-ALLs from haematopoietic cell specific deletion of Atm were of more mature thymocytes, revealing that the developmental timing and celluar origin of Atm inactivation influences the phenotype of ATM-deficient T-ALLs. Although it has been hypothesized that ATM suppresses cancer by preventing deletion and inactivation of TP53, we find that Atm inhibits T-ALL independent of Tp53 deletion. Finally, we demonstrate that the Cyclin D3 protein that drives immature T cell proliferation is essential for transformation of Atm-deficient thymocytes. Our study establishes a pre-clinical model for pediatric T-ALLs with acquired ATM inactivation and identifies the cell cycle machinery as a therapeutic target for this aggressive childhood T-ALL subtype. PMID:25659036

  14. Evidence for the Deregulation of Protein Turnover Pathways in Atm-Deficient Mouse Cerebellum: An Organotypic Study.

    PubMed

    Kim, Catherine D; Reed, Ryan E; Juncker, Meredith A; Fang, Zhide; Desai, Shyamal D

    2017-07-01

    Interferon-stimulated gene 15 (ISG15), an antagonist of the ubiquitin pathway, is elevated in cells and brain tissues obtained from ataxia telangiectasia (A-T) patients. Previous studies reveal that an elevated ISG15 pathway inhibits ubiquitin-dependent protein degradation, leading to activation of basal autophagy as a compensatory mechanism for protein turnover in A-T cells. Also, genotoxic stress (ultraviolet [UV] radiation) deregulates autophagy and induces aberrant degradation of ubiquitylated proteins in A-T cells. In the current study, we show that, as in A-T cells, ISG15 protein expression is elevated in cerebellums and various other tissues obtained from Atm-compromised mice in an Atm-allele-dependent manner (Atm+/+ < Atm+/- < Atm-/-). Notably, in cerebellums, the brain part primarily affected in A-T, levels of ISG15 were significantly greater (3-fold higher) than cerebrums obtained from the same set of mice. Moreover, as in A-T cell culture, UV induces aberrant degradation of ubiquitylated proteins and autophagy in Atm-deficient, but not in Atm-proficient, cerebellar brain slices grown in culture. Thus, the ex vivo organotypic A-T mouse brain culture model mimics that of an A-T human cell culture model and could be useful for studying the role of ISG15-dependent proteinopathy in cerebellar neurodegeneration, a hallmark of A-T in humans. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

  15. Homeostatic regulation of meiotic DSB formation by ATM/ATR

    SciTech Connect

    Cooper, Tim J.; Wardell, Kayleigh; Garcia, Valerie; Neale, Matthew J.

    2014-11-15

    Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

  16. Homeostatic regulation of meiotic DSB formation by ATM/ATR.

    PubMed

    Cooper, Tim J; Wardell, Kayleigh; Garcia, Valerie; Neale, Matthew J

    2014-11-15

    Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

  17. ATM promotes apoptosis and suppresses tumorigenesis in response to Myc

    NASA Astrophysics Data System (ADS)

    Pusapati, Raju V.; Rounbehler, Robert J.; Hong, Sungki; Powers, John T.; Yan, Mingshan; Kiguchi, Kaoru; McArthur, Mark J.; Wong, Paul K.; Johnson, David G.

    2006-01-01

    Overexpression of the c-myc oncogene contributes to the development of a significant number of human cancers. In response to deregulated Myc activity, the p53 tumor suppressor is activated to promote apoptosis and inhibit tumor formation. Here we demonstrate that p53 induction in response to Myc overexpression requires the ataxia-telangiectasia mutated (ATM) kinase, a major regulator of the cellular response to DNA double-strand breaks. In a transgenic mouse model overexpressing Myc in squamous epithelial tissues, inactivation of Atm suppresses apoptosis and accelerates tumorigenesis. Deregulated Myc expression induces DNA damage in primary transgenic keratinocytes and the formation of H2AX and phospho-SMC1 foci in transgenic tissue. These findings suggest that Myc overexpression causes DNA damage in vivo and that the ATM-dependent response to this damage is critical for p53 activation, apoptosis, and the suppression of tumor development. p53 | DNA damage

  18. Traffic Management for Satellite-ATM Networks

    NASA Technical Reports Server (NTRS)

    Goyal, Rohit; Jain, Raj; Fahmy, Sonia; Vandalore, Bobby; Goyal, Mukul

    1998-01-01

    Various issues associated with "Traffic Management for Satellite-ATM Networks" are presented in viewgraph form. Specific topics include: 1) Traffic management issues for TCP/IP based data services over satellite-ATM networks; 2) Design issues for TCP/IP over ATM; 3) Optimization of the performance of TCP/IP over ATM for long delay networks; and 4) Evaluation of ATM service categories for TCP/IP traffic.

  19. UBR5-mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function.

    PubMed

    Zhang, Tianyi; Cronshaw, Janet; Kanu, Nnennaya; Snijders, Ambrosius P; Behrens, Axel

    2014-08-19

    The Mre11/Rad50/NBS1 (MRN) protein complex and ATMIN protein mediate ATM kinase signaling in response to ionizing radiation (IR) and chromatin changes, respectively. NBS1 and ATMIN directly compete for ATM binding, but the molecular mechanism favoring either NBS1 or ATMIN in response to specific stimuli is enigmatic. Here, we identify the E3 ubiquitin ligase UBR5 as a key component of ATM activation in response to IR. UBR5 interacts with ATMIN and catalyzes ubiquitination of ATMIN at lysine 238 in an IR-stimulated manner, which decreases ATMIN interaction with ATM and promotes MRN-mediated signaling. We show that UBR5 deficiency, or mutation of ATMIN lysine 238, prevents ATMIN dissociation from ATM and inhibits ATM and NBS1 foci formation after IR, thereby impairing checkpoint activation and increasing radiosensitivity. Thus, UBR5-mediated ATMIN ubiquitination is a vital event for ATM pathway selection and activation in response to DNA damage.

  20. Absence of ERK5/MAPK7 delays tumorigenesis in Atm-/- mice.

    PubMed

    Granados-Jaén, Alba; Angulo-Ibáñez, Maria; Rovira-Clavé, Xavier; Gamez, Celina Paola Vasquez; Soriano, Francesc X; Reina, Manuel; Espel, Enric

    2016-11-15

    Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm-/- mice. Compared with Atm-/- thymocytes, Mapk7-/-Atm-/- thymocytes exhibited an improved response to DNA damage (increased phosphorylation of H2AX) and a restored apoptotic response after treatment of mice with ionizing radiation. These findings define an antagonistic function of ATM and MAPK7 in the thymocyte response to DNA damage, and suggest that the lack of MAPK7 inhibits thymic lymphoma growth in Atm-/- mice by partially restoring the DNA damage response in thymocytes.

  1. Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

    PubMed

    Kobayashi, Junya; Saito, Yuichiro; Okui, Michiyo; Miwa, Noriko; Komatsu, Kenshi

    2015-04-01

    Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

    SciTech Connect

    Takubo, Keiyo . E-mail: keiyot@gmail.com; Hirao, Atsushi; Ohmura, Masako; Azuma, Masaki; Arai, Fumio; Nagamatsu, Go; Suda, Toshio . E-mail: sudato@sc.itc.keio.ac.jp

    2006-12-29

    Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16{sup Ink4a} activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16{sup Ink4a}-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16{sup Ink4a}-mediated senescence.

  3. ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts

    PubMed Central

    Qi, Yongmei; Qiu, Qian; Gu, Xueyan; Tian, Yihong; Zhang, Yingmei

    2016-01-01

    The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy. PMID:27089984

  4. ATM Inhibition Potentiates Death of Androgen Receptor-inactivated Prostate Cancer Cells with Telomere Dysfunction

    PubMed Central

    Reddy, Vidyavathi; Wu, Min; Ciavattone, Nicholas; McKenty, Nathan; Menon, Mani; Barrack, Evelyn R.; Reddy, G. Prem-Veer; Kim, Sahn-Ho

    2015-01-01

    Androgen receptor (AR) plays a role in maintaining telomere stability in prostate cancer cells, as AR inactivation induces telomere dysfunction within 3 h. Since telomere dysfunction in other systems is known to activate ATM (ataxia telangiectasia mutated)-mediated DNA damage response (DDR) signaling pathways, we investigated the role of ATM-mediated DDR signaling in AR-inactivated prostate cancer cells. Indeed, the induction of telomere dysfunction in cells treated with AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation (activation) of ATM and its downstream effector Chk2 and the presenceof phosphorylated ATM at telomeres, indicating activation of DDR signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation, induced PARP cleavage, abrogated cell cycle checkpoint activation and attenuated the formation of γH2AX foci at telomeres in AR-inactivated cells, suggesting that ATM inhibitor induces apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration-resistant prostate cancer. PMID:26336104

  5. ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts.

    PubMed

    Qi, Yongmei; Qiu, Qian; Gu, Xueyan; Tian, Yihong; Zhang, Yingmei

    2016-04-19

    The ATM (ataxia telangiectasia mutated) protein has recently been proposed to play critical roles in the response to mitochondrial dysfunction by initiating mitophagy. Here, we have used ATM-proficient GM00637 cells and ATM-deficient GM05849 cells to investigate the mitophagic effect of spermidine and to elucidate the role of ATM in spermdine-induced mitophagy. Our results indicate that spermidine induces mitophagy by eliciting mitochondrial depolarization, which triggers the formation of mitophagosomes and mitolysosomes, thereby promoting the accumulation of PINK1 and translocation of Parkin to damaged mitochondria, finally leading to the decreased mitochondrial mass in GM00637 cells. However, in GM05849 cells or GM00637 cells pretreated with the ATM kinase inhibitor KU55933, the expression of full-length PINK1 and the translocation of Parkin are blocked, and the colocalization of Parkin with either LC3 or PINK1 is disrupted. These results suggest that ATM drives the initiation of the mitophagic cascade. Our study demonstrates that spermidine induces mitophagy through ATM-dependent activation of the PINK1/Parkin pathway. These findings underscore the importance of a mitophagy regulatory network of ATM and PINK1/Parkin and elucidate a novel mechanism by which ATM influences spermidine-induced mitophagy.

  6. Gigabit ATM: another technical mistake?

    NASA Astrophysics Data System (ADS)

    Christ, Paul

    1998-09-01

    Once upon a time, or more precisely during February 1988 at the CCITT Seoul plenary, and definitely arriving as a revolution, ATM hit the hard-core B-ISDN circuit-switching gang. Initiated by the Telecoms' camp, but, surprisingly, soon to be pushed by computer minded people, ATM's generic technological history is somewhat richer than single-sided stories. Here are two classical elements of that history: Firstly, together with X.25, ATM suffers from the connection versus datagram dichotomy, well known for more than twenty years. Secondly, and lesser known, ATM's use of cells in support of the 'I' of B-ISDN was questioned from the very beginning by the packet switching camp. Furthermore, in this context, there are two other essential elements to be considered: Firstly, the exponential growth of the Internet and later intranets, using Internet technology, sparked by the success of the Web and the WINTEL alliance, resulted in a corresponding demand for both aggregate and end-system network bandwidth. Secondly, servers, historically restricted to the exclusive club of HIPPI-equipped supercomputers, suddenly become ordinary high-end PCs with 64-bit wide PCI busses -- definitely aiming at the Gigabit. Here, if your aim is for Gigabit ATM with 5000-transactions per second classical supercomputers, a 65K ATM MTU -- as implemented by Cray -- might be okay. Following Clark and others, another part of the story is the adoption and redefinition, by the IETF, of the Telecoms' notion of 'Integrated Services' and QoS mechanisms. The quest for low-delay IP packet forwarding, perhaps possible over ATM cut-throughs, has resulted in the switching versus/or integrated-with-routing movement. However, a blow for ATM may be the recent results concerning fast routing table lookup algorithms. This, by making Gigabit routing possible using ordinary Pentium processors may eventually render the much prophesized ATM switching performance unnecessary. Recently, with the rise of Gigabit Ethernet

  7. ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression.

    PubMed

    Guarini, Anna; Marinelli, Marilisa; Tavolaro, Simona; Bellacchio, Emanuele; Magliozzi, Monia; Chiaretti, Sabina; De Propris, Maria Stefania; Peragine, Nadia; Santangelo, Simona; Paoloni, Francesca; Nanni, Mauro; Del Giudice, Ilaria; Mauro, Francesca Romana; Torrente, Isabella; Foà, Robin

    2012-01-01

    The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease. Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval. Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations. ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.

  8. ATM gene alterations in chronic lymphocytic leukemia patients induce a distinct gene expression profile and predict disease progression

    PubMed Central

    Guarini, Anna; Marinelli, Marilisa; Tavolaro, Simona; Bellacchio, Emanuele; Magliozzi, Monia; Chiaretti, Sabina; De Propris, Maria Stefania; Peragine, Nadia; Santangelo, Simona; Paoloni, Francesca; Nanni, Mauro; Del Giudice, Ilaria; Mauro, Francesca Romana; Torrente, Isabella; Foà, Robin

    2012-01-01

    Background The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease. Design and Methods Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval. Results Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations. Conclusions ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval. PMID:21993670

  9. ATM down-regulation is associated with poor prognosis in sporadic breast carcinomas.

    PubMed

    Bueno, R C; Canevari, R A; Villacis, R A R; Domingues, M A C; Caldeira, J R F; Rocha, R M; Drigo, S A; Rogatto, S R

    2014-01-01

    Ataxia telangiectasia-mutated (ATM) gene downexpression has been reported in sporadic breast carcinomas (BC); however, the prognostic value and mechanisms of ATM deregulation remain unclear. ATM and miRNAs (miR-26a, miR-26b, miR-203, miR-421, miR-664, miR-576-5p and miR-18a) expression levels were evaluated by quantitative real-time PCR (RT-qPCR) in 52 BC and 3 normal breast samples. ATM protein expression was assessed by immunohistochemistry in 968 BC and 35 adjacent normal breast tissues. ATM copy number alteration was detected by array comparative genomic hybridization (aCGH) in 42 tumours. Low ATM levels were associated with tumour grade. Absence of ATM protein expression was associated with distant metastasis (P < 0.001), reduced disease-free survival (DFS, P < 0.001) and cancer-specific survival (CSS, P < 0.001). Multivariate analysis indicated ATM protein expression as an independent prognostic marker for DFS (P = 0.001, HR = 0.579) and CSS (P = 0.001, HR = 0.554). ATM copy number loss was detected in 12% of tumours and associated with lower mRNA levels. miR-421 over-expression was detected in 36.5% of cases which exhibit lower ATM transcript levels (P = 0.075, r = -0.249). The data suggest that ATM protein expression is an independent prognostic marker in sporadic BC. Gene copy number loss and miR-421 over-expression may be involved in ATM deregulation in BC.

  10. Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

    PubMed

    Skowronska, Anna; Parker, Anton; Ahmed, Gulshanara; Oldreive, Ceri; Davis, Zadie; Richards, Sue; Dyer, Martin; Matutes, Estella; Gonzalez, David; Taylor, A Malcolm R; Moss, Paul; Thomas, Peter; Oscier, David; Stankovic, Tatjana

    2012-12-20

    The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

  11. Intrinsic mitochondrial dysfunction in ATM-deficient lymphoblastoid cells.

    PubMed

    Ambrose, Mark; Goldstine, Jimena V; Gatti, Richard A

    2007-09-15

    One of the characteristic features of cells from patients with ataxia telangiectasia (A-T) is that they are in a state of continuous oxidative stress and exhibit constitutive activation of pathways that normally respond to oxidative damage. In this report, we investigated whether the oxidative stress phenotype of A-T cells might be a reflection of an intrinsic mitochondrial dysfunction. Mitotracker Red staining showed that the structural organization of mitochondria in A-T cells was abnormal compared to wild-type. Moreover, A-T cells harbored a much larger population of mitochondria with decreased membrane potential (DeltaPsi) than control cells. In addition, the basal expression levels of several nuclear DNA-encoded oxidative damage responsive genes whose proteins are targeted to the mitochondria--polymerase gamma, mitochondrial topoisomerase I, peroxiredoxin 3 and manganese superoxide dismutase--are elevated in A-T cells. Consistent with these results, we found that overall mitochondrial respiratory activity was diminished in A-T compared to wild-type cells. Treating A-T cells with the antioxidant, alpha lipoic acid (ALA), restored mitochondrial respiration rates to levels approaching those of wild-type. When wild-type cells were transfected with ATM-targeted siRNA, we observed a small but significant reduction in the respiration rates of mitochondria. Moreover, mitochondria in A-T cells induced to stably express full-length ATM, exhibited respiration rates approaching those of wild-type cells. Taken together, our results provide evidence for an intrinsic mitochondrial dysfunction in A-T cells, and implicate a requirement for ATM in the regulation of mitochondrial function.

  12. Photonic ATM Front End Processors

    DTIC Science & Technology

    1998-01-01

    b. PostDoc: Dr. Zhijian Zhang (1/2 time) and Dr. Liji Wu . (1/2 time) c. Graduate Student: Mr. S. H. Yang, (to be graduated at Dec. 1998) Circuit...Y. Chai, F. S. Choa, H. J. Chao, Z. Zhang, L. Wu , S. Yang, "A photonic ATM front-end processor", Proc. ECOC󈨦, paper TuB27, 1998. 6. H.J.Chao, Z...Zhang, L. Wu , S.H. Yang, F.S. Choa and L. Wang," A photonic ATM front-end processor", IEEE Proc. LEOS󈨦 Annual Meeting, paper WR3, 1998. 7. H. J. Chao

  13. A new hemoglobin variant: Hb Meylan [β73(E17)Asp → Phe; HBB: c.220G>T; c.221A>T] with a double base mutation at the same codon.

    PubMed

    Renoux, Céline; Feray, Cécile; Joly, Philippe; Zanella-Cleon, Isabelle; Garcia, Caroline; Lacan, Philippe; Couprie, Nicole; Francina, Alain

    2015-01-01

    We report a new β-globin chain variant: Hb Meylan [β73(E17)Asp → Phe; HBB: c.220G>T; c.221A>T]. The new variant results from a double nucleotide mutation at the same codon. The possible molecular mechanisms are discussed.

  14. Complementary functions of ATM and H2AX in development and suppression of genomic instability

    PubMed Central

    Zha, Shan; Sekiguchi, JoAnn; Brush, James W.; Bassing, Craig H.; Alt, Frederick W.

    2008-01-01

    Upon DNA damage, histone H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other phosphoinositide 3-kinase-related protein kinases. To elucidate further the potential overlapping and unique functions of ATM and H2AX, we asked whether they have synergistic functions in the development and maintenance of genomic stability by inactivating both genes in mouse germ line. Combined ATM/H2AX deficiency caused embryonic lethality and dramatic cellular genomic instability. Mechanistically, severe genomic instability in the double-deficient cells is associated with a requirement for H2AX to repair oxidative DNA damage resulting from ATM deficiency. We discuss these findings in the context of synergies between ATM and other repair factors. PMID:18599436

  15. ATM deficiency generating genomic instability sensitizes pancreatic ductal adenocarcinoma cells to therapy-induced DNA damage.

    PubMed

    Perkhofer, Lukas; Schmitt, Anna; Romero Carrasco, Maria Carolina; Ihle, Michaela; Hampp, Stephanie; Ruess, Dietrich Alexander; Hessmann, Elisabeth; Russell, Ronan; Lechel, André; Azoitei, Ninel; Lin, Qiong; Liebau, Stefan; Hohwieler, Meike; Bohnenberger, Hanibal; Lesina, Marina; Algül, Hana; Gieldon, Laura; Schröck, Evelin; Gaedcke, Jochen; Wagner, Martin; Wiesmüller, Lisa; Sipos, Bence; Seufferlein, Thomas; Reinhardt, Hans Christian; Frappart, Pierre-Olivier; Kleger, Alexander

    2017-08-08

    Pancreatic adenocarcinomas (PDAC) harbour recurrent functional mutations of the master DNA damage response kinase ATM which has been shown to accelerate tumorigenesis and epithelial-mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Copyright ©2017, American Association for Cancer Research.

  16. [Progress of the ATM Crew

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Activities for each of the following programs are discussed in separate sections for the bimonthly reporting period: Airborne Oceanographic Lidar (AOL); Airborne Topographic Mapper (ATM); Other Mission Support Activities, including modeling activities, EAARL activities, and the Scanning Radar Altimeter (SAR); Tropical Rain Measuring Mission (TRMM). The tasks undertaken for each program are discussed in the pertinent section of the report.

  17. Block loss for ATM video

    NASA Astrophysics Data System (ADS)

    Chan, Sze K.; Leon-Garcia, Alberto

    1993-10-01

    In BISDN, the asynchronous transfer mode (ATM) requires all information to be represented as a sequence of standard data units called cells. Cell los is inherent in ATM networks due to the cell header corruption and buffer overflow in the network. Several studies have shown that cell losses are bursty for an ATM network. In this work, we encoded real video sequences with a variable bit-rate (VBR) version of the H.261 video encoder in order for us to determine the relationship between blocks in a video frame and the number of ATM cells generated. We then considered the impact of bursty cell losses on image block loss probability. Block loss distributions are given at different codec and channel parameters. We also obtained block loss results using a cell loss correction scheme. Three sequences were analyzed to obtain the cumulative block loss probability distribution. Similar maximum and minimum block loss probability values were obtained for each sequence. The block loss probability distribution varies according to the amount and type of motion present in each sequence. We show that the block loss is confined to one group of blocks (GOB). The maximum block loss probability can be two orders of magnitude larger than the channel cell loss probability. By using the cell loss correction scheme, block loss was reduced to a level equivalent to reducing cell loss probability by five orders of magnitude.

  18. Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

    PubMed

    Balestrini, Alessia; Nicolas, Laura; Yang-Lott, Katherine; Guryanova, Olga A; Levine, Ross L; Bassing, Craig H; Chaudhuri, Jayanta; Petrini, John H J

    2016-02-01

    The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice). Nbs1(ΔB/ΔB) Atm(-/-) hematopoietic cells derived by crossing to vav(cre) were viable in vivo. Nbs1(ΔB/ΔB) Atm(-/-) (VAV) mice exhibited a pronounced defect in double-strand break repair and completely penetrant early onset lymphomagenesis. In addition to repair defects observed, fragile site instability was noted, indicating that the Mre11 complex promotes genome stability upon replication stress in vivo. The data suggest combined influences of the Mre11 complex on DNA repair, as well as the responses to DNA damage and DNA replication stress. A novel mouse model was developed, by combining a vav(cre)-inducible ATM knockout mouse with an NBS1 hypomorphic mutation, to analyze ATM-independent functions of the Mre11 complex in vivo. These data show that the DNA repair, rather than DDR signaling functions of the complex, is acutely required in the context of ATM deficiency to suppress genome instability and lymphomagenesis. ©2015 American Association for Cancer Research.

  19. Evaluation of cytotoxicity and DNA damage response with analysis of intracellular ATM signaling pathways.

    PubMed

    Bandi, Sriram; Viswanathan, Preeti; Gupta, Sanjeev

    2014-06-01

    Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs.

  20. NPP ATMS Snowfall Rate Product

    NASA Technical Reports Server (NTRS)

    Meng, Huan; Ferraro, Ralph; Kongoli, Cezar; Wang, Nai-Yu; Dong, Jun; Zavodsky, Bradley; Yan, Banghua

    2015-01-01

    Passive microwave measurements at certain high frequencies are sensitive to the scattering effect of snow particles and can be utilized to retrieve snowfall properties. Some of the microwave sensors with snowfall sensitive channels are Advanced Microwave Sounding Unit (AMSU), Microwave Humidity Sounder (MHS) and Advance Technology Microwave Sounder (ATMS). ATMS is the follow-on sensor to AMSU and MHS. Currently, an AMSU and MHS based land snowfall rate (SFR) product is running operationally at NOAA/NESDIS. Based on the AMSU/MHS SFR, an ATMS SFR algorithm has been developed recently. The algorithm performs retrieval in three steps: snowfall detection, retrieval of cloud properties, and estimation of snow particle terminal velocity and snowfall rate. The snowfall detection component utilizes principal component analysis and a logistic regression model. The model employs a combination of temperature and water vapor sounding channels to detect the scattering signal from falling snow and derive the probability of snowfall (Kongoli et al., 2015). In addition, a set of NWP model based filters is also employed to improve the accuracy of snowfall detection. Cloud properties are retrieved using an inversion method with an iteration algorithm and a two-stream radiative transfer model (Yan et al., 2008). A method developed by Heymsfield and Westbrook (2010) is adopted to calculate snow particle terminal velocity. Finally, snowfall rate is computed by numerically solving a complex integral. NCEP CMORPH analysis has shown that integration of ATMS SFR has improved the performance of CMORPH-Snow. The ATMS SFR product is also being assessed at several NWS Weather Forecast Offices for its usefulness in weather forecast.

  1. ATM-dependent phosphorylation of MEF2D promotes neuronal survival after DNA damage.

    PubMed

    Chan, Shing Fai; Sances, Sam; Brill, Laurence M; Okamoto, Shu-Ichi; Zaidi, Rameez; McKercher, Scott R; Akhtar, Mohd W; Nakanishi, Nobuki; Lipton, Stuart A

    2014-03-26

    Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM-MEF2D pathway may contribute to neurodegeneration in AT.

  2. ATM deficiency induces oxidative stress and endoplasmic reticulum stress in astrocytes.

    PubMed

    Liu, Na; Stoica, George; Yan, Mingshan; Scofield, Virginia L; Qiang, Wenan; Lynn, William S; Wong, Paul K Y

    2005-12-01

    ATM kinase, the product of the ataxia telangiectasia mutated (Atm) gene, is activated by genomic damage. ATM plays a crucial role in cell growth and development. Here we report that primary astrocytes isolated from ATM-deficient mice grow slowly, become senescent, and die in culture. However, before reaching senescence, these primary Atm(-/-) astrocytes, like Atm(-/-) lymphocytes, show increased spontaneous DNA synthesis. These astrocytes also show markers of oxidative stress and endoplasmic reticulum (ER) stress, including increased levels of heat shock proteins (HSP70 and GRP78), malondialdehyde adducts, Cu/Zn superoxide dismutase, procaspase 12 cleavage, and redox-sensitive phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In addition, HSP70 and ERK1/2 phosphorylation are upregulated in the cerebella of ATM-deficient mice. This increase in ERK1/2 phosphorylation is seen primarily in cerebellar astrocytes, or Bergmann glia, near degenerating Purkinje cells. ERK1/2 activation and astrogliosis are also found in other parts of the brain, for example, the cortex. We conclude that ATM deficiency induces intrinsic growth defects, oxidative stress, ER stress, and ERKs activation in astrocytes.

  3. Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.

    PubMed

    Hirozane, Toru; Tohmonda, Takahide; Yoda, Masaki; Shimoda, Masayuki; Kanai, Yae; Matsumoto, Morio; Morioka, Hideo; Nakamura, Masaya; Horiuchi, Keisuke

    2016-09-28

    Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption.

  4. ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity.

    PubMed

    Vail, Graham; Cheng, Aifang; Han, Yu Ray; Zhao, Teng; Du, Shengwang; Loy, Michael M T; Herrup, Karl; Plummer, Mark R

    2016-07-01

    Ataxia telangiectasia is a multisystemic disorder that includes a devastating neurodegeneration phenotype. The ATM (ataxia-telangiectasia mutated) protein is well-known for its role in the DNA damage response, yet ATM is also found in association with cytoplasmic vesicular structures: endosomes and lysosomes, as well as neuronal synaptic vesicles. In keeping with this latter association, electrical stimulation of the Schaffer collateral pathway in hippocampal slices from ATM-deficient mice does not elicit normal long-term potentiation (LTP). The current study was undertaken to assess the nature of this deficit. Theta burst-induced LTP was reduced in Atm(-/-) animals, with the reduction most pronounced at burst stimuli that included 6 or greater trains. To assess whether the deficit was associated with a pre- or postsynaptic failure, we analyzed paired-pulse facilitation and found that it too was significantly reduced in Atm(-/-) mice. This indicates a deficit in presynaptic function. As further evidence that these synaptic effects of ATM deficiency were presynaptic, we used stochastic optical reconstruction microscopy. Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a presynaptic marker) than with Homer1 (a postsynaptic marker). These results underline how, in addition to its nuclear functions, ATM plays an important functional role in the neuronal synapse where it participates in the regulation of presynaptic vesicle physiology. Copyright © 2016 the American Physiological Society.

  5. Loss of caspase-2 augments lymphomagenesis and enhances genomic instability in Atm-deficient mice.

    PubMed

    Puccini, Joseph; Shalini, Sonia; Voss, Anne K; Gatei, Magtouf; Wilson, Claire H; Hiwase, Devendra K; Lavin, Martin F; Dorstyn, Loretta; Kumar, Sharad

    2013-12-03

    Caspase-2, the most evolutionarily conserved member of the caspase family, has been shown to be involved in apoptosis induced by various stimuli. Our recent work indicates that caspase-2 has putative functions in tumor suppression and protection against cellular stress. As such, the loss of caspase-2 enhances lymphomagenesis in Eµ-Myc transgenic mice, and caspase-2 KO (Casp2(-/-)) mice show characteristics of premature aging. However, the extent and specificity of caspase-2 function in tumor suppression is currently unclear. To further investigate this, ataxia telangiectasia mutated KO (Atm(-/-)) mice, which develop spontaneous thymic lymphomas, were used to generate Atm(-/-)Casp2(-/-) mice. Initial characterization revealed that caspase-2 deficiency enhanced growth retardation and caused synthetic perinatal lethality in Atm(-/-) mice. A comparison of tumor susceptibility demonstrated that Atm(-/-)Casp2(-/-) mice developed tumors with a dramatically increased incidence compared with Atm(-/-) mice. Atm(-/-)Casp2(-/-) tumor cells displayed an increased proliferative capacity and extensive aneuploidy that coincided with elevated oxidative damage. Furthermore, splenic and thymic T cells derived from premalignant Atm(-/-)Casp2(-/-) mice also showed increased levels of aneuploidy. These observations suggest that the tumor suppressor activity of caspase-2 is linked to its function in the maintenance of genomic stability and suppression of oxidative damage. Given that ATM and caspase-2 are important components of the DNA damage and antioxidant defense systems, which are essential for the maintenance of genomic stability, these proteins may synergistically function in tumor suppression by regulating these processes.

  6. Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass

    PubMed Central

    Hirozane, Toru; Tohmonda, Takahide; Yoda, Masaki; Shimoda, Masayuki; Kanai, Yae; Matsumoto, Morio; Morioka, Hideo; Nakamura, Masaya; Horiuchi, Keisuke

    2016-01-01

    Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption. PMID:27677594

  7. Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

    PubMed

    Bakr, A; Oing, C; Köcher, S; Borgmann, K; Dornreiter, I; Petersen, C; Dikomey, E; Mansour, W Y

    2015-03-31

    Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

  8. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors.

    PubMed

    Guo, Kexiao; Shelat, Anang A; Guy, R Kiplin; Kastan, Michael B

    2014-04-01

    The ATM (ataxia-telangiectasia, mutated) protein kinase is a major regulator of cellular responses to DNA double-strand breaks (DSBs), DNA lesions that can be caused by ionizing irradiation (IR), oxidative damage, or exposure to certain chemical agents. In response to DSBs, the ATM kinase is activated and subsequently phosphorylates numerous downstream substrates, including p53, Chk2, BRCA1, and KAP1, which affect processes such as cell cycle progression and DNA repair. Numerous studies have demonstrated that loss of ATM function results in enhanced sensitivity to ionizing irradiation in clinically relevant dose ranges, suggesting that ATM kinase is an attractive therapeutic target for enhancing tumor cell kill with radiotherapy. Previously identified small-molecule ATM kinase inhibitors, such as CP466722 and Ku55933, were identified using in vitro kinase assays carried out with recombinant ATM kinase isolated from mammalian cells. Since it has not been feasible to express full-length recombinant ATM in bacterial or baculovirus systems, a robust in vitro screening tool has been lacking. We have developed a cell-based assay that is robust, straightforward, and sensitive. Using this high-throughput assay, we screened more than 7000 compounds and discovered additional small molecules that inhibit the ATM kinase and further validated these hits by secondary assays.

  9. Adaptive Neural Network Controller for ATM Traffic

    DTIC Science & Technology

    1996-12-01

    IEEE Communications Magazine (October 1995). 2. Baum, Eric B...Adaptive Control in ATM Networks," IEEE Communications Magazine (October 1995). 9. Evanowsky, John B. "Information for the Warrior," IEEE Communications Magazine (October...Network Applications in ATM," IEEE Communications Magazine (October 1995). 78 16. Imrich, et al. "A counter based congestion control for ATM

  10. Running TCP/IP over ATM Networks.

    ERIC Educational Resources Information Center

    Witt, Michael

    1995-01-01

    Discusses Internet protocol (IP) and subnets and describes how IP may operate over asynchronous transfer mode (ATM). Topics include TCP (transmission control protocol), ATM cells and adaptation layers, a basic architectural model for IP over ATM, address resolution, mapping IP to a subnet technology, and connection management strategy. (LRW)

  11. Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM.

    PubMed

    McCabe, Nuala; Hanna, Conor; Walker, Steven M; Gonda, David; Li, Jie; Wikstrom, Katarina; Savage, Kienan I; Butterworth, Karl T; Chen, Clark; Harkin, D Paul; Prise, Kevin M; Kennedy, Richard D

    2015-06-01

    Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors. ©2015 American Association for Cancer Research.

  12. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    SciTech Connect

    Fukumoto, Yasunori Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  13. Phenotypic consequences of somatic mutations in the ataxia-telangiectasia mutated gene in non-small cell lung cancer

    PubMed Central

    Weber, Anika Maria; Drobnitzky, Neele; Devery, Aoife Maire; Bokobza, Sivan Mili; Adams, Richard A.; Maughan, Timothy S.; Ryan, Anderson Joseph

    2016-01-01

    Mutations in the Ataxia-telangiectasia mutated (ATM) gene are frequently found in human cancers, including non-small cell lung cancer (NSCLC). Loss of ATM function confers sensitivity to ionising radiation (IR) and topoisomerase inhibitors and may thus define a subset of cancer patients that could get increased benefit from these therapies. In this study, we evaluated the phenotypic consequences of ATM missense changes reported in seven NSCLC cell lines with regard to radiosensitivity and functionality of ATM signalling. Our data demonstrate that only 2/7 NSCLC cell lines (H1395 and H23) harbouring ATM missense mutations show a functional impairment of ATM signalling following IR-exposure. In these two cell lines, the missense mutations caused a significant reduction in ATM protein levels, impairment of ATM signalling and marked radiosensitivity. Of note, only cell lines with homozygous mutations in the ATM gene showed significant impairment of ATM function. Based on these observations, we developed an immunohistochemistry-based assay to identify patients with loss or reduction of ATM protein expression in a clinical setting. In a set of 137 NSCLC and 154 colorectal cancer specimens we identified tumoral loss of ATM protein expression in 9.5% and 3.9% of cases, respectively, demonstrating the potential utility of this method. PMID:27602502

  14. Profiling of UV-induced ATM/ATR signaling pathways

    PubMed Central

    Stokes, Matthew P.; Rush, John; MacNeill, Joan; Ren, Jian Min; Sprott, Kam; Nardone, Julie; Yang, Vicky; Beausoleil, Sean A.; Gygi, Steven P.; Livingstone, Mark; Zhang, Hui; Polakiewicz, Roberto D.; Comb, Michael J.

    2007-01-01

    To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response. PMID:18077418

  15. The ATM signaling network in development and disease

    PubMed Central

    Stracker, Travis H.; Roig, Ignasi; Knobel, Philip A.; Marjanović, Marko

    2013-01-01

    The DNA damage response (DDR) rapidly recognizes DNA lesions and initiates the appropriate cellular programs to maintain genome integrity. This includes the coordination of cell cycle checkpoints, transcription, translation, DNA repair, metabolism, and cell fate decisions, such as apoptosis or senescence (Jackson and Bartek, 2009). DNA double-strand breaks (DSBs) represent one of the most cytotoxic DNA lesions and defects in their metabolism underlie many human hereditary diseases characterized by genomic instability (Stracker and Petrini, 2011; McKinnon, 2012). Patients with hereditary defects in the DDR display defects in development, particularly affecting the central nervous system, the immune system and the germline, as well as aberrant metabolic regulation and cancer predisposition. Central to the DDR to DSBs is the ataxia-telangiectasia mutated (ATM) kinase, a master controller of signal transduction. Understanding how ATM signaling regulates various aspects of the DDR and its roles in vivo is critical for our understanding of human disease, its diagnosis and its treatment. This review will describe the general roles of ATM signaling and highlight some recent advances that have shed light on the diverse roles of ATM and related proteins in human disease. PMID:23532176

  16. The ATM signaling network in development and disease.

    PubMed

    Stracker, Travis H; Roig, Ignasi; Knobel, Philip A; Marjanović, Marko

    2013-01-01

    The DNA damage response (DDR) rapidly recognizes DNA lesions and initiates the appropriate cellular programs to maintain genome integrity. This includes the coordination of cell cycle checkpoints, transcription, translation, DNA repair, metabolism, and cell fate decisions, such as apoptosis or senescence (Jackson and Bartek, 2009). DNA double-strand breaks (DSBs) represent one of the most cytotoxic DNA lesions and defects in their metabolism underlie many human hereditary diseases characterized by genomic instability (Stracker and Petrini, 2011; McKinnon, 2012). Patients with hereditary defects in the DDR display defects in development, particularly affecting the central nervous system, the immune system and the germline, as well as aberrant metabolic regulation and cancer predisposition. Central to the DDR to DSBs is the ataxia-telangiectasia mutated (ATM) kinase, a master controller of signal transduction. Understanding how ATM signaling regulates various aspects of the DDR and its roles in vivo is critical for our understanding of human disease, its diagnosis and its treatment. This review will describe the general roles of ATM signaling and highlight some recent advances that have shed light on the diverse roles of ATM and related proteins in human disease.

  17. ATM mediates cytotoxicity of a mutant telomerase RNA in human cancer cells

    PubMed Central

    Stohr, Bradley A.; Blackburn, Elizabeth H.

    2008-01-01

    Telomeres are elongated by the enzyme telomerase, which contains a template-bearing RNA (TER or TERC) and a protein reverse transcriptase. Overexpression of a particular mutant human TER with a mutated template sequence (MT-hTer-47A) in telomerase-positive cancer cells causes incorporation of mutant telomeric sequences, telomere uncapping, and initiation of a DNA damage response, ultimately resulting in cell growth inhibition and apoptosis. The DNA damage pathways underlying these cellular effects are not well understood. Here, we show that the ataxia-telangiectasia-mutated (ATM) protein is activated and forms telomeric foci in response to MT-hTer-47A expression. Depletion of ATM from two cancer cell lines, including the p53-mutant UM-UC-3 bladder cancer line, rendered the cells largely unresponsive to MT-hTer-47A. Relative to ATM-competent controls, ATM-depleted cells showed increased proliferation and clonogenic survival and reduced cell death following MT-hTer-47A treatment. In contrast, ATM depletion sensitized the cancer cells to treatment with camptothecin, a topoisomerase inhibitor which induces DNA double-strand breaks. We show that the effects of ATM depletion on the MT-hTer-47A response were not due to decreased expression of MT-hTer-47A or reduced activity of telomerase at the telomere. Instead, ATM depletion allowed robust cancer cell growth despite the continued presence of dysfunctional telomeres containing mutant sequence. Notably, the number of end-to-end telomere fusions induced by MT-hTer-47A treatment was markedly reduced in ATM-depleted cells. Our results identify ATM as a key mediator of the MT-hTer-47A dysfunctional telomere response, even in cells lacking wild-type p53, and provide evidence that telomere fusions contribute to MT-hTer-47A cytotoxicity. PMID:18593932

  18. Genetic Interactions of the Aspergillus nidulans atmAATM Homolog With Different Components of the DNA Damage Response Pathway

    PubMed Central

    Malavazi, Iran; Lima, Joel Fernandes; de Castro, Patrícia Alves; Savoldi, Marcela; de Souza Goldman, Maria Helena; Goldman, Gustavo Henrique

    2008-01-01

    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 AtmAATM loss of function caused synthetic lethality when combined with mutation in UvsBATR. 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 chkACHK1 and chkBCHK2 genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for ΔatmA, ΔuvsB, ΔchkA, and ΔchkB. 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 bimEAPC1, ankAWEE1 and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents. PMID:18245360

  19. Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents.

    PubMed

    Cui, Yuxia; Palii, Stela S; Innes, Cynthia L; Paules, Richard S

    2014-01-01

    DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

  20. The role of ATM and 53BP1 as predictive markers in cervical cancer

    PubMed Central

    Roossink, Frank; Wieringa, Hylke W; Noordhuis, Maartje G; ten Hoor, Klaske A; Kok, Mirjam; Slagter-Menkema, Lorian; Hollema, Harry; de Bock, Geertruida H; Pras, Elisabeth; de Vries, Elisabeth GE; de Jong, Steven; van der Zee, Ate GJ; Schuuring, Ed; Wisman, G Bea A; van Vugt, Marcel ATM

    2012-01-01

    Treatment of advanced-stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the ‘DNA damage response’ (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in patients with cervical cancer treated with (chemo)radiation. In a panel of cervical cancer cell lines, inactivation of ataxia telangiectasia mutated (ATM) or its substrate p53-binding protein-1 (53BP1) clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radiosensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance. To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of patients with advanced-stage cervical cancer (n = 375) treated with (chemo)radiation. High levels of phosphorylated (p-)ATM [p = 0.006, hazard ratio (HR) = 1.817] were related to poor locoregional disease-free survival. Furthermore, high levels of p-ATM predicted shorter disease-specific survival (p = 0.038, HR = 1.418). The presence of phosphorylated 53BP1 was associated with p-ATM (p = 0.001, odds ratio = 2.206) but was not related to any clinicopathological features or survival. In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve the efficacy of (chemo)radiation. PMID:22323184

  1. The role of ATM and 53BP1 as predictive markers in cervical cancer.

    PubMed

    Roossink, Frank; Wieringa, Hylke W; Noordhuis, Maartje G; ten Hoor, Klaske A; Kok, Mirjam; Slagter-Menkema, Lorian; Hollema, Harry; de Bock, Geertruida H; Pras, Elisabeth; de Vries, Elisabeth G E; de Jong, Steven; van der Zee, Ate G J; Schuuring, Ed; Wisman, G Bea A; van Vugt, Marcel A T M

    2012-11-01

    Treatment of advanced-stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the 'DNA damage response' (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in patients with cervical cancer treated with (chemo)radiation. In a panel of cervical cancer cell lines, inactivation of ataxia telangiectasia mutated (ATM) or its substrate p53-binding protein-1 (53BP1) clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radiosensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance. To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of patients with advanced-stage cervical cancer (n = 375) treated with (chemo)radiation. High levels of phosphorylated (p-)ATM [p = 0.006, hazard ratio (HR) = 1.817] were related to poor locoregional disease-free survival. Furthermore, high levels of p-ATM predicted shorter disease-specific survival (p = 0.038, HR = 1.418). The presence of phosphorylated 53BP1 was associated with p-ATM (p = 0.001, odds ratio = 2.206) but was not related to any clinicopathological features or survival. In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve the efficacy of (chemo)radiation. Copyright © 2012 UICC.

  2. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    PubMed

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

  3. Functional activation of ATM by the prostate cancer suppressor NKX3.1.

    PubMed

    Bowen, Cai; Ju, Jeong-Ho; Lee, Ji-Hoon; Paull, Tanya T; Gelmann, Edward P

    2013-08-15

    The prostate tumor suppressor NKX3.1 augments response to DNA damage and enhances survival after DNA damage. Within minutes of DNA damage, NKX3.1 undergoes phosphorylation at tyrosine 222, which is required for a functional interaction with ataxia telangiectasia mutated (ATM) kinase. NKX3.1 binds to the N-terminal region of ATM, accelerates ATM activation, and hastens the formation of γhistone2AX. NKX3.1 enhances DNA-dependent ATM kinase activation by both the MRN complex and H2O2 in a DNA-damage-independent manner. ATM, bound to the NKX3.1 homeodomain, phosphorylates NKX3.1, leading to ubiquitination and degradation. Thus, NKX3.1 and ATM have a functional interaction leading to ATM activation and then NKX3.1 degradation in a tightly regulated DNA damage response specific to prostate epithelial cells. These findings demonstrate a mechanism for the tumor-suppressor properties of NKX3.1, demonstrate how NKX3.1 may enhance DNA integrity in prostate stem cells and may help to explain how cells differ in their sensitivity to DNA damage. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Functional Activation of ATM by the Prostate Cancer Suppressor NKX3.1

    PubMed Central

    Bowen, Cai; Ju, Jeong-Ho; Lee, Ji-Hoon; Paull, Tanya T.; Gelmann, Edward P.

    2013-01-01

    SUMMARY The prostate tumor suppressor NKX3.1 augments response to DNA damage and enhances survival after DNA damage. Within minutes of DNA damage, NKX3.1 undergoes phosphorylation at tyrosine 222, which is required for a functional interaction with ataxia telangiectasia mutated (ATM) kinase. NKX3.1 binds to the N-terminal region of ATM, accelerates ATM activation, and hastens the formation of γhistone2AX. NKX3.1 enhances DNA-dependent ATM kinase activation by both the MRN complex and H2O2 in a DNA-damage-independent manner. ATM, bound to the NKX3.1 homeodomain, phosphorylates NKX3.1, leading to ubiquitination and degradation. Thus, NKX3.1 and ATM have a functional interaction leading to ATM activation and then NKX3.1 degradation in a tightly regulated DNA damage response specific to prostate epithelial cells. These findings demonstrate a mechanism for the tumor-suppressor properties of NKX3.1, demonstrate how NKX3.1 may enhance DNA integrity in prostate stem cells and may help to explain how cells differ in their sensitivity to DNA damage. PMID:23890999

  5. Identification of p32 as a novel substrate for ATM in heart

    SciTech Connect

    Kato, Hisakazu; Takashima, Seiji Asano, Yoshihiro; Shintani, Yasunori; Yamazaki, Satoru; Seguchi, Osamu; Yamamoto, Hiroyuki; Nakano, Atsushi; Higo, Shuichiro; Ogai, Akiko; Minamino, Tetsuo; Kitakaze, Masafumi; Hori, Masatsugu

    2008-02-22

    Chemotherapeutic agents to induce DNA damage have been limited to use due to severe side effects of cardiotoxicity. ATM (Ataxia-telangiectasia mutated) is an essential protein kinase in triggering DNA damage responses. However, it is unclear how the ATM-mediated DNA damage responses are involved in the cardiac cell damage. To elucidate these functions in heart, we searched for specific substrates of ATM from mouse heart homogenate. Combining an in vitro phosphorylation following anion-exchange chromatography with purification by reverse-phase high-performance liquid chromatography (HPLC), we successfully identified p32, an ASF/SF2-associated protein, as a novel substrate for ATM. An in vitro kinase assay using recombinant p32 revealed that ATM directly phosphorylated p32. Furthermore, we determined Ser 148 of p32 as an ATM phosphorylation site. Since p32 is known to regulate mRNA splicing and transcription, p32 phosphorylation by ATM might be a new transcriptional regulatory pathway for specific DNA damage responses in heart.

  6. ATM-Dependent Phosphorylation of MEF2D Promotes Neuronal Survival after DNA Damage

    PubMed Central

    Chan, Shing Fai; Sances, Sam; Brill, Laurence M.; Okamoto, Shu-ichi; Zaidi, Rameez; McKercher, Scott R.; Akhtar, Mohd W.; Nakanishi, Nobuki

    2014-01-01

    Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT. PMID:24672010

  7. ATM Deficiency Is Associated with Sensitivity to PARP1- and ATR Inhibitors in Lung Adenocarcinoma.

    PubMed

    Schmitt, Anna; Knittel, Gero; Welcker, Daniela; Yang, Tsun-Po; George, Julie; Nowak, Michael; Leeser, Uschi; Büttner, Reinhard; Perner, Sven; Peifer, Martin; Reinhardt, Hans Christian

    2017-06-01

    Defects in maintaining genome integrity are a hallmark of cancer. The DNA damage response kinase ATM is frequently mutated in human cancer, but the significance of these events to chemotherapeutic efficacy has not been examined deeply in whole organism models. Here we demonstrate that bi-allelic Atm deletion in mouse models of Kras-mutant lung adenocarcinoma does not affect cisplatin responses. In marked contrast, Atm-deficient tumors displayed an enhanced response to the topoisomerase-II poison etoposide. Moreover, Atm-deficient cells and tumors were sensitive to the PARP inhibitor olaparib. This actionable molecular addiction to functional PARP1 signaling was preserved in models that were proficient or deficient in p53, resembling standard or high-risk genetic constellations, respectively. Atm deficiency also markedly enhanced sensitivity to the ATR inhibitor VE-822. Taken together, our results provide a functional rationale to profile human tumors for disabling ATM mutations, particularly given their impact on PARP1 and ATR inhibitors. Cancer Res; 77(11); 3040-56. ©2017 AACR. ©2017 American Association for Cancer Research.

  8. DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions.

    PubMed

    Sordet, Olivier; Nakamura, Asako J; Redon, Christophe E; Pommier, Yves

    2010-01-15

    A taxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA double-strand break (DSB) response. We recently showed that transcription-blocking topoisomerase I cleavage complexes (TOP1cc) produce DSBs related to R-loop formation and activate ATM in post-mitotic neurons and lymphocytes. Here we discuss how TOP1cc can produce transcription arrest with R-loop formation and generate DSBs that activate ATM, as well as data suggesting that those transcription-dependent DSBs tend to form at the IgH locus and at specific genomic sites. We also address the potential roles of ATM in response to transcription-blocking TOP1cc.

  9. Statistical Analysis of ATM-Dependent Signaling in Quantitative Mass Spectrometry Phosphoproteomics.

    PubMed

    Waardenberg, Ashley J

    2017-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase, which when perturbed is associated with modified protein signaling that ultimately leads to a range of neurological and DNA repair defects. Recent advances in phospho-proteomics coupled with high-resolution mass-spectrometry provide new opportunities to dissect signaling pathways that ATM utilize under a number of conditions. This chapter begins by providing a brief overview of ATM function, its various regulatory roles and then leads into a workflow focused on the use of the statistical programming language R, together with code, for the identification of ATM-dependent substrates in the cytoplasm. This chapter cannot cover statistical properties in depth nor the range of possible methods in great detail, but instead aims to equip researchers with a set of tools to perform analysis between two conditions through examples with R functions.

  10. Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies.

    PubMed

    Gaudio, Eugenio; Spizzo, Riccardo; Paduano, Francesco; Luo, Zhenghua; Efanov, Alexey; Palamarchuk, Alexey; Leber, Amanda S; Kaou, Mohamed; Zanesi, Nicola; Bottoni, Arianna; Costinean, Stefan; Rassenti, Laura Z; Nakamura, Tatsuya; Kipps, Thomas J; Aqeilan, Rami I; Pekarsky, Yuri; Trapasso, Francesco; Croce, Carlo M

    2012-01-05

    The T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas.

  11. The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response

    PubMed Central

    Zhang, Xinna; Wan, Guohui; Berger, Franklin G.; He, Xiaoming; Lu, Xiongbin

    2011-01-01

    SUMMARY The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one fourth of miRNAs are significantly up-regulated after DNA damage, while loss of ATM abolishes their induction. KSRP (KH-type splicing regulatory protein) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis. PMID:21329876

  12. ATM functions at the peroxisome to induce pexophagy in response to ROS.

    PubMed

    Zhang, Jiangwei; Tripathi, Durga Nand; Jing, Ji; Alexander, Angela; Kim, Jinhee; Powell, Reid T; Dere, Ruhee; Tait-Mulder, Jacqueline; Lee, Ji-Hoon; Paull, Tanya T; Pandita, Raj K; Charaka, Vijaya K; Pandita, Tej K; Kastan, Michael B; Walker, Cheryl Lyn

    2015-10-01

    Peroxisomes are highly metabolic, autonomously replicating organelles that generate reactive oxygen species (ROS) as a by-product of fatty acid β-oxidation. Consequently, cells must maintain peroxisome homeostasis, or risk pathologies associated with too few peroxisomes, such as peroxisome biogenesis disorders, or too many peroxisomes, inducing oxidative damage and promoting diseases such as cancer. We report that the PEX5 peroxisome import receptor binds ataxia-telangiectasia mutated (ATM) and localizes this kinase to the peroxisome. In response to ROS, ATM signalling activates ULK1 and inhibits mTORC1 to induce autophagy. Specificity for autophagy of peroxisomes (pexophagy) is provided by ATM phosphorylation of PEX5 at Ser 141, which promotes PEX5 monoubiquitylation at Lys 209, and recognition of ubiquitylated PEX5 by the autophagy adaptor protein p62, directing the autophagosome to peroxisomes to induce pexophagy. These data reveal an important new role for ATM in metabolism as a sensor of ROS that regulates pexophagy.

  13. ATM specifically mediates repair of double-strand breaks with blocked DNA ends

    PubMed Central

    Álvarez-Quilón, Alejandro; Serrano-Benítez, Almudena; Ariel Lieberman, Jenna; Quintero, Cristina; Sánchez-Gutiérrez, Daniel; Escudero, Luis M.; Cortés-Ledesma, Felipe

    2014-01-01

    Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders. PMID:24572510

  14. ATM specifically mediates repair of double-strand breaks with blocked DNA ends.

    PubMed

    Álvarez-Quilón, Alejandro; Serrano-Benítez, Almudena; Lieberman, Jenna Ariel; Quintero, Cristina; Sánchez-Gutiérrez, Daniel; Escudero, Luis M; Cortés-Ledesma, Felipe

    2014-02-27

    Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.

  15. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    PubMed

    Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-01-01

    Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

  16. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase

    PubMed Central

    Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-01-01

    Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression. PMID:26824362

  17. Potential role of ATM in hepatocyte endocytosis of ApoE-deficient, ApoB48-containing lipoprotein in ApoE-deficient mice.

    PubMed

    Wu, Jianhua; Xiao, Yanhong; Liu, Juang; Yang, Hong; Dong, Xiaomin; Hu, San; Jin, Shanrui; Wu, Dongfang

    2014-02-01

    Individuals carrying mutations at both ataxia telangiectasia mutated (ATM) gene alleles reportedly have increased plasma cholesterol and triglyceride levels. Previous studies have demonstrated that defective ATM function promotes atherosclerosis. We previously demonstrated that ATM facilitates the clearance of plasma apolipoprotein (Apo)E-deficient, ApoB48-containing (E(-)/B(48)) lipoproteins in ApoE-deficient mice (ApoE(-/-) mice). However, to date there is no exact explanation available as to the mechanism(s) through which ATM is involved in the removal of E(-)/B(48) lipoprotein in ApoE(-/-) mice. In this study, to our knowledge, we demonstrate for the first time that heterozygous ATM mutation reduces the hepatocyte uptake of E(-)/B(48) lipoproteins in ApoE(-/-) mice; however, heterozygous ATM mutation did not affect hepatocyte binding to E(-)/B(48) lipoproteins. Moreover, our results revealed that ATM proteins were localized in the nucleus, early endosomes and late endosomes, but not in the plasma membrane in the hepatocytes of ApoE(-/-) mice. In addition, following treatment with the ATM activator, chloroquine, and E(-)/B(48) lipoproteins, ATM interacted with class III phosphatidylinositol-3-kinases (PI3Ks) and the activated ATM protein enhanced class III PI3K activity. Furthermore, treatment with a class III PI3K inhibitor (LY290042 and 3-MA) attenuated the intracellular total cholesterol accumulation induced by ATM activation. These results provide insight into the mechanisms behind the involvment of ATM in the process of endocytosis of E(-)/B(48) lipoprotein in ApoE(-/-) mice, demonstrating the role of class III PI3K protein.

  18. Mutation of ataxia-telangiectasia mutated is associated with dysfunctional glutathione homeostasis in cerebellar astroglia.

    PubMed

    Campbell, Andrew; Bushman, Jared; Munger, Joshua; Noble, Mark; Pröschel, Christoph; Mayer-Pröschel, Margot

    2016-02-01

    Astroglial dysfunction plays an important role in neurodegenerative diseases otherwise attributed to neuronal loss of function. Here we focus on the role of astroglia in ataxia-telangiectasia (A-T), a disease caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. A hallmark of A-T pathology is progressive loss of cerebellar neurons, but the mechanisms that impact neuronal survival are unclear. We now provide a possible mechanism by which A-T astroglia affect the survival of cerebellar neurons. As astroglial functions are difficult to study in an in vivo setting, particularly in the cerebellum where these cells are intertwined with the far more numerous neurons, we conducted in vitro coculture experiments that allow for the generation and pharmacological manipulation of purified cell populations. Our analyses revealed that cerebellar astroglia isolated from Atm mutant mice show decreased expression of the cystine/glutamate exchanger subunit xCT, glutathione (GSH) reductase, and glutathione-S-transferase. We also found decreased levels of intercellular and secreted GSH in A-T astroglia. Metabolic labeling of l-cystine, the major precursor for GSH, revealed that a key component of the defect in A-T astroglia is an impaired ability to import this rate-limiting precursor for the production of GSH. This impairment resulted in suboptimal extracellular GSH supply, which in turn impaired survival of cerebellar neurons. We show that by circumventing the xCT-dependent import of L-cystine through addition of N-acetyl-L-cysteine (NAC) as an alternative cysteine source, we were able to restore GSH levels in A-T mutant astroglia providing a possible future avenue for targeted therapeutic intervention. © 2015 Wiley Periodicals, Inc.

  19. The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain.

    PubMed

    Kanu, Nnennaya; Penicud, Kay; Hristova, Mariya; Wong, Barnaby; Irvine, Elaine; Plattner, Florian; Raivich, Gennadij; Behrens, Axel

    2010-12-03

    Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.

  20. Ultrafast networks (ATM): first clinical experiences.

    PubMed

    Duerinckx, A J; Valentino, D J; Hayrapetian, A; Hagan, G; Grant, E G

    1996-06-01

    Ultrafast networks using asynchronous transfer mode (ATM) technology can provide the bandwidth and throughput that may be sufficient to satisfy the medical imaging community. Several trials are underway to assess the effect of ATM network capabilities on the clinical practice of radiology, by providing immediate interactive radiology consultations between subspecialists and general radiologists at affiliated academic institutions. The hardware to build such networks is now commercially available and its cost is decreasing steadily, but the monthly charges for ATM bandwidth use are still high. Nevertheless, given the tremendous increase in communication capability and data transfer rates possible with ATM networks, cost alone should not be the determining factor for selecting this technology. The ATM concept in general is first reviewed, followed by a description of early clinical ATM network installation in four medical environments worldwide. These medical clusters include: the UCLA affiliated hospitals (UCLA Medical Center, West LA VAMC and Olive-View UCLA Medical Center), the UCSF affiliated hospitals, Duke University Hospitals and a cluster of medical centers in Berlin which have all been connected via ATM networks. The use of ATM technology in these realistic clinical environments is discussed and evaluated for its potential impact on patient care and clinical teaching within radiology departments. From this preliminary study it is concluded that image communications over a regional PACS using an ATM network can allow interactive consultations between different subspecialist and general radiologists or other specialized radiologists spread over different medical centers.

  1. 53BP1 depletion causes PARP inhibitor resistance in ATM-deficient breast cancer cells.

    PubMed

    Hong, Ruoxi; Ma, Fei; Zhang, Weimin; Yu, Xiying; Li, Qing; Luo, Yang; Zhu, Changjun; Jiang, Wei; Xu, Binghe

    2016-09-09

    Mutations in DNA damage response factors BRCA1 and BRCA2 confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors in breast and ovarian cancers. BRCA1/BRCA2-defective tumors can exhibit resistance to PARP inhibitors via multiple mechanisms, one of which involves loss of 53BP1. Deficiency in the DNA damage response factor ataxia-telangiectasia mutated (ATM) can also sensitize tumors to PARP inhibitors, raising the question of whether the presence or absence of 53BP1 can predict sensitivity of ATM-deficient breast cancer to these inhibitors. Cytotoxicity of PARP inhibitor and ATM inhibitor in breast cancer cell lines was assessed by MTS, colony formation and apoptosis assays. ShRNA lentiviral vectors were used to knockdown 53BP1 expression in breast cancer cell lines. Phospho-ATM and 53BP1 protein expressions were determined in human breast cancer tissues by immunohistochemistry (IHC). We show that inhibiting ATM increased cytotoxicity of PARP inhibitor in triple-negative and non-triple-negative breast cancer cell lines, and depleting the cells of 53BP1 reduced this cytotoxicity. Inhibiting ATM abrogated homologous recombination induced by PARP inhibitor, and down-regulating 53BP1 partially reversed this effect. Further, overall survival was significantly better in triple-negative breast cancer patients with lower levels of phospho-ATM and tended to be better in patients with negative 53BP1. These results suggest that 53BP1 may be a predictor of PARP inhibitor resistance in patients with ATM-deficient tumors.

  2. ATM Inhibition Potentiates Death of Androgen Receptor-inactivated Prostate Cancer Cells with Telomere Dysfunction.

    PubMed

    Reddy, Vidyavathi; Wu, Min; Ciavattone, Nicholas; McKenty, Nathan; Menon, Mani; Barrack, Evelyn R; Reddy, G Prem-Veer; Kim, Sahn-Ho

    2015-10-16

    Androgen receptor (AR) plays a role in maintaining telomere stability in prostate cancer cells, as AR inactivation induces telomere dysfunction within 3 h. Since telomere dysfunction in other systems is known to activate ATM (ataxia telangiectasia mutated)-mediated DNA damage response (DDR) signaling pathways, we investigated the role of ATM-mediated DDR signaling in AR-inactivated prostate cancer cells. Indeed, the induction of telomere dysfunction in cells treated with AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation (activation) of ATM and its downstream effector Chk2 and the presenceof phosphorylated ATM at telomeres, indicating activation of DDR signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation, induced PARP cleavage, abrogated cell cycle checkpoint activation and attenuated the formation of γH2AX foci at telomeres in AR-inactivated cells, suggesting that ATM inhibitor induces apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration-resistant prostate cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The depletion of ATM inhibits colon cancer proliferation and migration via B56γ2-mediated Chk1/p53/CD44 cascades.

    PubMed

    Liu, Rui; Tang, Jiajia; Ding, Chaodong; Liang, Weicheng; Zhang, Li; Chen, Tianke; Xiong, Yan; Dai, Xiaowei; Li, Wenfeng; Xu, Yunsheng; Hu, Jin; Lu, Liting; Liao, Wanqin; Lu, Xincheng

    2017-04-01

    Ataxia-telangiectasia mutated (ATM) protein kinase is a major guardian of genomic stability, and its well-established function in cancer is tumor suppression. Here, we report an oncogenic role of ATM. Using two isogenic sets of human colon cancer cell lines that differed only in their ATM status, we demonstrated that ATM deficiency significantly inhibits cancer cell proliferation, migration, and invasion. The tumor-suppressive function of ATM depletion is not modulated by the compensatory activation of ATR, but it is associated with B56γ2-mediated Chk1/p53/CD44 signaling pathways. Under normal growth conditions, the depletion of ATM prevents B56γ2 ubiquitination and degradation, which activates PP2A-mediated Chk1/p53/p21 signaling pathways, leading to senescence and cell cycle arrest. CD44 was validated as a novel ATM target based on its ability to rescue cell migration and invasion defects in ATM-depleted cells. The activation of p53 induced by ATM depletion suppresses CD44 transcription, thus resulting in epithelial-mesenchymal transition (EMT) and cell migration suppression. Our study suggests that ATM has tumorigenic potential in post-formed colon neoplasia, and it supports ATM as an appealing target for improving cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Multifunctional Role of ATM/Tel1 Kinase in Genome Stability: From the DNA Damage Response to Telomere Maintenance

    PubMed Central

    2014-01-01

    The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development. PMID:25247188

  5. Buffer Management Simulation in ATM Networks

    NASA Technical Reports Server (NTRS)

    Yaprak, E.; Xiao, Y.; Chronopoulos, A.; Chow, E.; Anneberg, L.

    1998-01-01

    This paper presents a simulation of a new dynamic buffer allocation management scheme in ATM networks. To achieve this objective, an algorithm that detects congestion and updates the dynamic buffer allocation scheme was developed for the OPNET simulation package via the creation of a new ATM module.

  6. Buffer Management Simulation in ATM Networks

    NASA Technical Reports Server (NTRS)

    Yaprak, E.; Xiao, Y.; Chronopoulos, A.; Chow, E.; Anneberg, L.

    1998-01-01

    This paper presents a simulation of a new dynamic buffer allocation management scheme in ATM networks. To achieve this objective, an algorithm that detects congestion and updates the dynamic buffer allocation scheme was developed for the OPNET simulation package via the creation of a new ATM module.

  7. ATM: Restructing Learning for Deaf Students.

    ERIC Educational Resources Information Center

    Keefe, Barbara; Stockford, David

    Governor Baxter School for the Deaf is one of six Maine pilot sites chosen by NYNEX to showcase asynchronous transfer mode (ATM) technology. ATM is a network connection that allows high bandwidth transmission of data, voice, and video. Its high speed capability allows for high quality two-way full-motion video, which is especially beneficial to a…

  8. Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder.

    PubMed

    Scott, John W; Park, Elizabeth; Rodriguiz, Ramona M; Oakhill, Jonathan S; Issa, Samah M A; O'Brien, Matthew T; Dite, Toby A; Langendorf, Christopher G; Wetsel, William C; Means, Anthony R; Kemp, Bruce E

    2015-09-23

    Mutations that reduce expression or give rise to a Thr85Ser (T85S) mutation of Ca(2+)-CaM-dependent protein kinase kinase-2 (CaMKK2) have been implicated in behavioural disorders such as anxiety, bipolar and schizophrenia in humans. Here we report that Thr85 is an autophosphorylation site that endows CaMKK2 with a molecular memory that enables sustained autonomous activation following an initial, transient Ca(2+) signal. Conversely, autophosphorylation of Ser85 in the T85S mutant fails to generate autonomous activity but instead causes a partial loss of CaMKK2 activity. The loss of autonomous activity in the mutant can be rescued by blocking glycogen synthase kinase-3 (GSK3) phosphorylation of CaMKK2 with the anti-mania drug lithium. Furthermore, CaMKK2 null mice representing a loss of function model the human behavioural phenotypes, displaying anxiety and manic-like behavioural disturbances. Our data provide a novel insight into CaMKK2 regulation and its perturbation by a mutation associated with behavioural disorders.

  9. Autophosphorylation of CaMKK2 generates autonomous activity that is disrupted by a T85S mutation linked to anxiety and bipolar disorder

    PubMed Central

    Scott, John W.; Park, Elizabeth; Rodriguiz, Ramona M.; Oakhill, Jonathan S.; Issa, Samah M. A.; O’Brien, Matthew T.; Dite, Toby A.; Langendorf, Christopher G.; Wetsel, William C.; Means, Anthony R.; Kemp, Bruce E.

    2015-01-01

    Mutations that reduce expression or give rise to a Thr85Ser (T85S) mutation of Ca2+-CaM-dependent protein kinase kinase-2 (CaMKK2) have been implicated in behavioural disorders such as anxiety, bipolar and schizophrenia in humans. Here we report that Thr85 is an autophosphorylation site that endows CaMKK2 with a molecular memory that enables sustained autonomous activation following an initial, transient Ca2+ signal. Conversely, autophosphorylation of Ser85 in the T85S mutant fails to generate autonomous activity but instead causes a partial loss of CaMKK2 activity. The loss of autonomous activity in the mutant can be rescued by blocking glycogen synthase kinase-3 (GSK3) phosphorylation of CaMKK2 with the anti-mania drug lithium. Furthermore, CaMKK2 null mice representing a loss of function model the human behavioural phenotypes, displaying anxiety and manic-like behavioural disturbances. Our data provide a novel insight into CaMKK2 regulation and its perturbation by a mutation associated with behavioural disorders. PMID:26395653

  10. Dynamics of TCP traffic over ATM networks

    SciTech Connect

    Floyd, S.; Romanow, A.

    1994-08-01

    The authors investigate the performance of TCP (Transport Control Protocol) connections over ATM (Asynchronous Transfer Mode) networks without ATM-level congestion control, and compare it to the performance of TCP over packet-based networks. For simulations of congested networks, the effective throughput of TCP over ATM can be quite low when cells are dropped at the congested ATM switch. The low throughput is due to wasted bandwidth as the congested link transmits cells from ``corrupted`` packets, i.e., packets in which at least one cell is dropped by the switch. This fragmentation effect can be corrected and high throughput can be achieved if the switch drops whole packets prior to buffer overflow; they call this strategy Early Packet Discard. They also discuss general issues of congestion avoidance for best-effort traffic in ATM networks.

  11. The Aspergillus nidulans ATM Kinase Regulates Mitochondrial Function, Glucose Uptake and the Carbon Starvation Response

    PubMed Central

    Krohn, Nadia Graciele; Brown, Neil Andrew; Colabardini, Ana Cristina; Reis, Thaila; Savoldi, Marcela; Dinamarco, Taísa Magnani; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2013-01-01

    Mitochondria supply cellular energy and also perform a role in the adaptation to metabolic stress. In mammals, the ataxia-telangiectasia mutated (ATM) kinase acts as a redox sensor controlling mitochondrial function. Subsequently, transcriptomic and genetic studies were utilized to elucidate the role played by a fungal ATM homolog during carbon starvation. In Aspergillus nidulans, AtmA was shown to control mitochondrial function and glucose uptake. Carbon starvation responses that are regulated by target of rapamycin (TOR) were shown to be AtmA-dependent, including autophagy and hydrolytic enzyme secretion. AtmA also regulated a p53-like transcription factor, XprG, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Thus, AtmA possibly represents a direct or indirect link between mitochondrial stress, metabolism, and growth through the influence of TOR and XprG function. The coordination of cell growth and division with nutrient availability is crucial for all microorganisms to successfully proliferate in a heterogeneous environment. Mitochondria supply cellular energy but also perform a role in the adaptation to metabolic stress and the cross-talk between prosurvival and prodeath pathways. The present study of Aspergillus nidulans demonstrated that AtmA also controlled mitochondrial mass, function, and oxidative phosphorylation, which directly or indirectly influenced glucose uptake. Carbon starvation responses, including autophagy, shifting metabolism to the glyoxylate cycle, and the secretion of carbon scavenging enzymes were AtmA-dependent. Transcriptomic profiling of the carbon starvation response demonstrated how TOR signaling and the retrograde response, which signals mitochondrial dysfunction, were directly or indirectly influenced by AtmA. The AtmA kinase was also shown to influence a p53-like transcription factor, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Therefore, in response to metabolic

  12. The Aspergillus nidulans ATM kinase regulates mitochondrial function, glucose uptake and the carbon starvation response.

    PubMed

    Krohn, Nadia Graciele; Brown, Neil Andrew; Colabardini, Ana Cristina; Reis, Thaila; Savoldi, Marcela; Dinamarco, Taísa Magnani; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2014-01-10

    Mitochondria supply cellular energy and also perform a role in the adaptation to metabolic stress. In mammals, the ataxia-telangiectasia mutated (ATM) kinase acts as a redox sensor controlling mitochondrial function. Subsequently, transcriptomic and genetic studies were utilized to elucidate the role played by a fungal ATM homolog during carbon starvation. In Aspergillus nidulans, AtmA was shown to control mitochondrial function and glucose uptake. Carbon starvation responses that are regulated by target of rapamycin (TOR) were shown to be AtmA-dependent, including autophagy and hydrolytic enzyme secretion. AtmA also regulated a p53-like transcription factor, XprG, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Thus, AtmA possibly represents a direct or indirect link between mitochondrial stress, metabolism, and growth through the influence of TOR and XprG function. The coordination of cell growth and division with nutrient availability is crucial for all microorganisms to successfully proliferate in a heterogeneous environment. Mitochondria supply cellular energy but also perform a role in the adaptation to metabolic stress and the cross-talk between prosurvival and prodeath pathways. The present study of Aspergillus nidulans demonstrated that AtmA also controlled mitochondrial mass, function, and oxidative phosphorylation, which directly or indirectly influenced glucose uptake. Carbon starvation responses, including autophagy, shifting metabolism to the glyoxylate cycle, and the secretion of carbon scavenging enzymes were AtmA-dependent. Transcriptomic profiling of the carbon starvation response demonstrated how TOR signaling and the retrograde response, which signals mitochondrial dysfunction, were directly or indirectly influenced by AtmA. The AtmA kinase was also shown to influence a p53-like transcription factor, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Therefore, in response to metabolic

  13. Satellite ATM Networks: Architectures and Guidelines Developed

    NASA Technical Reports Server (NTRS)

    vonDeak, Thomas C.; Yegendu, Ferit

    1999-01-01

    An important element of satellite-supported asynchronous transfer mode (ATM) networking will involve support for the routing and rerouting of active connections. Work published under the auspices of the Telecommunications Industry Association (http://www.tiaonline.org), describes basic architectures and routing protocol issues for satellite ATM (SATATM) networks. The architectures and issues identified will serve as a basis for further development of technical specifications for these SATATM networks. Three ATM network architectures for bent pipe satellites and three ATM network architectures for satellites with onboard ATM switches were developed. The architectures differ from one another in terms of required level of mobility, supported data rates, supported terrestrial interfaces, and onboard processing and switching requirements. The documentation addresses low-, middle-, and geosynchronous-Earth-orbit satellite configurations. The satellite environment may require real-time routing to support the mobility of end devices and nodes of the ATM network itself. This requires the network to be able to reroute active circuits in real time. In addition to supporting mobility, rerouting can also be used to (1) optimize network routing, (2) respond to changing quality-of-service requirements, and (3) provide a fault tolerance mechanism. Traffic management and control functions are necessary in ATM to ensure that the quality-of-service requirements associated with each connection are not violated and also to provide flow and congestion control functions. Functions related to traffic management were identified and described. Most of these traffic management functions will be supported by on-ground ATM switches, but in a hybrid terrestrial-satellite ATM network, some of the traffic management functions may have to be supported by the onboard satellite ATM switch. Future work is planned to examine the tradeoffs of placing traffic management functions onboard a satellite as

  14. Having pancreatic cancer with tumoral loss of ATM and normal TP53 protein expression is associated with a poorer prognosis.

    PubMed

    Kim, Haeryoung; Saka, Burcu; Knight, Spencer; Borges, Michael; Childs, Erica; Klein, Alison; Wolfgang, Christopher; Herman, Joseph; Adsay, Volkan N; Hruban, Ralph H; Goggins, Michael

    2014-04-01

    To determine how often loss of ataxia-telangiectasia-mutated (ATM) protein expression occurs in primary pancreatic ductal adenocarcinomas and to determine its prognostic significance. The expression of ATM and TP53 was determined by immunohistochemistry in 397 surgically resected pancreatic ductal adenocarcinomas (Hopkins; Johns Hopkins Medical Institutions, Baltimore, MD), a second set of 159 cases (Emory; Emory University Hospital, Atlanta, GA), and 21 cancers after neoadjuvant chemoradiotherapy. Expression was correlated with the clinicopathologic parameters, including survival. Tumoral ATM loss was observed in one cancer known to have biallelic inactivation of ATM and 50 of the first 396 (12.8%) cases, significantly more often in patients with a family history of pancreatic cancer (12/49; 24.5%) than in those without (38/347; 11.0%; P = 0.019). In the Hopkins series, ATM loss was associated with a significantly decreased overall survival in patients whose cancers had normal TP53 expression (P = 0.019) and was a significant independent predictor of decreased overall survival (P = 0.014). Seventeen (10.7%) of 159 Emory cases had tumoral ATM loss and tumoral ATM loss/normal TP53 was associated with poorer overall survival (P = 0.1). Multivariate analysis of the combined Hopkins/Emory cases found that tumoral ATM loss/normal TP53 was an independent predictor of decreased overall survival [HR = 2.61; confidence interval (CI), 1.27-5.37; P = 0.009]. Of 21 cancers examined after neoadjuvant chemoradiotherapy, one had tumoral loss of ATM; it had no histologic evidence of tumor response. Tumoral loss of ATM protein was detected more often in patients with a family history of pancreatic cancer than in those without. Patients whose pancreatic cancers had loss of ATM but normal TP53 had worse overall survival after pancreatic resection. ©2014 AACR.

  15. ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents.

    PubMed

    Agnihotri, Sameer; Burrell, Kelly; Buczkowicz, Pawel; Remke, Marc; Golbourn, Brian; Chornenkyy, Yevgen; Gajadhar, Aaron; Fernandez, Nestor A; Clarke, Ian D; Barszczyk, Mark S; Pajovic, Sanja; Ternamian, Christian; Head, Renee; Sabha, Nesrin; Sobol, Robert W; Taylor, Michael D; Rutka, James T; Jones, Chris; Dirks, Peter B; Zadeh, Gelareh; Hawkins, Cynthia

    2014-10-01

    Alkylating agents are a first-line therapy for the treatment of several aggressive cancers, including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed, increasing therapeutic response while minimizing toxicity. Using an siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular, the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM), were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. Inhibition of ATM and MPG-mediated BER cooperate to sensitize tumor cells to alkylating agents, impairing tumor growth in vitro and in vivo with no toxicity to normal cells, providing an ideal therapeutic window. ©2014 American Association for Cancer Research.

  16. Prognostic significance of ATM and cyclin B1 in pancreatic neuroendocrine tumor.

    PubMed

    Shin, Jae Uk; Lee, Chang Hoon; Lee, Kyu Taek; Lee, Jong Kyun; Lee, Kwang Hyuck; Kim, Kwang Min; Kim, Kyoung-Mee; Park, Sang-Mo; Rhee, Jong Chul

    2012-10-01

    Ataxia telangiectasia mutated kinase (ATM) and cyclin B1 are involved in cell cycle control. The prognostic significance of both molecules has not yet been investigated in pancreatic neuroendocrine tumors. The aim of this study was to evaluate the clinical and prognostic significance of ATM and cyclin B1 in patients with pancreatic neuroendocrine tumors. A total of 107 pancreatic neuroendocrine tumor specimens that were surgically resected were immunohistochemically investigated using the tissue microarray technique. Clinicopathologic results and survival were evaluated retrospectively. High expression of ATM and cyclin B1 was related to well-differentiated endocrine tumors of the World Health Organization (WHO) classification, but not related to TNM stages. The high ATM expression group (ATM ≥ 4) had a significantly smaller tumor size, lower recurrence rate, more number of functioning tumor, and well differentiation of WHO classification. The high cyclin B1 expression group (cyclin B1 ≥ 5) was related to smaller tumor size, less vascular invasion, less recurrence rate, and less death rate. However, cyclin B1 was the only significant factor for survival following multivariate analysis (p = 0.008; OR, 0.54; 95 % CI, 0.35-0.85). The current results suggested that expression of ATM and cyclin B1 may be useful markers to identify patients with poor prognosis who may benefit from close follow-up and aggressive therapy in pancreatic neuroendocrine tumors.

  17. A putative "hepitype" in the ATM gene associated with chronic lymphocytic leukemia risk.

    PubMed

    Martín-Guerrero, Idoia; Enjuanes, Anna; Richter, Julia; Ammerpohl, Ole; Colomer, Dolors; Ardanaz, Maite; Marco, Fernando; Salas, Antonio; Campo, Elias; Siebert, Reiner; García-Orad, Africa

    2011-11-01

    Chronic lymphocytic leukemia (CLL) cells are characterized by several chromosomal lesions. Some of these aberrations imply chromosome breaks as a result of unrepaired double strand breaks (DSBs) in the DNA. The ATM (ataxia telangiectasia-mutated) protein is the principal integrator of cellular responses to DSBs. ATM deletion is also an adverse prognostic factor in CLL. Taking this into account, we evaluated if genetic and/or epigenetic variation in the ATM gene may modulate the individual susceptibility to develop CLL. Our case-control association study was performed in a large Spanish population of 1,503 individuals, including 742 patients with CLL and 761 controls. We identified one haplotype within the ATM gene that confers an increased risk of CLL development (OR = 1.33; 95% CI: 1.10-1.60). Two polymorphisms of this ATM haplotype eliminated one CpG site each in Introns 15 and 61, causing changes in DNA methylation pattern. These data provide the first evidence for the existence of a putative "hepitype" in the ATM gene associated with CLL risk. Copyright © 2011 Wiley-Liss, Inc.

  18. Lower expression of ATM and gene deletion is more frequent in adrenocortical carcinomas than adrenocortical adenomas.

    PubMed

    Ye, Junna; Qi, Yan; Wang, Weiqing; Sun, Fukang; Wei, Qin; Su, Tingwei; Zhou, Weiwei; Jiang, Yiran; Yuan, Wenqi; Cai, Jianfei; Cui, Bin; Ning, Guang

    2012-06-01

    Adrenocortical carcinoma (ACC) is a rare endocrine malignancy accounting for approximately 0.02-0.2% of all cancer deaths. The molecular pathogenesis of ACC has been the hot topic of recent reviews but it is still poorly understood. It is imperative to have a better understanding on the pathophysiology of ACC so as to establish precise diagnosis and effective treatment. This study aims to identify the molecular markers between ACCs and adrenocortical adenomas (ACAs). With MLPA, we checked on 10 ACA and 9 ACC tissue samples. The MLPA results showed deletion on chromosomes 18q, 11q, 11p, and 13q and duplication on chromosomes 3q, 4q, 6p, and 19p. There was a significant difference in the number of aberration copies of the ataxia telangiectasia-mutated (ATM) gene located on chromosome 11q22-q23 between ACCs and ACAs. Five out of 9 (56%) ACC specimens had deletion of ATM (P = 0.011). RT-PCR result then demonstrated that ATM mRNA level is lower in ACCs than in ACAs (P < 0.001). In addition, immunohistochemistry (IHC) study of the 19 ACA and 18 ACC samples confirmed lower expression of ATM protein in ACCs than in ACAs (P < 0.001). The study demonstrated that ATM expression was diminished in ACC than in ACA, suggesting an important role of ATM in the tumorigenesis of ACC.

  19. Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

    PubMed

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  20. ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation

    PubMed Central

    Barlow, Carrolee; Ribaut-Barassin, Catherine; Zwingman, Theresa A.; Pope, Amber J.; Brown, Kevin D.; Owens, Jennie W.; Larson, Denise; Harrington, Elizabeth A.; Haeberle, Anne-Marie; Mariani, Jean; Eckhaus, Michael; Herrup, Karl; Bailly, Yannick; Wynshaw-Boris, Anthony

    2000-01-01

    We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm−/−) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm−/− mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm−/− mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm−/− mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers. PMID:10639172

  1. What is a microsatellite: a computational and experimental definition based upon repeat mutational behavior at A/T and GT/AC repeats.

    PubMed

    Kelkar, Yogeshwar D; Strubczewski, Noelle; Hile, Suzanne E; Chiaromonte, Francesca; Eckert, Kristin A; Makova, Kateryna D

    2010-01-01

    Microsatellites are abundant in eukaryotic genomes and have high rates of strand slippage-induced repeat number alterations. They are popular genetic markers, and their mutations are associated with numerous neurological diseases. However, the minimal number of repeats required to constitute a microsatellite has been debated, and a definition of a microsatellite that considers its mutational behavior has been lacking. To define a microsatellite, we investigated slippage dynamics for a range of repeat sizes, utilizing two approaches. Computationally, we assessed length polymorphism at repeat loci in ten ENCODE regions resequenced in four human populations, assuming that the occurrence of polymorphism reflects strand slippage rates. Experimentally, we determined the in vitro DNA polymerase-mediated strand slippage error rates as a function of repeat number. In both approaches, we compared strand slippage rates at tandem repeats with the background slippage rates. We observed two distinct modes of mutational behavior. At small repeat numbers, slippage rates were low and indistinguishable from background measurements. A marked transition in mutability was observed as the repeat array lengthened, such that slippage rates at large repeat numbers were significantly higher than the background rates. For both mononucleotide and dinucleotide microsatellites studied, the transition length corresponded to a similar number of nucleotides (approximately 10). Thus, microsatellite threshold is determined not by the presence/absence of strand slippage at repeats but by an abrupt alteration in slippage rates relative to background. These findings have implications for understanding microsatellite mutagenesis, standardization of genome-wide microsatellite analyses, and predicting polymorphism levels of individual microsatellite loci.

  2. The checkpoint kinase ATM protects against stress-induced elevation of cyclin D1 and potential cell death in neurons.

    PubMed

    Hitomi, Masahiro; Stacey, Dennis W

    2010-06-01

    Quantitative cytometric studies show that cyclin D1 levels must decline during S phase for proper cell cycle progression, and that cyclin D1 decline follows phosphorylation induced by the checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). ATM is mutated in ataxia telangiectasia (AT), a disease characterized by progressive neurodegeneration. Importantly, neurodegeneration in many cases has been linked to the increased expression of cyclin D1 in neurons leading to inappropriate cell cycle entry. These facts prompted us to test the possibility that ATM normally protects against neural degeneration by suppressing cyclin D1 levels, particularly following genotoxic stress. For this purpose, neural stem cells were induced to differentiate into mature neural cells, including neurons. ATM activity in these cultures was inhibited with a specific chemical inhibitor in the presence or absence of hydrogen peroxide treatment, and the effect on cyclin D1 expression was determined by quantitative, single cell cytometric analyses. As predicted, inhibition of ATM did promote elevation of cyclin D1 in differentiated neurons, particularly under conditions of oxidative stress. The survival of differentiated neurons and of neural stem cells was reduced by such treatments. These data support our suggestion that ATM functions to maintain low levels of cyclin D1 expression in differentiated neurons; and may provide important clues in understanding neural degeneration in general. Copyright 2010 International Society for Advancement of Cytometry.

  3. Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines.

    PubMed

    Kubota, Eiji; Williamson, Christopher T; Ye, Ruiqiong; Elegbede, Anifat; Peterson, Lars; Lees-Miller, Susan P; Bebb, D Gwyn

    2014-01-01

    Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption.

  4. Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

    PubMed Central

    Kubota, Eiji; Williamson, Christopher T; Ye, Ruiqiong; Elegbede, Anifat; Peterson, Lars; Lees-Miller, Susan P; Bebb, D Gwyn

    2014-01-01

    Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption. PMID:24841718

  5. Targeting ATM-deficient CLL through interference with DNA repair pathways.

    PubMed

    Knittel, Gero; Liedgens, Paul; Reinhardt, Hans C

    2015-01-01

    Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world and accounts for approximately 30% of adult leukemias and 25% of non-Hodgkin lymphomas. The median age at diagnosis is 72 years. During recent years numerous genetic aberrations have been identified that are associated with an aggressive course of the disease and resistance against genotoxic chemotherapies. The DNA damage-responsive proapoptotic ATM-CHK2-p53 signaling pathway is frequently mutationally inactivated in CLL either through large deletions on chromosome 11q (ATM) or 17p (TP53), or through protein-damaging mutations. Here, we focus on the role of ATM signaling for the immediate DNA damage response, DNA repair and leukemogenesis. We further discuss novel therapeutic concepts for the targeted treatment of ATM-defective CLLs. We specifically highlight the potential use of PARP1 and DNA-PKcs inhibitors for the treatment of ATM-mutant CLL clones. Lastly, we briefly discuss the current state of genetically engineered mouse models of the disease and emphasize the use of these preclinical tools as a common platform for the development and validation of novel therapeutic agents.

  6. Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia.

    PubMed

    Li, Jiali; Chen, Jianmin; Ricupero, Christopher L; Hart, Ronald P; Schwartz, Melanie S; Kusnecov, Alexander; Herrup, Karl

    2012-05-01

    Ataxia telangiectasia is a neurodegenerative disease caused by mutation of the Atm gene. Here we report that ataxia telangiectasia mutated (ATM) deficiency causes nuclear accumulation of histone deacetylase 4 (HDAC4) in neurons and promotes neurodegeneration. Nuclear HDAC4 binds to chromatin, as well as to myocyte enhancer factor 2A (MEF2A) and cAMP-responsive element binding protein (CREB), leading to histone deacetylation and altered neuronal gene expression. Blocking either HDAC4 activity or its nuclear accumulation blunts these neurodegenerative changes and rescues several behavioral abnormalities of ATM-deficient mice. Full rescue of the neurodegeneration, however, also requires the presence of HDAC4 in the cytoplasm, suggesting that the ataxia telangiectasia phenotype results both from a loss of cytoplasmic HDAC4 as well as its nuclear accumulation. To remain cytoplasmic, HDAC4 must be phosphorylated. The activity of the HDAC4 phosphatase, protein phosphatase 2A (PP2A), is downregulated by ATM-mediated phosphorylation. In ATM deficiency, enhanced PP2A activity leads to HDAC4 dephosphorylation and the nuclear accumulation of HDAC4. Our results define a crucial role of the cellular localization of HDAC4 in the events leading to ataxia telangiectasia neurodegeneration.

  7. ATM Damage Response and XLF Repair Factor are Functionally Redundant In Joining DNA Breaks

    PubMed Central

    Zha, Shan; Guo, Chunguang; Boboila, Cristian; Oksenych, Valentyn; Cheng, Hwei-Ling; Zhang, Yu; Wesemann, Duane R.; Yuen, Grace; Patel, Harin; Goff, Peter H.; Dubois, Richard L.; Alt, Frederick W.

    2010-01-01

    Classical non-homologous DNA end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Deficiencies for C-NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for C-NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF) is mutated in certain immunodeficient human patients and has been implicated in C-NHEJ3,4,5,6. Yet, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The Ataxia Telangiectasia-Mutated protein (“ATM”) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM-deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX-deficiency does not measurably impact these processes7,8,9. Here, we show that XLF, ATM, and H2AX all have fundamental roles in processing and joining ends during V(D)J recombination; but that these roles have been masked by unanticipated functional redundancies. Thus, combined ATM/XLF-deficiency nearly blocks mouse lymphocyte development due inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs C-NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in C-NHEJ are mediated via ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, suggesting a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX indeed has a role in this process. PMID:21160472

  8. MEK inhibitors block growth of lung tumours with mutations in ataxia–telangiectasia mutated

    PubMed Central

    Smida, Michal; Fece de la Cruz, Ferran; Kerzendorfer, Claudia; Uras, Iris Z.; Mair, Barbara; Mazouzi, Abdelghani; Suchankova, Tereza; Konopka, Tomasz; Katz, Amanda M.; Paz, Keren; Nagy-Bojarszky, Katalin; Muellner, Markus K.; Bago-Horvath, Zsuzsanna; Haura, Eric B.; Loizou, Joanna I.; Nijman, Sebastian M. B.

    2016-01-01

    Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours. PMID:27922010

  9. Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency.

    PubMed

    Bhargava, Ragini; Carson, Caree R; Lee, Gabriella; Stark, Jeremy M

    2017-01-24

    A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3' exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.

  10. ATM Polymorphisms Are Associated With Risk of Radiation-Induced Pneumonitis

    SciTech Connect

    Zhang Li; Yang Ming; Bi Nan; Fang Mingjing; Sun Tong; Ji Wei; Tan Wen; Zhao Lujun; Yu Dianke; Lin Dongxin; Wang Luhua

    2010-08-01

    Purpose: Since the ataxia telangiectasia mutated (ATM) protein plays crucial roles in repair of double-stranded DNA breaks, control of cell cycle checkpoints, and radiosensitivity, we hypothesized that variations in this gene might be associated with radiation-induced pneumonitis (RP). Methods and Materials: A total of 253 lung cancer patients receiving thoracic irradiation between 2004 and 2006 were included in this study. Common Terminology Criteria for Adverse Events version 3.0 was used to grade RP. Five haplotype-tagging single nucleotide polymorphisms (SNPs) in the ATM gene were genotyped using DNA from blood lymphocytes. Hazard ratios (HRs) and 95% confidence intervals (CIs) of RP for genotypes were computed by the Cox model, adjusted for clinical factors. The function of the ATM SNP associated with RP was examined by biochemical assays. Results: During the median 22-month follow-up, 44 (17.4%) patients developed grade {>=} 2 RP. In multivariate Cox regression models adjusted for other clinical predictors, we found two ATM variants were independently associated with increased RP risk. They were an 111G > A) polymorphism (HR, 2.49; 95% CI, 1.07-5.80) and an ATM 126713G > A polymorphism (HR, 2.47; 95% CI, 1.16-5.28). Furthermore, genotype-dependent differences in ATM expression were demonstrated both in cell lines (p < 0.001) and in individual lung tissue samples (p = 0.003), which supported the results of the association study. Conclusions: Genetic polymorphisms of ATM are significantly associated with RP risk. These variants might exert their effect through regulation of ATM expression and serve as independent biomarkers for prediction of RP in patients treated with thoracic radiotherapy.

  11. Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

    PubMed

    Zhao, Long; Chen, Hui; Zhan, Yi-Qun; Li, Chang-Yan; Ge, Chang-Hui; Zhang, Jian-Hong; Wang, Xiao-Hui; Yu, Miao; Yang, Xiao-Ming

    2014-07-01

    Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

  12. MSFC institutional area network and ATM technology

    NASA Technical Reports Server (NTRS)

    Amin, Ashok T.

    1994-01-01

    The New Institutional Area Network (NEWIAN) at Marshall supports over 5000 end users with access to 26 file servers providing work presentation services. It is comprised of some 150 Ethernet LAN's interconnected by bridges/routers which are in turn connected to servers over two dual FDDI rings. The network supports various higher level protocols such as IP, IPX, AppleTalk (AT), and DECNet. At present IPX and AT protocols packets are routed, and IP protocol packets are bridged; however, work is in progress to route all IP packets. The impact of routing IP packets on network operation is examined. Broadband Integrated Services Data Network (BISDN), presently at various stages of development, is intended to provide voice, video, and data transfer services over a single network. BISDN will use asynchronous transfer mode (ATM) as a data transfer technique which provides for transmission, multiplexing, switching, and relaying of small size data units called cells. Limited ATM Wide Area Network (WAN) services are offered by Wiltel, AT&T, Sprint, and others. NASA is testing a pilot ATM WAN with a view to provide Program Support Communication Network services using ATM. ATM supports wide range of data rates and quality of service requirements. It is expected that ATM switches will penetrate campus networks as well. However, presently products in these areas are at various stages of development and standards are not yet complete. We examine development of ATM to help assess its role in the evolution of NEWIAN.

  13. ATM kinase promotes both caspase-8 and caspase-9 activation during TNF-α-induced apoptosis of HeLa cells.

    PubMed

    Liu, Linhua; Yim, Hyungshin; Choi, Jae Hyuk; Kim, Seung-Tak; Jin, Yinghua; Lee, Seung Ki

    2014-03-18

    In this study, we show that atraxia telangiectasia mutated kinase (ATM) activity is generally upregulated by different apoptotic stimuli, i.e. TNF-α, TRAIL, paclitaxel, or UV. Apoptotic progression is markedly attenuated by siATM-RNA through down regulation of caspase-8 and caspase-9 in parallel with decreases in FLIP-S (short form of cellular FLICE inhibitory protein) protein levels and Bid cleavage. In addition, ATM activity is upregulated through t-Cdc6 while caspase-8 and caspase-9 activities increase. Taken together, we suggest that ATM regulates caspase-8 activation by influencing levels of FLIP-S, ATM kinase activity is upregulated by t-Cdc6, and increased ATM activity plays an essential role in the amplification of apoptosis in TNF-α-stimulated HeLa cells.

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

    PubMed

    Poletto, Mattia; Yang, Di; Fletcher, Sally C; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J; Dianov, Grigory L

    2017-09-29

    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. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Low ATM protein expression in malignant tumor as well as cancer-associated stroma are independent prognostic factors in a retrospective study of early-stage hormone-negative breast cancer.

    PubMed

    Feng, Xiaolan; Li, Haocheng; Dean, Michelle; Wilson, Holly E; Kornaga, Elizabeth; Enwere, Emeka K; Tang, Patricia; Paterson, Alexander; Lees-Miller, Susan P; Magliocco, Anthony M; Bebb, Gwyn

    2015-05-03

    The serine/threonine protein kinase ataxia telangiectasia mutated (ATM) is critical in maintaining genomic integrity. Upon DNA double-strand breaks, ATM phosphorylates key downstream proteins including p53 and BRCA1/2, thereby orchestrating complex signaling pathways involved in cell cycle arrest, DNA repair, senescence and apoptosis. Although sporadic mutation of ATM occurs rarely in breast cancer, the status of its protein expression and its clinical significance in breast cancer remain not well established. Our study was designed to investigate the influence of ATM protein in both tumor and cancer-associated stroma on clinical outcome in hormone-positive (HPBC) and hormone-negative (HNBC) early-stage breast cancer (EBC). Tissue microarrays (TMAs), containing formalin-fixed, paraffin-embedded resected tumors from two cohorts of patients (HPBC cohort: n=130; HNBC cohort: n=168) diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, were analyzed for ATM protein expression using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA). ATM expression levels were measured within the tumor as a whole (tATM) as indicated by pan-cytokeratin expression, tumor nuclear compartment (nATM) as indicated by both DAPI and pan-cytokeratin-positive results, and cancer-associated stroma (csATM) as indicated by vimentin-positive and pan-cytokeratin-negative results. ATM expression levels within these compartments were correlated with clinical outcome. While tATM and nATM were significantly lower in tumors compared to normal breast epithelial tissues, csATM was significantly higher than the corresponding normal tissue compartment. In addition, the median expression level of both tATM and nATM were two- to threefold lower (P<0.001) in HNBC than in HPBC. In both HNBC and HPBC cohorts, patients with low tATM, nATM and csATM tumors had significantly poorer survival outcomes than those with a high tATM, nATM and csATM, but this effect was more pronounced in

  16. Genomic organization of the ATM gene

    SciTech Connect

    Uziel, T.; Savitsky, K.; Platzer, M.; Rosenthal, A.

    1996-04-15

    The ATM gene was recently identified and found to be responsible for the human genetic disorder ataxia-telangiectasia. The major ATM transcript is 13 kb. Using long-distance PCR, we determined the genomic structure of this gene and identified all of its exon-intron boundaries. The ATM gene spans approximately 150 kb of genomic DNA and consists of 66 exons. The initiation codon falls within exon 4. The last exon is 3.8 kb and contains the stop codon and a 3{prime}-untranslated region of about 3600 nucleotides. 19 refs., 2 figs., 1 tab.

  17. Is the poly A (T>C) mutation a causative factor for misdiagnosis in second trimester prenatal diagnosis of β-thalassemia by fetal blood analysis on high performance liquid chromatography?

    PubMed

    Italia, Khushnooma Y; Sawant, Pratibha M; Nadkarni, Anita H; Ghosh, Kanjaksha; Colah, Roshan B

    2012-01-01

    We report the problems in diagnosis faced by two families referred for prenatal diagnosis of thalassemia where cordocentesis and fetal blood analysis by high performance liquid chromatography (HPLC) had to be done. The Hb A levels of the fetal blood measured by HPLC on the VARIANT™ Hemoglobin Testing System were 1.2 and 6.7%, respectively, suggestive of a heterozygous β-thalassemia (β-thal) fetus in the first case and a normal fetus in the second case. In one family, one of the parents had a borderline Hb A(2) level and in the other, one parent had normal RBC indices. However, DNA sequencing, done later, showed that in the first case the fetus was a compound heterozygote for the IVS-I-5 (G>C) and the polyadenylation signal site [poly A (T>C)] mutation, while in the second case, the fetus was homozygous for the poly A mutation. This emphasizes that characterization of β-thal mutations must be done whenever one of the parents has a borderline Hb A(2) level or normal RBC indices, and one should not rely on fetal blood analysis by HPLC for prenatal diagnosis of β-thal so as to avoid misdiagnosis.

  18. Incorporating truncating variants in PALB2, CHEK2, and ATM into the BOADICEA breast cancer risk model.

    PubMed

    Lee, Andrew J; Cunningham, Alex P; Tischkowitz, Marc; Simard, Jacques; Pharoah, Paul D; Easton, Douglas F; Antoniou, Antonis C

    2016-12-01

    The proliferation of gene panel testing precipitates the need for a breast cancer (BC) risk model that incorporates the effects of mutations in several genes and family history (FH). We extended the BOADICEA model to incorporate the effects of truncating variants in PALB2, CHEK2, and ATM. The BC incidence was modeled via the explicit effects of truncating variants in BRCA1/2, PALB2, CHEK2, and ATM and other unobserved genetic effects using segregation analysis methods. The predicted average BC risk by age 80 for an ATM mutation carrier is 28%, 30% for CHEK2, 50% for PALB2, and 74% for BRCA1 and BRCA2. However, the BC risks are predicted to increase with FH burden. In families with mutations, predicted risks for mutation-negative members depend on both FH and the specific mutation. The reduction in BC risk after negative predictive testing is greatest when a BRCA1 mutation is identified in the family, but for women whose relatives carry a CHEK2 or ATM mutation, the risks decrease slightly. The model may be a valuable tool for counseling women who have undergone gene panel testing for providing consistent risks and harmonizing their clinical management. A Web application can be used to obtain BC risks in clinical practice (http://ccge.medschl.cam.ac.uk/boadicea/).Genet Med 18 12, 1190-1198.

  19. Incorporating Truncating Variants in PALB2, CHEK2 and ATM into the BOADICEA Breast Cancer Risk Model

    PubMed Central

    Lee, Andrew J.; Cunningham, Alex P.; Tischkowitz, Marc; Simard, Jacques; Pharoah, Paul D.; Easton, Douglas F.; Antoniou, Antonis C.

    2016-01-01

    Purpose The proliferation of gene-panel testing precipitates the need for a breast cancer (BC) risk model that incorporates the effects of mutations in several genes and family history (FH). We extended the BOADICEA model to incorporate the effects of truncating variants in PALB2, CHEK2 and ATM. Methods The BC incidence was modelled via the explicit effects of truncating variants in BRCA1/2, PALB2, CHEK2 and ATM and other unobserved genetic effects using segregation analysis methods. Results The predicted average BC risk by age 80 for an ATM mutation carrier is 28%, 30% for CHEK2, 50% for PALB2, 74% for BRCA1 and BRCA2. However, the BC risks are predicted to increase with FH-burden. In families with mutations, predicted risks for mutation-negative members depend on both FH and the specific mutation. The reduction in BC risk after negative predictive-testing is greatest when a BRCA1 mutation is identified in the family, but for women whose relatives carry a CHEK2 or ATM mutation, the risks decrease slightly. Conclusions The model may be a valuable tool for counselling women who have undergone gene-panel testing for providing consistent risks and harmonizing their clinical management. A web-application can be used to obtain BC- risks in clinical practice (http://ccge.medschl.cam.ac.uk/boadicea/). PMID:27464310

  20. SASIL. Sandia ATM SONET Interface Logic

    SciTech Connect

    Kitta, J P

    1994-07-01

    SASIL is used to program the EPLD`s (Erasable Programmable Logic Devices) and PAL`s (Programmable Array Logic) that make up a large percentage of the Sandia ATM SONET Interface (OC3 version) for the INTEL Paragon.

  1. A survey of IP over ATM architectures

    SciTech Connect

    Chen, H.; Tsang, R.; Brandt, J.; Hutchins, J.

    1997-07-01

    Over the past decade, the Internet has burgeoned into a worldwide information highway consisting of approximately 5 million hosts on over 45,000 interconnected networks. This unprecedented growth, together with the introduction of multimedia workstations, has spurred the development of innovative applications that require high speed, low latency, and real-time transport. Today`s Internet can neither scale in its bandwidth nor guarantee the Quality of Services (QoS) necessary to meet these performance requirements. Many network researchers propose to use the Asynchronous Transfer Mode (ATM) technology as the underlying infrastructure for the next generation of workgroup, campus, and enterprise IP networks. Since ATM is significantly different from today`s legacy network technologies, efficient implementation of IP over ATM is especially challenging. This tutorial paper covers several existing proposals that integrate IP over ATM.

  2. Cisplatin-mediated radiosensitization of non-small cell lung cancer cells is stimulated by ATM inhibition.

    PubMed

    Toulany, Mahmoud; Mihatsch, Julia; Holler, Marina; Chaachouay, Hassan; Rodemann, H Peter

    2014-05-01

    Cisplatin activates ataxia-telangiectasia-mutated (ATM), a protein with roles in DNA repair, cell cycle progression and autophagy. We investigated the radiosensitizing effect of cisplatin with respect to its effect on ATM pathway activation. Non-small cell lung cancer cells (NSCLC) cell lines (A549, H460) and human fibroblast (ATM-deficient AT5, ATM-proficient 1BR3) cells were used. The effects of cisplatin combined with irradiation on ATM pathway activity, clonogenicity, DNA double-strand break (DNA-DSB) repair and cell cycle progression were analyzed with Western blotting, colony formation and γ-H2AX foci assays as well as FACS analysis, respectively. Cisplatin radiosensitized H460 cells, but not A549 cells. Radiosensitization of H460 cells was not due to impaired DNA-DSB repair, increased apoptosis or cell cycle dysregulation. The lack of radiosensitization demonstrated for A549 cells was associated with cisplatin-mediated stimulation of ATM (S1981) and AMPKα (T172) phosphorylation and autophagy. However, in both cell lines inhibition of ATM and autophagy by KU-55933 and chloroquine diphosphate (CQ) respectively resulted in a significant radiosensitization. Combined treatment with the AMPK inhibitor compound-C led to radiosensitization of A549 but not of H460 cells. As compared to the treatment with KU-55933 alone, radiosensitivity of A549 cells was markedly stimulated by the combination of KU-55933 and cisplatin. However, the combination of CQ and cisplatin did not modulate the pattern of radiation sensitivity of A549 or H460 cells. In accordance with the results that cisplatin via stimulation of ATM activity can abrogate its radiosensitizing effect, ATM deficient cells were significantly sensitized to ionizing radiation by cisplatin. The results obtained indicate that ATM targeting can potentiate cisplatin-induced radiosensitization. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells.

    PubMed

    Kwok, Marwan; Davies, Nicholas; Agathanggelou, Angelo; Smith, Edward; Oldreive, Ceri; Petermann, Eva; Stewart, Grant; Brown, Jeff; Lau, Alan; Pratt, Guy; Parry, Helen; Taylor, Malcolm; Moss, Paul; Hillmen, Peter; Stankovic, Tatjana

    2016-02-04

    TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation. © 2016 by The American Society of Hematology.

  4. A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon.

    PubMed

    Vain, Philippe; Thole, Vera; Worland, Barbara; Opanowicz, Magdalena; Bush, Max S; Doonan, John H

    2011-06-01

    In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.

  5. ATM Dependent Silencing Links Nucleolar Chromatin Reorganization to DNA Damage Recognition.

    PubMed

    Harding, Shane M; Boiarsky, Jonathan A; Greenberg, Roger A

    2015-10-13

    Resolution of DNA double-strand breaks (DSBs) is essential for the suppression of genome instability. DSB repair in transcriptionally active genomic regions represents a unique challenge that is associated with ataxia telangiectasia mutated (ATM) kinase-mediated transcriptional silencing. Despite emerging insights into the underlying mechanisms, how DSB silencing connects to DNA repair remains undefined. We observe that silencing within the rDNA depends on persistent DSBs. Non-homologous end-joining was the predominant mode of DSB repair allowing transcription to resume. ATM-dependent rDNA silencing in the presence of persistent DSBs led to the large-scale reorganization of nucleolar architecture, with movement of damaged chromatin to nucleolar cap regions. These findings identify ATM-dependent temporal and spatial control of DNA repair and provide insights into how communication between DSB signaling and ongoing transcription promotes genome integrity.

  6. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  7. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  8. Asynchronous Transfer Mode (ATM) Switch Technology and Vendor Survey

    NASA Technical Reports Server (NTRS)

    Berry, Noemi

    1995-01-01

    Asynchronous Transfer Mode (ATM) switch and software features are described and compared in order to make switch comparisons meaningful. An ATM switch's performance cannot be measured solely based on its claimed switching capacity; traffic management and congestion control are emerging as the determining factors in an ATM network's ultimate throughput. Non-switch ATM products and experiences with actual installations of ATM networks are described. A compilation of select vendor offerings as of October 1994 is provided in chart form.

  9. The ATM- and ATR-related SCD domain is over-represented in proteins involved in nervous system development

    PubMed Central

    Cara, Lukas; Baitemirova, Medina; Follis, Jack; Larios-Sanz, Maia; Ribes-Zamora, Albert

    2016-01-01

    ATM and ATR are cellular kinases with a well-characterized role in the DNA-damage response. Although the complete set of ATM/ATR targets is unknown, they often contain clusters of S/TQ motifs that constitute an SCD domain. In this study, we identified putative ATM/ATR targets that have a conserved SCD domain across vertebrates. Using this approach, we have identified novel putative ATM/ATR targets in pathways known to be under direct control of these kinases. Our analysis has also unveiled significant enrichment of SCD-containing proteins in cellular pathways, such as vesicle trafficking and actin cytoskeleton, where a regulating role for ATM/ATR is either unknown or poorly understood, hinting at a much broader and overarching role for these kinases in the cell. Of particular note is the overrepresentation of conserved SCD-containing proteins involved in pathways related to neural development. This finding suggests that ATM/ATR could be directly involved in controlling this process, which may be linked to the adverse neurological effects observed in patients with mutations in ATM. PMID:26743489

  10. The ATM- and ATR-related SCD domain is over-represented in proteins involved in nervous system development.

    PubMed

    Cara, Lukas; Baitemirova, Medina; Follis, Jack; Larios-Sanz, Maia; Ribes-Zamora, Albert

    2016-01-08

    ATM and ATR are cellular kinases with a well-characterized role in the DNA-damage response. Although the complete set of ATM/ATR targets is unknown, they often contain clusters of S/TQ motifs that constitute an SCD domain. In this study, we identified putative ATM/ATR targets that have a conserved SCD domain across vertebrates. Using this approach, we have identified novel putative ATM/ATR targets in pathways known to be under direct control of these kinases. Our analysis has also unveiled significant enrichment of SCD-containing proteins in cellular pathways, such as vesicle trafficking and actin cytoskeleton, where a regulating role for ATM/ATR is either unknown or poorly understood, hinting at a much broader and overarching role for these kinases in the cell. Of particular note is the overrepresentation of conserved SCD-containing proteins involved in pathways related to neural development. This finding suggests that ATM/ATR could be directly involved in controlling this process, which may be linked to the adverse neurological effects observed in patients with mutations in ATM.

  11. Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

    PubMed Central

    Lumsden, Joanne M.; McCarty, Thomas; Petiniot, Lisa K.; Shen, Rhuna; Barlow, Carrolee; Wynn, Thomas A.; Morse, Herbert C.; Gearhart, Patricia J.; Wynshaw-Boris, Anthony; Max, Edward E.; Hodes, Richard J.

    2004-01-01

    Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell–dependent antigen (Ag), Atm−/− mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm−/− B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm−/− cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm−/− B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm−/− B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm−/− B cells occurs at the level of genomic DNA recombination as measured by digestion–circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the μ–γ1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR. PMID:15504820

  12. ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

    PubMed Central

    Renou, Jean-Pierre; Pichon, Olivier; Fochesato, Sylvain; Ortet, Philippe; Montané, Marie-Hélène

    2007-01-01

    ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of γ-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that

  13. ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis.

    PubMed

    Ricaud, Lilian; Proux, Caroline; Renou, Jean-Pierre; Pichon, Olivier; Fochesato, Sylvain; Ortet, Philippe; Montané, Marie-Hélène

    2007-05-09

    ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases

  14. ATM regulates 3-Methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents

    PubMed Central

    Agnihotri, Sameer; Burrell, Kelly; Buczkowicz, Pawel; Remke, Marc; Golbourn, Brian; Chornenkyy, Yevgen; Gajadhar, Aaron; Fernandez, Nestor A.; Clarke, Ian D.; Barszczyk, Mark S.; Pajovic, Sanja; Ternamian, Christian; Head, Renee; Sabha, Nesrin; Sobol, Robert W.; Taylor, Michael D; Rutka, James T.; Jones, Chris; Dirks, Peter B.; Zadeh, Gelareh; Hawkins, Cynthia

    2014-01-01

    Alkylating agents are a frontline therapy for the treatment of several aggressive cancers including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed; increasing therapeutic response while minimizing toxicity. Using a siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM) were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. PMID:25100205

  15. [MiR-181a Promotes Proliferation of Human Acute Myeloid Leukemia Cells by Targeting ATM].

    PubMed

    Hua, Jia-Ye; Feng, Ying; Pang, Ying; Zhou, Xu-Hong; Xu, Bing; Yan, Mu-Xia

    2016-04-01

    To investigate miR-181a function and regulation mechanism by identifying miR-181a target genes in acute myeloid leukemia (AML). The HL-60 cells of human AML was transfected by small molecular analog miR-181a, the cell proliferation was detected by CCK-8 method after electroporation in HL-60 cell lines. Target genes of miR-181a were predicted and analyzed by the bioinformatics software and database. Target genes were confirmed by HL-60 cell line and the patient leukemia cells. Overexpressed miR-181a in HL-60 cell line significantly enhanced cell proliferation compared with that in control (P < 0.05). Dual luciferase reporter gene assay showed that miR-181a significantly suppressed the reporter gene activity containing ATM 3'-UTR by about 56.8% (P < 0.05), but it didn't suppress the reporter gene activity containing 3'-UTR ATM mutation. Western blot showed that miR-181a significantly downregulated the expression of ATM in human leukemia cells. It is also found that miR-181a was significantly increased in AML, which showed a negative correlation with ATM expression. miR-181a promotes cell proliferation in AML by regulating the tumor suppressor ATM, thus it plays the role as oncogene in pathogenesis of AML.

  16. ATM modulates the loading of recombination proteins onto a chromosomal translocation breakpoint hotspot.

    PubMed

    Sun, Jiying; Oma, Yukako; Harata, Masahiko; Kono, Kazuteru; Shima, Hiroki; Kinomura, Aiko; Ikura, Tsuyoshi; Suzuki, Hidekazu; Mizutani, Shuki; Kanaar, Roland; Tashiro, Satoshi

    2010-10-27

    Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.

  17. Activation and Inhibition of ATM by Phytochemicals: Awakening and Sleeping the Guardian Angel Naturally.

    PubMed

    Farooqi, Ammad Ahmad; Wu, Shyh-Jong; Chang, Yung-Ting; Tang, Jen-Yang; Li, Kun-Tzu; Ismail, Muhammad; Liaw, Chih-Chuang; Li, Ruei-Nian; Chang, Hsueh-Wei

    2015-10-01

    Double-stranded breaks (DSBs) are cytotoxic DNA lesions caused by oxygen radicals, ionizing radiation, and radiomimetic chemicals. Increasing understanding of DNA damage signaling has provided an ever-expanding list of modulators reported to orchestrate DNA damage repair and ataxia telangiectasia mutated (ATM) is the master regulator and main transducer of the DSB response. Increasingly, it is being realized that DNA damage response is a synchronized and branched network that functionalizes different molecular cascades to activate special checkpoints, thus temporarily arresting progression of the cell cycle while damage is being assessed and processed. It is noteworthy that both nutrigenetics and nutrigenomics have revolutionized the field of molecular biology and rapidly accumulating experimental evidence has started to shed light on biological activities of a wide range of phytochemicals reported to modulate cell cycle, DNA repair, cell growth, differentiation and apoptosis as evidenced by cell-based studies. In this review, we have attempted to provide an overview of DNA damage signaling, how ATM signaling regulates tumor necrosis factors-related apoptosis inducing ligand (TRAIL)-induced intracellular network. We also illuminate on how resveratrol, epigallocatechin gallate, curcumin, jaceosidin, cucurbitacin, apigenin, genistein, and others trigger activation of ATM in different cancer cells as well as agents for ATM inactivation. Understanding the interplay of TRAIL-induced intracellular signaling and ATM modulation of downstream effectors is very important. This holds particularly for a reconceptualization of the apparently paradoxical roles and therapeutically targetable for enhancing the response to DNA damage-inducing therapy.

  18. Haploinsufficiency of Bcl11b suppresses the progression of ATM-deficient T cell lymphomas.

    PubMed

    Pinkney, Kerice A; Jiang, Wenxia; Lee, Brian J; Loredan, Denis G; Li, Chen; Bhagat, Govind; Zha, Shan

    2015-07-30

    Bcl11b is a transcription factor important for T cell development and also a tumor-suppressor gene that is hemizygously inactivated in ~10% human T cell acute lymphoblastic leukemia (T-ALL) and several murine T-ALL models, including ATM(-/-) thymic lymphomas. Here we report that heterozygous loss of Bcl11b (Bcl11b(+/-)) unexpectedly reduced lethal thymic lymphoma in ATM(-/-) mice by suppressing lymphoma progression, but not initiation. The suppression was associated with a T cell-mediated immune response in ATM(-/-)Bcl11b(+/-) mice, revealing a haploid insufficient function of Bcl11b in immune modulation against lymphoma and offering an explanation for the complex relationship between Bcl11b status with T-ALL prognosis.

  19. Health ATMs in Saudi Arabia: A Perspective

    PubMed Central

    Aldosari, Bakheet

    2017-01-01

    Background: Health ATMs are terminals which are connected to a centrally located database storing patients’ electronic healthcare records (EHR). These machines are capable of collecting information in a far superior fashion than humans and are also able to rectify obsolete data in a manner that humans are generally not inclined to. Objectives: The main goal of this study is to assess the importance of adopting health ATMs in the Kingdom of Saudi Arabia (KSA), which can improve the confidence of patients, reward health self-management, and achieve positive health outcomes through their easy-to-use applications that are secure and accessible through various devices. Methods: Strength, Weakness, Opportunity, and Threat (SWOT) analysis was used to assess the efficiency of adopting health ATMs in KSA and reveal the said characteristics. Three focus groups assembled in the cities of Riyadh, Jeddah and Dammam during the period 2013-2014. The groups consisted of individuals experienced in the function of health ATMs. Results: It was found that the sector possessed a number of strengths that would help it in reaching the goals outlined therein, thereby achieving successful outcomes. Conclusions: Health ATMs could be a promising new advancement in the field of health if the project were to be planned and implemented correctly. Their benefits would consequently reach organizational and national levels. It is, therefore, crucial to educate the project managers about the benefits of learning from others as well as educating them about the needs and the requirements of the concerned organization. PMID:28883680

  20. ATM kinase sustains HER2 tumorigenicity in breast cancer.

    PubMed

    Stagni, Venturina; Manni, Isabella; Oropallo, Veronica; Mottolese, Marcella; Di Benedetto, Anna; Piaggio, Giulia; Falcioni, Rita; Giaccari, Danilo; Di Carlo, Selene; Sperati, Francesca; Cencioni, Maria Teresa; Barilà, Daniela

    2015-04-16

    ATM kinase preserves genomic stability by acting as a tumour suppressor. However, its identification as a component of several signalling networks suggests a dualism for ATM in cancer. Here we report that ATM expression and activity promotes HER2-dependent tumorigenicity in vitro and in vivo. We reveal a correlation between ATM activation and the reduced time to recurrence in patients diagnosed with invasive HER2-positive breast cancer. Furthermore, we identify ATM as a novel modulator of HER2 protein stability that acts by promoting a complex of HER2 with the chaperone HSP90, therefore preventing HER2 ubiquitination and degradation. As a consequence, ATM sustains AKT activation downstream of HER2 and may modulate the response to therapeutic approaches, suggesting that the status of ATM activity may be informative for the treatment and prognosis of HER2-positive tumours. Our findings provide evidence for ATM's tumorigenic potential revising the canonical role of ATM as a pure tumour suppressor.

  1. Neutrophil oxidative burst activates ATM to regulate cytokine production and apoptosis.

    PubMed

    Harbort, C J; Soeiro-Pereira, Paulo Vitor; von Bernuth, Horst; Kaindl, Angela M; Costa-Carvalho, Beatriz Tavares; Condino-Neto, Antonio; Reichenbach, Janine; Roesler, Joachim; Zychlinsky, Arturo; Amulic, Borko

    2015-12-24

    Neutrophils play an essential role in the initial stages of inflammation by balancing pro- and antiinflammatory signals. Among these signals are the production of proinflammatory cytokines and the timely initiation of antiinflammatory cell death via constitutive apoptosis. Here we identify ataxia-telangiectasia mutated (ATM) kinase as a modulator of these neutrophil functions. Ataxia-telangiectasia (AT) is a pleiotropic multisystem disorder caused by mutations in the gene-encoding ATM, a master regulator of the DNA damage response. In addition to progressive neurodegeneration and high rates of cancer, AT patients have numerous symptoms that can be linked to chronic inflammation. We report that neutrophils isolated from patients with AT overproduce proinflammatory cytokines and have a prolonged lifespan compared with healthy controls. This effect is partly mediated by increases in activation of p38 MAP kinase. Furthermore, we show that the oxidative burst, catalyzed by nicotinamide adenine dinucleotide phosphate oxidase, can activate ATM in neutrophils. Finally, activation of ATM and DNA damage signaling suppress cytokine production and can abrogate the overproduction of IL-8 in ROS-deficient cells. This reveals a novel mechanism for the regulation of cytokine production and apoptosis, establishing DNA damage as a downstream mediator of immune regulation by reactive oxygen species. We propose that deficiencies in the DNA damage response, like deficiencies in the oxidative burst seen in chronic granulomatous disease, could lead to pathologic inflammation. © 2015 by The American Society of Hematology.

  2. DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression.

    PubMed

    Carlessi, L; De Filippis, L; Lecis, D; Vescovi, A; Delia, D

    2009-06-01

    Ataxia-telangiectasia (A-T) is a neurodegenerative disorder caused by defects in the ATM kinase, a component of the DNA-damage response (DDR). Here, we employed an immortalized human neural stem-cell line (ihNSC) capable of differentiating in vitro into neurons, oligodendrocytes and astrocytes to assess the ATM-dependent response and outcome of ATM ablation. The time-dependent differentiation of ihNSC was accompanied by an upregulation of ATM and DNA-PK, sharp downregulation of ATR and Chk1, transient induction of p53 and by the onset of apoptosis in a fraction of cells. The response to ionizing radiation (IR)-induced DNA lesions was normal, as attested by the phosphorylation of ATM and some of its substrates (e.g., Nbs1, Smc1, Chk2 and p53), and by the kinetics of gamma-H2AX nuclear foci formation. Depletion in these cells of ATM by shRNA interference (shATM) attenuated the differentiation-associated apoptosis and response to IR, but left unaffected the growth, self-renewal and genomic stability. shATM cells generated a normal number of MAP2/beta-tubulin III+ neurons, but a reduced number of GalC+ oligodendrocytes, which were nevertheless more susceptible to oxidative stress. Altogether, these findings highlight the potential of ihNSCs as an in vitro model system to thoroughly assess, besides ATM, the role of DDR genes in neurogenesis and/or neurodegeneration.

  3. Methods for detection of ataxia telangiectasia mutations

    DOEpatents

    Gatti, Richard A.

    2005-10-04

    The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

  4. NPP After Launch: Characterizing ATMS Performance

    NASA Technical Reports Server (NTRS)

    Lambrigtsen, Bjorn

    2011-01-01

    The NPOESS Preparatory Project (NPP) mission is scheduled to launch in the fall of 2011. Although several teams from the government and the instrument contractor will be assessing and characterizing the performance of the Advanced Technology Microwave Sounder (ATMS) and the Cross-track Infrared Sounder (CrIS) sounding suite, the NASA NPP Science Team will be paying particular attention to the aspects of these sensors that affect their utility for atmospheric and climate research. In this talk we discuss relevant aspects of ATMS and our post launch analysis approach.

  5. Mystery of DNA repair: the role of the MRN complex and ATM kinase in DNA damage repair.

    PubMed

    Czornak, Kamila; Chughtai, Sanaullah; Chrzanowska, Krystyna H

    2008-01-01

    Genomes are subject to a number of exogenous or endogenous DNA-damaging agents that cause DNA double-strand breaks (DSBs). These critical DNA lesions can result in cell death or a wide variety of genetic alterations, including deletions, translocations, loss of heterozygosity, chromosome loss, or chromosome fusions, which enhance genome instability and can trigger carcinogenesis. The cells have developed an efficient mechanism to cope with DNA damages by evolving the DNA repair machinery. There are 2 major DSB repair mechanisms: nonhomologous end joining (NHEJ) and homologous recombination (HR). One element of the repair machinery is the MRN complex, consisting of MRE11, RAD50 and NBN (previously described as NBS1), which is involved in DNA replication, DNA repair, and signaling to the cell cycle checkpoints. A number of kinases, like ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad-3-related), and DNA PKcs (DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage. If the damage cannot be repaired, they direct the cell to apoptosis. The MRN complex as well as repair kinases are also involved in telomere maintenance and genome stability. The dysfunction of particular elements involved in the repair mechanisms leads to genome instability disorders, like ataxia telangiectasia (A-T), A-T-like disorder (ATLD) and Nijmegen breakage syndrome (NBS). The mutated genes responsible for these disorders code for proteins that play key roles in the process of DNA repair. Here we present a detailed review of current knowledge on the MRN complex, kinases engaged in DNA repair, and genome instability disorders.

  6. Asynchronous Transfer Mode (ATM) Technology: An Overview

    DTIC Science & Technology

    1993-06-04

    IEEE Communications Magazine , April 1992, pp. 60 - 68. [5] Stallings, W.: Handbook of Computer-Communications Standards, Volume 1: The Open Systems...Conf. Digest, paper ThH3, San Jose. CA, Feb. 2-7, 1992, p.225. [14] Newman, P.: ATM Technology for Corporate Networks. IEEE Communications Magazine . April

  7. Terminal Area ATM Research at NASA Ames

    NASA Technical Reports Server (NTRS)

    Tobias, Leonard

    1997-01-01

    The presentation will highlight the following: (1) A brief review of ATC research underway 15 years ago; (2) A summary of Terminal Area ATM Tool Development ongoing at NASA Ames; and (3) A projection of research activities 10-15 years from now.

  8. Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors.

    PubMed

    Wang, W-T; Catto, J W F; Meuth, M

    2015-05-28

    While DNA damage response pathways are well characterized in cancer cells, much less is known about their status in normal cells. These pathways protect tumour cells from DNA damage and replication stress and consequently present potential therapeutic targets. Here we characterize the response of human telomerase reverse transcriptase (hTERT)-immortalized normal human urothelial (NHU) and bladder cancer cell lines to agents that disrupt the DNA damage response. Effects of replication and DNA damage response inhibitors on cell cycle progression, checkpoint induction and apoptosis were analysed in hTERT-NHU and bladder cancer cell lines. The primary signalling cascade responding to replication stress in malignant cells (ataxia telangiectasia-mutated (ATM) and Rad3-related-checkpoint kinase 1 (ATR-CHK1)) is not activated in hTERT-NHU cells after treatment with a replication inhibitor and these cells do not depend upon CHK1 for protection from apoptosis during replication stress. Instead, ATM signalling is rapidly activated under these conditions. Intriguingly, an ATM inhibitor suppressed S-phase checkpoint activation after exposure to replication inhibitors and stopped entry of cells into S-phase indicating G1 checkpoint activation. Consistent with this, hTERT-NHU cells treated with the ATM inhibitor showed increased levels of cyclin-dependent kinase inhibitor p19(INK4D), reduced levels of cyclin D1 and CDK4, and reduced phosphorylation of the retinoblastoma protein. In contrast, a bladder cancer cell line cotreated with ATM and replication inhibitors progressed more slowly through S phase and showed a marked increase in apoptosis. Taken together, our findings suggest that ATM and CHK1 signalling cascades have different roles in tumour and normal epithelial cells, confirming these as promising therapeutic targets.

  9. DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

    PubMed

    Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

    2017-01-25

    The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system.

  10. ATMs, Coffee Shops Ideal Spots for Heart Defibrillators

    MedlinePlus

    ... Hortons and Starbucks, and ATMs connected to large banks. In fact, those businesses accounted for eight of ... Starbucks and Second Cup -- along with five large-bank ATMs -- made up most of the remaining top ...

  11. Remote facility sharing with ATM networks [PC based ATM Link Delay Simulator (LDS)]. Final report

    SciTech Connect

    Kung, H. T.

    2001-06-01

    The ATM Link Delay Simulator (LDS) adds propagation delay to the ATM link on which it is installed, to allow control of link propagation delay in network protocol experiments simulating an adjustable piece of optical fiber. Our LDS simulates a delay of between 1.5 and 500 milliseconds and is built with commodity PC hardware, only the ATM network interface card is not generally available. Our implementation is special in that it preserves the exact spacing of ATM data cells a feature that requires sustained high performance. Our implementation shows that applications demanding sustained high performance are possible on commodity PC hardware. This illustrates the promise that PC hardware has for adaptability to demanding specialized testing of high speed network.

  12. FACET: Future ATM Concepts Evaluation Tool

    NASA Technical Reports Server (NTRS)

    Bilmoria, Karl D.; Banavar, Sridhar; Chatterji, Gano B.; Sheth, Kapil S.; Grabbe, Shon

    2000-01-01

    FACET (Future ATM Concepts Evaluation Tool) is an Air Traffic Management research tool being developed at the NASA Ames Research Center. This paper describes the design, architecture and functionalities of FACET. The purpose of FACET is to provide E simulation environment for exploration, development and evaluation of advanced ATM concepts. Examples of these concepts include new ATM paradigms such as Distributed Air-Ground Traffic Management, airspace redesign and new Decision Support Tools (DSTs) for controllers working within the operational procedures of the existing air traffic control system. FACET is currently capable of modeling system-wide en route airspace operations over the contiguous United States. Airspace models (e.g., Center/sector boundaries, airways, locations of navigation aids and airports) are available from databases. A core capability of FACET is the modeling of aircraft trajectories. Using round-earth kinematic equations, aircraft can be flown along flight plan routes or great circle routes as they climb, cruise and descend according to their individual aircraft-type performance models. Performance parameters (e.g., climb/descent rates and speeds, cruise speeds) are obtained from data table lookups. Heading, airspeed and altitude-rate dynamics are also modeled. Additional functionalities will be added as necessary for specific applications. FACET software is written in Java and C programming languages. It is platform-independent, and can be run on a variety of computers. FACET has been designed with a modular software architecture to enable rapid integration of research prototype implementations of new ATM concepts. There are several advanced ATM concepts that are currently being implemented in FACET airborne separation assurance, dynamic density predictions, airspace redesign (re-sectorization), benefits of a controller DST for direct-routing, and the integration of commercial space transportation system operations into the U.S. National

  13. FACET: Future ATM Concepts Evaluation Tool

    NASA Technical Reports Server (NTRS)

    Bilmoria, Karl D.; Banavar, Sridhar; Chatterji, Gano B.; Sheth, Kapil S.; Grabbe, Shon

    2000-01-01

    FACET (Future ATM Concepts Evaluation Tool) is an Air Traffic Management research tool being developed at the NASA Ames Research Center. This paper describes the design, architecture and functionalities of FACET. The purpose of FACET is to provide E simulation environment for exploration, development and evaluation of advanced ATM concepts. Examples of these concepts include new ATM paradigms such as Distributed Air-Ground Traffic Management, airspace redesign and new Decision Support Tools (DSTs) for controllers working within the operational procedures of the existing air traffic control system. FACET is currently capable of modeling system-wide en route airspace operations over the contiguous United States. Airspace models (e.g., Center/sector boundaries, airways, locations of navigation aids and airports) are available from databases. A core capability of FACET is the modeling of aircraft trajectories. Using round-earth kinematic equations, aircraft can be flown along flight plan routes or great circle routes as they climb, cruise and descend according to their individual aircraft-type performance models. Performance parameters (e.g., climb/descent rates and speeds, cruise speeds) are obtained from data table lookups. Heading, airspeed and altitude-rate dynamics are also modeled. Additional functionalities will be added as necessary for specific applications. FACET software is written in Java and C programming languages. It is platform-independent, and can be run on a variety of computers. FACET has been designed with a modular software architecture to enable rapid integration of research prototype implementations of new ATM concepts. There are several advanced ATM concepts that are currently being implemented in FACET airborne separation assurance, dynamic density predictions, airspace redesign (re-sectorization), benefits of a controller DST for direct-routing, and the integration of commercial space transportation system operations into the U.S. National

  14. Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors.

    PubMed

    Bardelle, Catherine; Boros, Joanna

    2012-08-01

    The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

  15. The Association between ATM IVS 22-77 T>C and Cancer Risk: A Meta-Analysis

    PubMed Central

    Ji, Guixiang; Zou, Peng; Zhao, Peng; Lu, Ailin

    2012-01-01

    Background and Objectives It has become increasingly clear that ATM (ataxia-telangiectasia-mutated) safeguards genome stability, which is a cornerstone of cellular homeostasis, and ATM IVS 22-77 T>C affects the normal activity of ATM proteins. However, the association between the ATM IVS 22-77 T>C genetic variant and cancer risk is controversial. Therefore, we conducted a systematic meta-analysis to estimate the overall cancer risk associated with the polymorphism and to quantify any potential between-study heterogeneity. Methods A total of nine studies including 4,470 cases and 4,862 controls were analyzed for ATM IVS 22-77 T>C association with cancer risk in this meta-analysis. Heterogeneity among articles and their publication bias were also tested. Results Our results showed that no association reached the level of statistical significance in the overall risk. Interestingly, in the stratified analyses, we observed an inverse relationship in lung and breast cancer. Conclusion Further functional research on the ATM mechanism should be performed to explain the inconsistent results in different cancer types. PMID:22276117

  16. The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation.

    PubMed

    Alao, John P; Sunnerhagen, Per

    2009-11-10

    The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles.

  17. ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

    PubMed

    Kijas, Amanda W; Lim, Yi Chieh; Bolderson, Emma; Cerosaletti, Karen; Gatei, Magtouf; Jakob, Burkhard; Tobias, Frank; Taucher-Scholz, Gisela; Gueven, Nuri; Oakley, Greg; Concannon, Patrick; Wolvetang, Ernst; Khanna, Kum Kum; Wiesmüller, Lisa; Lavin, Martin F

    2015-09-30

    The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

  18. A synthetic lethal screen reveals enhanced sensitivity to ATR inhibitor treatment in mantle cell lymphoma with ATM loss-of-function.

    PubMed

    Menezes, Daniel L; Holt, Jenny; Tang, Yan; Feng, Jiajia; Barsanti, Paul; Pan, Yue; Ghoddusi, Majid; Zhang, Wei; Thomas, George; Holash, Jocelyn; Lees, Emma; Taricani, Lorena

    2015-01-01

    Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost. These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited. ©2014 American Association for Cancer Research.

  19. Early B-cell-specific inactivation of ATM synergizes with ectopic CyclinD1 expression to promote pre-germinal center B-cell lymphomas in mice.

    PubMed

    Yamamoto, K; Lee, B J; Li, C; Dubois, R L; Hobeika, E; Bhagat, G; Zha, S

    2015-06-01

    Ataxia telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin lymphomas, including ~50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B cells causes cell autonomous, clonal mature B-cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly, naive B-cell-specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. Although EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increases the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas toward pre-GC-derived small lymphocytic neoplasms, sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naive B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.e. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-GC B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL.

  20. Early B-cell Specific Inactivation of ATM Synergizes with Ectopic CyclinD1 Expression to Promote Pre-germinal center B-cell Lymphomas in Mice

    PubMed Central

    Yamamoto, Kenta; Lee, Brian J.; Li, Chen; Dubois, Richard L.; Hobeika, Elias; Bhagat, Govind; Zha, Shan

    2017-01-01

    Ataxia Telangiectasia Mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin Lymphomas (B-NHL), including ~50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B-cells causes cell-autonomous, clonal mature B cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly naïve B cell specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. While EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increased the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas towards pre-GC derived small lymphocytic neoplasms sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naïve B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.g. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-germinal center B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL. PMID:25676421

  1. Regulator of G protein signaling 6 mediates doxorubicin-induced ATM and p53 activation by a reactive oxygen species-dependent mechanism.

    PubMed

    Huang, Jie; Yang, Jianqi; Maity, Biswanath; Mayuzumi, Daisuke; Fisher, Rory A

    2011-10-15

    Doxorubicin (DXR), among the most widely used cancer chemotherapy agents, promotes cancer cell death via activation of ataxia telangiectasia mutated (ATM) and the resultant upregulation of tumor suppressor p53. The exact mechanism by which DXR activates ATM is not fully understood. Here, we discovered a novel role for regulator of G protein signaling 6 (RGS6) in mediating activation of ATM and p53 by DXR. RGS6 was robustly induced by DXR, and genetic loss of RGS6 dramatically impaired DXR-induced activation of ATM and p53, as well as its in vivo apoptotic actions in heart. The ability of RGS6 to promote p53 activation in response to DXR was independent of RGS6 interaction with G proteins but required ATM. RGS6 mediated activation of ATM and p53 by DXR via a reactive oxygen species (ROS)-dependent and DNA damage-independent mechanism. This mechanism represents the primary means by which DXR promotes activation of the ATM-p53 apoptosis pathway that underlies its cytotoxic activity. Our findings contradict the canonical theories that DXR activates ATM primarily by promoting DNA damage either directly or indirectly (via ROS) and that RGS6 function is mediated by its interactions with G proteins. These findings reveal a new mechanism for the chemotherapeutic actions of DXR and identify RGS6 as a novel target for cancer chemotherapy.

  2. Automated transportation management system (ATMS) software project management plan (SPMP)

    SciTech Connect

    Weidert, R.S., Westinghouse Hanford

    1996-05-20

    The Automated Transportation Management System (ATMS) Software Project Management plan (SPMP) is the lead planning document governing the life cycle of the ATMS and its integration into the Transportation Information Network (TIN). This SPMP defines the project tasks, deliverables, and high level schedules involved in developing the client/server ATMS software.

  3. ATM protein is deficient in over 40% of lung adenocarcinomas.

    PubMed

    Villaruz, Liza C; Jones, Helen; Dacic, Sanja; Abberbock, Shira; Kurland, Brenda F; Stabile, Laura P; Siegfried, Jill M; Conrads, Thomas P; Smith, Neil R; O'Connor, Mark J; Pierce, Andrew J; Bakkenist, Christopher J

    2016-09-06

    Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide, and of the estimated 1.2 million new cases of lung cancer diagnosed every year, over 30% are lung adenocarcinomas. The backbone of 1st-line systemic therapy in the metastatic setting, in the absence of an actionable oncogenic driver, is platinum-based chemotherapy. ATM and ATR are DNA damage signaling kinases activated at DNA double-strand breaks (DSBs) and stalled and collapsed replication forks, respectively. ATM protein is lost in a number of cancer cell lines and ATR kinase inhibitors synergize with cisplatin to resolve xenograft models of ATM-deficient lung cancer. We therefore sought to determine the frequency of ATM loss in a tissue microarray (TMA) of lung adenocarcinoma. Here we report the validation of a commercial antibody (ab32420) for the identification of ATM by immunohistochemistry and estimate that 61 of 147 (41%, 95% CI 34%-50%) cases of lung adenocarcinoma are negative for ATM protein expression. As a positive control for ATM staining, nuclear ATM protein was identified in stroma and immune infiltrate in all evaluable cases. ATM loss in lung adenocarcinoma was not associated with overall survival. However, our preclinical findings in ATM-deficient cell lines suggest that ATM could be a predictive biomarker for synergy of an ATR kinase inhibitor with standard-of-care cisplatin. This could improve clinical outcome in 100,000's of patients with ATM-deficient lung adenocarcinoma every year.

  4. A Managerial Analysis of ATM in Facilitating Distance Education.

    ERIC Educational Resources Information Center

    Littman, Marlyn Kemper

    In this paper, the fundamental characteristics and capabilities of ATM (Asynchronous Transfer Mode) networks in a distance learning environment are examined. Current and projected ATM applications are described, and issues and challenges associated with developing ATM networking solutions for instructional delivery are explored. Other topics…

  5. Do Structural Missense Variants in the ATM Gene Found in Women With Breast Cancer Cause Breast Cancer in "Knock-in" Mouse Strains?

    DTIC Science & Technology

    2006-04-01

    W81XWH-05-1-0282 TITLE: Do Structural Missense Variants in the ATM Gene Found in Women with Breast Cancer Cause Breast Cancer in "Knock-in...5a. CONTRACT NUMBER Do Structural Missense Variants in the ATM Gene Found in Women with Breast Cancer Cause Breast Cancer in "Knock-in" Mouse...human cohort-specific missense mutations will develop breast cancer with dominant inheritance in a subset of animals. It also is hypothesized that

  6. WRN Is Required for ATM Activation and the S-Phase Checkpoint in Response to Interstrand Cross-Link–Induced DNA Double-Strand Breaks

    PubMed Central

    Cheng, Wen-Hsing; Muftic, Diana; Muftuoglu, Meltem; Dawut, Lale; Morris, Christa; Helleday, Thomas; Shiloh, Yosef

    2008-01-01

    Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks. PMID:18596239

  7. DNA sequence analysis of spontaneous and gamma-radiation (anoxic)-induced lacId mutations in Escherichia coli umuC122::Tn5: differential requirement for umuC at G.C vs. A.T sites and for the production of transversions vs. transitions.

    PubMed

    Sargentini, N J; Smith, K C

    1994-12-01

    Escherichia coli umuC122::Tn5 cells were gamma-irradiated (137Cs, 750 Gy, under N2), and lac-constitutive mutants were produced at 36% of the wild-type level (the umuC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacId mutations was very similar to that for the wild-type strain). The specific nature of the umuC strain's partial radiation mutability was determined by sequencing 325 radiation-induced lacId mutations. The yields of radiation-induced mutation classes in the umuC strain (as a percentage of the wild-type yield) were: 80% for A.T-->G.C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A.T-->C.G transversions, 36% for G.C-->A.T transitions, 25% for multi-base deletions, 21% for A.T-->T.A transversions, 11% for G.C-->C.G transversions, 9% for G.C-->T.A transversions, and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umuC strain is near-normal for A.T-->G.C. transitions, single-base deletions and possibly A.T-->C.G transversions; is generally deficient for mutagenesis at G.C sites and for transversions, and is grossly deficient in multiple mutations. Damage at G.C sites seems more difficult for translesion DNA synthesis to bypass than damage at A.T sites, and especially when trying to produce a transversion. The yield of G.C-->A.T transitions in the umuC strain (36% of the wild-type level) argues that abasic sites are involved in no more than 64% of gamma-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD' proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G.C vs. A.T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.

  8. Analysis of Chromosomal Aberrations after Low and High Dose Rate Gamma Irradiation in ATM or NBS Suppressed Human Fibroblast Cells

    NASA Technical Reports Server (NTRS)

    Hada, M.; Huff, J. L.; Patel, Z.; Pluth, J. M.; George, K. A.; Cucinotta, F. A.

    2009-01-01

    A detailed understanding of the biological effects of heavy nuclei is needed for space radiation protection and for cancer therapy. High-LET radiation produces more complex DNA lesions that may be non-repairable or that may require additional processing steps compared to endogenous DSBs, increasing the possibility of misrepair. Interplay between radiation sensitivity, dose, and radiation quality has not been studied extensively. Previously we studied chromosome aberrations induced by low- and high- LET radiation in several cell lines deficient in ATM (ataxia telangactasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. We found that the yields of both simple and complex chromosomal aberrations were significantly increased in the DSB repair defective cells compared to normal cells. The increased aberrations observed for the ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex aberrations, while the linear dose-response term was significantly higher in NBS cells only for simple exchanges. These results point to the importance of the functions of ATM and NBS in chromatin modifications that function to facilitate correct DSB repair and minimize aberration formation. To further understand the sensitivity differences that were observed in ATM and NBS deficient cells, in this study, chromosomal aberration analysis was performed in normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, or Mirin, an MRN complex inhibitor involved in activation of ATM. We are also testing siRNA knockdown of these proteins. Normal and ATM or NBS suppressed cells were irradiated with gamma-rays and chromosomes were collected with a premature chromosome

  9. Analysis of Chromosomal Aberrations after Low and High Dose Rate Gamma Irradiation in ATM or NBS Suppressed Human Fibroblast Cells

    NASA Technical Reports Server (NTRS)

    Hada, M.; Huff, J. L.; Patel, Z.; Pluth, J. M.; George, K. A.; Cucinotta, F. A.

    2009-01-01

    A detailed understanding of the biological effects of heavy nuclei is needed for space radiation protection and for cancer therapy. High-LET radiation produces more complex DNA lesions that may be non-repairable or that may require additional processing steps compared to endogenous DSBs, increasing the possibility of misrepair. Interplay between radiation sensitivity, dose, and radiation quality has not been studied extensively. Previously we studied chromosome aberrations induced by low- and high- LET radiation in several cell lines deficient in ATM (ataxia telangactasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. We found that the yields of both simple and complex chromosomal aberrations were significantly increased in the DSB repair defective cells compared to normal cells. The increased aberrations observed for the ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex aberrations, while the linear dose-response term was significantly higher in NBS cells only for simple exchanges. These results point to the importance of the functions of ATM and NBS in chromatin modifications that function to facilitate correct DSB repair and minimize aberration formation. To further understand the sensitivity differences that were observed in ATM and NBS deficient cells, in this study, chromosomal aberration analysis was performed in normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, or Mirin, an MRN complex inhibitor involved in activation of ATM. We are also testing siRNA knockdown of these proteins. Normal and ATM or NBS suppressed cells were irradiated with gamma-rays and chromosomes were collected with a premature chromosome

  10. Inhibition of TGFbeta1 Signaling Attenutates ATM Activity inResponse to Genotoxic Stress

    SciTech Connect

    Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose; Ravani, Shraddha A.; Glick, Adam B.; Lavin, Martin J.; Koslov, Sergei; Shiloh, Yosef; Barcellos-Hoff, Mary Helen

    2006-09-15

    Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta}1 (TGF{beta}), which is activated by radiation, is a potent and pleiotropic mediator of physiological and pathological processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}1 null murine epithelial cells or human epithelial cells treated with a small molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17 and p53, reduced {gamma}H2AX radiation-induced foci, and increased radiosensitivity compared to TGF{beta} competent cells. We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM that directs epithelial cell stress responses, cell fate and tissue integrity. Thus, TGF{beta}1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.

  11. ATM prevents DSB formation by coordinating SSB repair and cell cycle progression.

    PubMed

    Khoronenkova, Svetlana V; Dianov, Grigory L

    2015-03-31

    DNA single-strand breaks (SSBs) arise as a consequence of spontaneous DNA instability and are also formed as DNA repair intermediates. Their repair is critical because they otherwise terminate gene transcription and generate toxic DNA double-strand breaks (DSBs) on replication. To prevent the formation of DSBs, SSB repair must be completed before DNA replication. To accomplish this, cells should be able to detect unrepaired SSBs, and then delay cell cycle progression to allow more time for repair; however, to date there is no evidence supporting the coordination of SSB repair and replication in human cells. Here we report that ataxia-telangiectasia mutated kinase (ATM) plays a major role in restricting the replication of SSB-containing DNA and thus prevents DSB formation. We show that ATM is activated by SSBs and coordinates their repair with DNA replication. SSB-mediated ATM activation is followed by a G1 cell cycle delay that allows more time for repair and thus prevents the replication of damaged DNA and DSB accrual. These findings establish an unanticipated role for ATM in the signaling of DNA SSBs and provide important insight into the molecular defects leading to genetic instability in patients with ataxia-telangiectasia.

  12. Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies

    PubMed Central

    Gaudio, Eugenio; Spizzo, Riccardo; Paduano, Francesco; Luo, Zhenghua; Efanov, Alexey; Palamarchuk, Alexey; Leber, Amanda S.; Kaou, Mohamed; Zanesi, Nicola; Bottoni, Arianna; Costinean, Stefan; Rassenti, Laura Z.; Nakamura, Tatsuya; Kipps, Thomas J.; Aqeilan, Rami I.; Pekarsky, Yuri; Trapasso, Francesco

    2012-01-01

    The T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas. PMID:22065599

  13. ATM Functions at the Peroxisome to Induce Pexophagy in Response to ROS

    PubMed Central

    Alexander, Angela; Kim, Jinhee; Powell, Reid T.; Dere, Ruhee; Tait-Mulder, Jacqueline; Lee, Ji-Hoon; Paull, Tanya T.; Pandita, Raj K.; Charaka, Vijaya K.; Pandita, Tej K.; Kastan, Michael B.; Walker, Cheryl Lyn

    2015-01-01

    Peroxisomes are highly metabolic, autonomously replicating organelles that generate ROS as a by product of fatty acid β-oxidation. Consequently, cells must maintain peroxisome homeostasis, or risk pathologies associated with too few peroxisomes, such as peroxisome biogenesis disorders, or too many peroxisomes, inducing oxidative damage and promoting diseases such as cancer. We report that the PEX5 peroxisome import receptor binds ataxia-telangiectasia mutated (ATM) and localizes this kinase to the peroxisome. In response to reactive oxygen species (ROS), ATM signaling activates ULK1 and inhibits mTORC1 to induce autophagy. Specificity for autophagy of peroxisomes (pexophagy) is provided by ATM phosphorylation of PEX5 at Ser141, which promotes PEX5 mono-ubiquitination at K209, and recognition of ubiquitinated PEX5 by the autophagy adapter protein p62, directing the autophagosome to peroxisomes to induce pexophagy. These data reveal an important new role for ATM in metabolism as a sensor of ROS that regulates pexophagy. PMID:26344566

  14. Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

    PubMed Central

    Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

    2002-01-01

    It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM+/−) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM+/− cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage. PMID:12119422

  15. Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

    NASA Technical Reports Server (NTRS)

    Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

    2002-01-01

    It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

  16. Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

    NASA Technical Reports Server (NTRS)

    Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

    2002-01-01

    It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

  17. Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer

    PubMed Central

    Tavtigian, Sean V.; Oefner, Peter J.; Babikyan, Davit; Hartmann, Anne; Healey, Sue; Le Calvez-Kelm, Florence; Lesueur, Fabienne; Byrnes, Graham B.; Chuang, Shu-Chun; Forey, Nathalie; Feuchtinger, Corinna; Gioia, Lydie; Hall, Janet; Hashibe, Mia; Herte, Barbara; McKay-Chopin, Sandrine; Thomas, Alun; Vallée, Maxime P.; Voegele, Catherine; Webb, Penelope M.; Whiteman, David C.; Sangrajrang, Suleeporn; Hopper, John L.; Southey, Melissa C.; Andrulis, Irene L.; John, Esther M.; Chenevix-Trench, Georgia

    2009-01-01

    The susceptibility gene for ataxia telangiectasia, ATM, is also an intermediate-risk breast-cancer-susceptibility gene. However, the spectrum and frequency distribution of ATM mutations that confer increased risk of breast cancer have been controversial. To assess the contribution of rare variants in this gene to risk of breast cancer, we pooled data from seven published ATM case-control mutation-screening studies, including a total of 1544 breast cancer cases and 1224 controls, with data from our own mutation screening of an additional 987 breast cancer cases and 1021 controls. Using an in silico missense-substitution analysis that provides a ranking of missense substitutions from evolutionarily most likely to least likely, we carried out analyses of protein-truncating variants, splice-junction variants, and rare missense variants. We found marginal evidence that the combination of ATM protein-truncating and splice-junction variants contribute to breast cancer risk. There was stronger evidence that a subset of rare, evolutionarily unlikely missense substitutions confer increased risk. On the basis of subset analyses, we hypothesize that rare missense substitutions falling in and around the FAT, kinase, and FATC domains of the protein may be disproportionately responsible for that risk and that a subset of these may confer higher risk than do protein-truncating variants. We conclude that a comparison between the graded distributions of missense substitutions in cases versus controls can complement analyses of truncating variants and help identify susceptibility genes and that this approach will aid interpretation of the data emerging from new sequencing technologies. PMID:19781682

  18. ATM photoheliograph. [at a solar observatory

    NASA Technical Reports Server (NTRS)

    Prout, R. A.

    1975-01-01

    The design and fabrication are presented of a 65 cm photoheliograph functional verification unit (FVU) installed in a major solar observatory. The telescope is used in a daily program of solar observation while serving as a test bed for the development of instrumentation to be included in early space shuttle launched solar telescopes. The 65 cm FVU was designed to be mechanically compatible with the ATM spar/canister and would be adaptable to a second ATM flight utilizing the existing spar/canister configuration. An image motion compensation breadboard and a space-hardened, remotely tuned H alpha filter, as well as solar telescopes of different optical configurations or increased aperture are discussed.

  19. Experiences with the AEROnet/PSCN ATM Prototype

    NASA Technical Reports Server (NTRS)

    Kurak, Richard S.; Lisotta, Anthony J.; McCabe, James D.; Nothaft, Alfred E.; Russell, Kelly R.; Lasinski, T. A. (Technical Monitor)

    1995-01-01

    This paper discusses the experience gained by the AEROnet/PSCN networking team in deploying a prototype Asynchronous Transfer Mode (ATM) based network as part of the wide-area network for the Numerical Aerodynamic Simulation (NAS) Program at NASA Ames Research Center. The objectives of this prototype were to test concepts in using ATM over wide-area Internet Protocol (IP) networks and measure end-to-end system performance. This testbed showed that end-to-end ATM over a DS3 reaches approximately 80% of the throughput achieved from a FDDI to DS3 network. The 20% reduction in through-put can be attributed to the overhead associated with running ATM. As a result, we conclude that if the loss in capacity due to ATM overhead is balanced by the reduction in cost of ATM services, as compared to dedicated circuits, then ATM can be a viable alternative.

  20. Activation of ATM by DNA Damaging Agents

    DTIC Science & Technology

    2004-09-01

    all cases, observe a dependence on ATM for phosphorylation. These phosphorylation events are attenuated by pretreatment of cells with N-acetyl cysteine...Chemistry requested that I evaluate the effect of N-acetyl cysteine pretreatment on doxorubicin-induced phosphorylation of histone H2AX at serine 139. In...S33/35, T68) in response to doxorubicin treatment * I detennined that phosphorylation at these sites can be partially attenuated by pretreatment of

  1. Preservation of methane hydrate at 1 atm

    USGS Publications Warehouse

    Stern, L.A.; Circone, S.; Kirby, S.H.; Durham, W.B.

    2001-01-01

    A "pressure-release" method that enables reproducible bulk preservation of pure, porous, methane hydrate at conditions 50 to 75 K above its equilibrium T (193 K) at 1 atm is refined. The amount of hydrate preserved by this method appears to be greatly in excess of that reported in the previous citations, and is likely the result of a mechanism different from ice shielding.

  2. ATM Coastal Topography-Mississippi, 2001

    USGS Publications Warehouse

    Nayegandhi, Amar; Yates, Xan; Brock, John C.; Sallenger, A.H.; Klipp, Emily S.; Wright, C. Wayne

    2009-01-01

    These remotely sensed, geographically referenced elevation measurements of lidar-derived first-surface (FS) topography were produced collaboratively by the U.S. Geological Survey (USGS), Florida Integrated Science Center (FISC), St. Petersburg, FL, and the National Aeronautics and Space Administration (NASA), Wallops Flight Facility, VA. This project provides highly detailed and accurate datasets of the Mississippi coastline, from Lakeshore to Petit Bois Island, acquired September 9-10, 2001. The datasets are made available for use as a management tool to research scientists and natural-resource managers. An innovative scanning lidar instrument originally developed by NASA, and known as the Airborne Topographic Mapper (ATM), was used during data acquisition. The ATM system is a scanning lidar system that measures high-resolution topography of the land surface and incorporates a green-wavelength laser operating at pulse rates of 2 to 10 kilohertz. Measurements from the laser-ranging device are coupled with data acquired from inertial navigation system (INS) attitude sensors and differentially corrected global positioning system (GPS) receivers to measure topography of the surface at accuracies of +/-15 centimeters. The nominal ATM platform is a Twin Otter or P-3 Orion aircraft, but the instrument may be deployed on a range of light aircraft. Elevation measurements were collected over the survey area using the ATM system, and the resulting data were then processed using the Airborne Lidar Processing System (ALPS), a custom-built processing system developed in a NASA-USGS collaboration. ALPS supports the exploration and processing of lidar data in an interactive or batch mode. Modules for presurvey flight-line definition, flight-path plotting, lidar raster and waveform investigation, and digital camera image playback have been developed. Processing algorithms have been developed to extract the range to the first and last significant return within each waveform. ALPS

  3. ATM Coastal Topography-Alabama 2001

    USGS Publications Warehouse

    Nayegandhi, Amar; Yates, Xan; Brock, John C.; Sallenger, A.H.; Bonisteel, Jamie M.; Klipp, Emily S.; Wright, C. Wayne

    2009-01-01

    These remotely sensed, geographically referenced elevation measurements of Lidar-derived first surface (FS) topography were produced collaboratively by the U.S. Geological Survey (USGS), Florida Integrated Science Center (FISC), St. Petersburg, FL, and the National Aeronautics and Space Administration (NASA), Wallops Flight Facility, VA. This project provides highly detailed and accurate datasets of the Alabama coastline, acquired October 3-4, 2001. The datasets are made available for use as a management tool to research scientists and natural resource managers. An innovative scanning Lidar instrument originally developed by NASA, and known as the Airborne Topographic Mapper (ATM), was used during data acquisition. The ATM system is a scanning Lidar system that measures high-resolution topography of the land surface, and incorporates a green-wavelength laser operating at pulse rates of 2 to 10 kilohertz. Measurements from the laser ranging device are coupled with data acquired from inertial navigation system (INS) attitude sensors and differentially corrected global positioning system (GPS) receivers to measure topography of the surface at accuracies of +/-15 centimeters. The nominal ATM platform is a Twin Otter or P-3 Orion aircraft, but the instrument may be deployed on a range of light aircraft. Elevation measurements were collected over the survey area using the ATM system, and the resulting data were then processed using the Airborne Lidar Processing System (ALPS), a custom-built processing system developed in a NASA-USGS collaboration. ALPS supports the exploration and processing of Lidar data in an interactive or batch mode. Modules for pre-survey flight line definition, flight path plotting, Lidar raster and waveform investigation, and digital camera image playback have been developed. Processing algorithms have been developed to extract the range to the first and last significant return within each waveform. ALPS is routinely used to create maps that

  4. MSFC Skylab ATM calibration rocket project

    NASA Technical Reports Server (NTRS)

    1974-01-01

    The Apollo Telescope Mount (ATM) Calibration Rocket (CALROC) performances and anomalies encountered are discussed. The performance period included six CALROC flights during the Skylab 2, 3 and 4 missions as well as those rocket flights prior to the Skylab mission which carried CALROC hardware for test purposes. Background material such as project purpose, management, launch facilities, booster and payload configuration is included for better understanding of the CALROC payload and its mission objectives.

  5. Intercalibrating and Validating Saphir and Atms Observations

    NASA Astrophysics Data System (ADS)

    Moradi, I.; Ferraro, R. R.

    2014-12-01

    We present the results of evaluating observations from microwave instruments aboard the Suomi National Polar-orbiting Partnership (NPP, ATMS instrument) and Megha-Tropiques (SAPHIR instrument) satellites. ATMS is a cross-track microwave sounder that currently flying on the Suomi National Polar-orbiting Partnership (S-NPP) satellite, launched in October 2011, which is in a Sun-synchronous orbit with the ascending equatorial crossing time at 01:30 a.m. Megha-Tropiques, launched in Nov 2011, is a low-inclination satellite meaning that the satellite only visits the tropical band between 30 S and 30 N. SAPHIR is a microwave humidity sounder with 6 channels operating at the frequencies close to the water vapor absorption line at 183 GHz. Megha-Tropiques revisits the tropical regions several times a day and provide a great capability for inter-calibrating the observations with the polar orbiting satellites. The study includes inter-comparison and inter-calibration of observations of similar channels from the two instruments, evaluation of the satellite data using high-quality radiosonde data from Atmospheric Radiation Measurement Program and GPS Radio Occultaion Observations from COSMIC mission, as well as geolocation error correction. The results of this study are valuable for generating climate data records from these instruments as well as for extending current climate data records from similar instruments such as AMSU-B and MHS to the ATMS and SAPHIR instruments.

  6. ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia

    PubMed Central

    D’Alessandro, Angelo; Alvarez-Calderon, Francesca; Kim, Jihye; Nemkov, Travis; Adane, Biniam; Rozhok, Andrii I.; Kumar, Amit; Kumar, Vijay; Pollyea, Daniel A.; Wempe, Michael F.; Jordan, Craig T.; Serkova, Natalie J.; Tan, Aik Choon; Hansen, Kirk C.; DeGregori, James

    2016-01-01

    Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML. PMID:27791036

  7. ATM has a major role in the double-strand break repair pathway dysregulation in sporadic breast carcinomas and is an independent prognostic marker at both mRNA and protein levels.

    PubMed

    Rondeau, S; Vacher, S; De Koning, L; Briaux, A; Schnitzler, A; Chemlali, W; Callens, C; Lidereau, R; Bièche, I

    2015-03-17

    Ataxia telangiectasia mutated (ATM) is a kinase that has a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks (DSB). In breast cancer, a low level of ATM was correlated with poor outcome; however, the molecular mechanism of this downregulation is still unclear. We used qRT-PCR assay to quantify mRNA levels of ATM gene in 454 breast tumours from patients with known clinical/pathological status and outcome; reverse phase protein arrays (RPPA) were used to assess the levels of ATM and 14 proteins in 233 breast tumours. ATM mRNA was associated with poor metastasis-free survival (MFS) (P=0.00012) on univariate analysis. ATM mRNA and protein levels were positively correlated (P=0.00040). A low level of ATM protein was correlated with poorer MFS (P=0.000025). ATM expression at mRNA or protein levels are independent prognostic factors on multivariate analysis (P=0.00046 and P=0.00037, respectively). The ATM protein level was positively correlated with the levels of six proteins of the DSB repair pathway: H2AX (P<0.0000001), XRCC5 (P<0.0000001), NBN (P<0.0000001), Mre11 (P=0.0000029), Rad50 (P=0.0064), and TP53BP1 (P=0.026), but not with proteins involved in other pathways that are altered in cancer. Low expression of ATM protein was significantly associated with high miR-203 expression (P=0.011). We confirmed that ATM expression is an independent prognostic marker at both RNA and protein levels. We showed that alteration of ATM is involved in dysregulation of the DSB repair pathway. Finally, miR-203 may be responsible for downregulation of ATM in breast cancers.

  8. ATM has a major role in the double-strand break repair pathway dysregulation in sporadic breast carcinomas and is an independent prognostic marker at both mRNA and protein levels

    PubMed Central

    Rondeau, S; Vacher, S; De Koning, L; Briaux, A; Schnitzler, A; Chemlali, W; Callens, C; Lidereau, R; Bièche, I

    2015-01-01

    Background: Ataxia telangiectasia mutated (ATM) is a kinase that has a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks (DSB). In breast cancer, a low level of ATM was correlated with poor outcome; however, the molecular mechanism of this downregulation is still unclear. Methods: We used qRT–PCR assay to quantify mRNA levels of ATM gene in 454 breast tumours from patients with known clinical/pathological status and outcome; reverse phase protein arrays (RPPA) were used to assess the levels of ATM and 14 proteins in 233 breast tumours. Results: ATM mRNA was associated with poor metastasis-free survival (MFS) (P=0.00012) on univariate analysis. ATM mRNA and protein levels were positively correlated (P=0.00040). A low level of ATM protein was correlated with poorer MFS (P=0.000025). ATM expression at mRNA or protein levels are independent prognostic factors on multivariate analysis (P=0.00046 and P=0.00037, respectively). The ATM protein level was positively correlated with the levels of six proteins of the DSB repair pathway: H2AX (P<0.0000001), XRCC5 (P<0.0000001), NBN (P<0.0000001), Mre11 (P=0.0000029), Rad50 (P=0.0064), and TP53BP1 (P=0.026), but not with proteins involved in other pathways that are altered in cancer. Low expression of ATM protein was significantly associated with high miR-203 expression (P=0.011). Conclusion: We confirmed that ATM expression is an independent prognostic marker at both RNA and protein levels. We showed that alteration of ATM is involved in dysregulation of the DSB repair pathway. Finally, miR-203 may be responsible for downregulation of ATM in breast cancers. PMID:25742469

  9. 53BP1 is limiting for NHEJ repair in ATM-deficient model systems that are subjected to oncogenic stress or radiation.

    PubMed

    Rybanska-Spaeder, Ivana; Reynolds, Taylor L; Chou, Jeremy; Prakash, Mansi; Jefferson, Tameca; Huso, David L; Desiderio, Stephen; Franco, Sonia

    2013-10-01

    The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1(-/-)/Atm(-/-)), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm(-/-) mice (average survival, 14 and 23 weeks for Trp53bp1(-/-)/Atm(-/-) and Atm(-/-) mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)-mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1(-/-)/Atm(-/-) primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm(-/-) thymocytes but is required to suppress large deletions in a subset of hybrid joints. The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.

  10. A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease

    PubMed Central

    Tidball, Andrew M.; Bryan, Miles R.; Uhouse, Michael A.; Kumar, Kevin K.; Aboud, Asad A.; Feist, Jack E.; Ess, Kevin C.; Neely, M. Diana; Aschner, Michael; Bowman, Aaron B.

    2015-01-01

    The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling. PMID:25489053

  11. A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

    PubMed

    Tidball, Andrew M; Bryan, Miles R; Uhouse, Michael A; Kumar, Kevin K; Aboud, Asad A; Feist, Jack E; Ess, Kevin C; Neely, M Diana; Aschner, Michael; Bowman, Aaron B

    2015-04-01

    The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling.

  12. Regulation of ATM in DNA double strand break repair accounts for the radiosensitivity in human cells exposed to high linear energy transfer ionizing radiation.

    PubMed

    Xue, Lian; Yu, Dong; Furusawa, Yoshiya; Okayasu, Ryuichi; Tong, Jian; Cao, Jianping; Fan, Saijun

    2009-11-02

    High linear energy transfer (LET) radiation shows different biological effects from low-LET radiation. The complex nature of high LET radiation-induced damage, especially the clustered DNA damage, brings about slow repair of DNA double strand breaks (DSBs), which finally lead to higher lethality and chromosome aberration. Ionizing radiation (IR) induced DNA DSBs are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) pathways in mammalian cells. The novel function of ataxia telangiectasia-mutated (ATM) protein is its involvement in the DSB repair of slow kinetics for "dirty" breaks rejoining by NHEJ, this suggests that ATM may play a more important role in high LET radiation-induced DNA damage. We show here that KU55933, an ATM inhibitor could distinctly lower the clonogenic survival in normal human skin fibroblast cells exposed to carbon ion radiation and dramatically impair the normal process for DSB repair. We also implicated the involvement of ATM in the two pathways of DNA DSB repair, with DNA-PKcs and Rad51 as the representative proteins. The phosphorylation of DNA-PKcs at Thr-2609 with both immunoblotting and immunofluorescent staining indicated an ATM-dependent change, while for Rad51, KU55933 pretreatment could postpone the formation of nuclear Rad51 foci. Interestingly, we also found that pretreatment with chloroquine, an ATM stimulator could protect cells from carbon ion radiation only at lower doses. For doses over 1Gy, protection was no longer observed. There was a dose-dependent increase for ATM kinase activity, with saturation at about 1Gy. Chloroquine pretreatment prior to 1Gy of carbon ion radiation did not enhance the autophosphorylation of ATM at serine 1981. The function of ATM in G2/M checkpoint arrest facilitated DSB repair in high-LET irradiation. Our results provide a possible mechanism for the direct involvement of ATM in DSB repair by high-LET irradiation.

  13. Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint

    PubMed Central

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T.C.; Fu, Li; Xu, Bo

    2011-01-01

    Summary The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically-activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. PMID:22099307

  14. Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint.

    PubMed

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T C; Fu, Li; Xu, Bo

    2011-11-18

    The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Deoxycytidine Kinase Augments ATM-Mediated DNA Repair and Contributes to Radiation Resistance

    PubMed Central

    Bunimovich, Yuri L.; Nair-Gill, Evan; Riedinger, Mireille; McCracken, Melissa N.; Cheng, Donghui; McLaughlin, Jami; Radu, Caius G.; Witte, Owen N.

    2014-01-01

    Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy. PMID:25101980

  16. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.

    PubMed

    Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver; Williams, R Scott; Kuhnlein, Jeff; Lee, Ji-Hoon; Classen, Scott; Guenther, Grant; Russell, Paul; Tainer, John A; Paull, Tanya T

    2014-03-03

    The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

  17. ATM Quality of Service Tests for Digitized Video Using ATM Over Satellite: Laboratory Tests

    NASA Technical Reports Server (NTRS)

    Ivancic, William D.; Brooks, David E.; Frantz, Brian D.

    1997-01-01

    A digitized video application was used to help determine minimum quality of service parameters for asynchronous transfer mode (ATM) over satellite. For these tests, binomially distributed and other errors were digitally inserted in an intermediate frequency link via a satellite modem and a commercial gaussian noise generator. In this paper, the relation- ship between the ATM cell error and cell loss parameter specifications is discussed with regard to this application. In addition, the video-encoding algorithms, test configurations, and results are presented in detail.

  18. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells

    PubMed Central

    Bakkenist, Christopher J.; Czambel, R. Kenneth; Hershberger, Pamela A.; Tawbi, Hussein; Beumer, Jan H.; Schmitz, John C.

    2015-01-01

    Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy. PMID:26097887

  19. Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair

    PubMed Central

    Soares, Daniele G.; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M.; Henriques, João A. P.; Larsen, Annette K.; Escargueil, Alexandre E.

    2016-01-01

    Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors. PMID:27029031

  20. Preclinical evaluation of a novel ATM inhibitor, KU59403, in vitro and in vivo in p53 functional and dysfunctional models of human cancer.

    PubMed

    Batey, Michael A; Zhao, Yan; Kyle, Suzanne; Richardson, Caroline; Slade, Andrew; Martin, Niall M B; Lau, Alan; Newell, David R; Curtin, Nicola J

    2013-06-01

    Ataxia telangiectasia mutated (ATM) kinase signals DNA double-strand breaks (DSB) to cell-cycle arrest via p53 and DNA repair. ATM-defective cells are sensitive to DSB-inducing agents, making ATM an attractive target for anticancer chemo- and radiosensitization. KU59403 is an ATM inhibitor with the potency, selectivity, and solubility for advanced preclinical evaluation. KU59403 was not cytotoxic to human cancer cell lines (SW620, LoVo, HCT116, and MDA-MB-231) per se but significantly increased the cytotoxicity of topoisomerase I and II poisons: camptothecin, etoposide, and doxorubicin. Chemo- and radiosensitization by ATM inhibition was not p53-dependent. Following administration to mice, KU59403 distributed to tissues and concentrations exceeding those required for in vitro activity were maintained for at least 4 hours in tumor xenografts. KU59403 significantly enhanced the antitumor activity of topoisomerase poisons in mice bearing human colon cancer xenografts (SW620 and HCT116) at doses that were nontoxic alone and well-tolerated in combination. Chemosensitization was both dose- and schedule-dependent. KU59403 represents a major advance in ATM inhibitor development, being the first compound to show good tissue distribution and significant chemosensitization in in vivo models of human cancer, without major toxicity. KU59403 provides the first proof-of-principle preclinical data to support the future clinical development of ATM inhibitors. ©2013 AACR

  1. Suppressing the high-level expression and function of ATM in advanced-stage melanomas does not sensitize the cells to ionizing radiation.

    PubMed

    Moschos, Stergios J; Dodd, Nicole R; Jukic, Drazen M; Fayewicz, Shelley L; Wang, Xiaolei; Becker, Dorothea

    2009-10-01

    Melanoma in its advanced stages is resistant not only to chemotherapy but also to radiation treatment. In line with efforts to identify genes that are key regulators of the disease and as such, may prove valuable targets for adjuvant and neo-adjuvant therapy of melanomas, we previously reported the presence of Serial Analysis of Gene Expression (SAGE) tags, corresponding to the Ataxia Telangiectasia Mutated (ATM) gene, in SAGE libraries generated from tissues representing primary and metastatic melanomas. In the present study, we document that ATM is expressed at high levels in advanced-stage melanomas. Given its crucial role in the cellular response to DNA double-strand breaks (DSB), ionizing radiation, and UV damage, we pursued a series of functional studies involving the targeting of ATM by way of RNA interference or an ATM-specific small-molecule inhibitor, followed by exposure of the cells to ionizing radiation or radiation combined with a DNA-intercalating drug, to test the hypothesis that the high-level expression of ATM prevents melanoma cells from undergoing apoptosis in response to DNA DSB-inducing treatments. However, unlike as demonstrated in the case of other malignancies, our findings summarized herein do not point to ATM as a pivotal DNA damage sensor for advanced-stage melanomas, raising the possibility that in these cells, genes other than ATM regulate and control the repair of DNA DSB.

  2. Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair.

    PubMed

    Lima, Michelle; Bouzid, Hana; Soares, Daniele G; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M; Henriques, João A P; Larsen, Annette K; Escargueil, Alexandre E

    2016-05-03

    Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors.

  3. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells.

    PubMed

    Bakkenist, Christopher J; Czambel, R Kenneth; Hershberger, Pamela A; Tawbi, Hussein; Beumer, Jan H; Schmitz, John C

    2015-01-01

    Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

  4. Is Increased Low-dose somatic Radiosensitivity Associated with Increased Transgenerational Germline Mutation

    SciTech Connect

    Brenner, David J.

    2008-10-02

    Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm+/–) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm+/– males did not significantly differ from that in wild-type BALB/c mice. Acute gamma-ray exposure did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm+/– and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analyzed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

  5. Frequent alterations of MCPH1 and ATM are associated with primary breast carcinoma: clinical and prognostic implications.

    PubMed

    Bhattacharya, Nilanjana; Mukherjee, Nupur; Singh, Ratnesh K; Sinha, Satyabrata; Alam, Neyaz; Roy, Anup; Roychoudhury, Susanta; Panda, Chinmay Kumar

    2013-12-01

    MCPH1 is a proximal regulator of DNA damage response pathway that is involved in recruitment of phosphorylated ATM to double-stranded DNA breaks. To understand the importance of MCPH1 and ATM in deregulation of DNA damage response pathway in breast carcinoma, we studied m-RNA expression and genetic/epigenetic alterations of these genes in primary breast carcinoma samples. Our study revealed reduced expression (mRNA/protein) and high alterations (deletion/methylation) (96 %, 121 of 126) of MCPH1 and ATM. Mutation was, however, rare in inactivation of MCPH1. In immunohistochemical analysis, reduced protein expression of MCPH1, ATM and p-ATM were concordant with their molecular alterations (P = 0.03-0.01). Alterations of MCPH1 and deletion of ATM were significantly high in estrogen/progesterone receptor-negative than estrogen/progesterone receptor-positive breast carcinoma samples compared to early or late age of onset tumors, indicating differences in pathogenesis of the molecular subtypes (P = 0.004-0.01). These genes also showed differential association with tumor stage, grade and lymph node status in different subtypes of breast carcinoma (P = 0.00001-0.01). Their coalterations showed significant association with tumor progression and prognosis (P = 0.003-0.05). Interestingly, patients with alterations of these genes or MCPH1 alone had poor outcome after treatment with DNA-interacting drugs and/or radiation (P = 0.01-0.05). Inactivation of MCPH1-ATM-associated DNA damage response pathway might have an important role in the development of breast carcinoma with diagnostic, prognostic and therapeutic implications.

  6. Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining.

    PubMed

    Sastre-Moreno, Guillermo; Pryor, John M; Moreno-Oñate, Marta; Herrero-Ruiz, Andrés M; Cortés-Ledesma, Felipe; Blanco, Luis; Ramsden, Dale A; Ruiz, Jose F

    2017-03-01

    DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

  7. The ATM-SMC1 pathway is essential for activation of the chromium[VI]-induced S-phase checkpoint.

    PubMed

    Wakeman, Timothy P; Kim, Wan-Ju; Callens, Shannon; Chiu, Arthur; Brown, Kevin D; Xu, Bo

    2004-10-04

    Hexavalent chromium (Cr[VI]) is a common industrial waste product, an environmental pollutant, and a recognized human carcinogen. Following cellular uptake, Cr[VI] can cause DNA damage, however, the mechanisms by which mammalian cells respond to Cr-induced DNA damage remain to be elucidated. Using single cell gel electrophoresis (e.g., Comet Assay) and immunofluoresence microscopy to detect the presence of gamma-H2AX foci, we find that Cr[VI] induces DNA double-strand breaks similar to ionizing radiation (IR). We also demonstrated that ataxia telangiectasia mutated (ATM) is activated in response to Cr[VI] and exposure to Cr[VI] triggers a dose and ATM-dependent S-phase arrest. Further, we document that ATM is required for phosphorylation of the structural maintenance of chromosome protein 1 (SMC1). Finally, we find that ATM-dependent phosphorylation of SMC1 is required to facilitate S-phase cell-cycle arrest in response to Cr[VI] exposure. Collectively, these results indicate that the ATM-SMC1 pathway plays a critical role in cellular response to Cr[VI].

  8. The E3 ubiquitin ligase Mule acts through the ATM-p53 axis to maintain B lymphocyte homeostasis.

    PubMed

    Hao, Zhenyue; Duncan, Gordon S; Su, Yu-Wen; Li, Wanda Y; Silvester, Jennifer; Hong, Claire; You, Han; Brenner, Dirk; Gorrini, Chiara; Haight, Jillian; Wakeham, Andrew; You-Ten, Annick; McCracken, Susan; Elia, Andrew; Li, Qinxi; Detmar, Jacqui; Jurisicova, Andrea; Hobeika, Elias; Reth, Michael; Sheng, Yi; Lang, Philipp A; Ohashi, Pamela S; Zhong, Qing; Wang, Xiaodong; Mak, Tak W

    2012-01-16

    Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell-specific Mule knockout (BMKO) mice were generated using the Cre-LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho-ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM-p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.

  9. Concurrent V(D)J recombination and DNA end instability increase interchromosomal trans-rearrangements in ATM-deficient thymocytes.

    PubMed

    Bowen, Steven; Wangsa, Darawalee; Ried, Thomas; Livak, Ferenc; Hodes, Richard J

    2013-04-01

    During the CD4(-)CD8(-) (DN) stage of T-cell development, RAG-dependent DNA breaks and V(D)J recombination occur at three T-cell receptor (TCR) loci: TCRβ, TCRγ and TCRδ. During this stage, abnormal trans-rearrangements also take place between TCR loci, occurring at increased frequency in absence of the DNA damage response mediator ataxia telangiectasia mutated (ATM). Here, we use this model of physiologic trans-rearrangement to study factors that predispose to rearrangement and the role of ATM in preventing chromosomal translocations. The frequency of DN thymocytes with DNA damage foci at multiple TCR loci simultaneously is increased 2- to 3-fold in the absence of ATM. However, trans-rearrangement is increased 10 000- to 100 000-fold, indicating that ATM function extends beyond timely resolution of DNA breaks. RAG-mediated synaptic complex formation occurs between recombination signal sequences with unequal 12 and 23 base spacer sequences (12/23 rule). TCR trans-rearrangements violate this rule, as we observed similar frequencies of 12/23 and aberrant 12/12 or 23/23 recombination products. This suggests that trans-rearrangements are not the result of trans-synaptic complex formation, but they are instead because of unstable cis synaptic complexes that form simultaneously at distinct TCR loci. Thus, ATM suppresses trans-rearrangement primarily through stabilization of DNA breaks at TCR loci.

  10. Traffic Management in ATM Networks Over Satellite Links

    NASA Technical Reports Server (NTRS)

    Goyal, Rohit; Jain, Raj; Goyal, Mukul; Fahmy, Sonia; Vandalore, Bobby; vonDeak, Thomas

    1999-01-01

    This report presents a survey of the traffic management Issues in the design and implementation of satellite Asynchronous Transfer Mode (ATM) networks. The report focuses on the efficient transport of Transmission Control Protocol (TCP) traffic over satellite ATM. First, a reference satellite ATM network architecture is presented along with an overview of the service categories available in ATM networks. A delay model for satellite networks and the major components of delay and delay variation are described. A survey of design options for TCP over Unspecified Bit Rate (UBR), Guaranteed Frame Rate (GFR) and Available Bit Rate (ABR) services in ATM is presented. The main focus is on traffic management issues. Several recommendations on the design options for efficiently carrying data services over satellite ATM networks are presented. Most of the results are based on experiments performed on Geosynchronous (GEO) latencies. Some results for Low Earth Orbits (LEO) and Medium Earth Orbit (MEO) latencies are also provided.

  11. ATMS Snowfall Rate Product and Its Applications

    NASA Astrophysics Data System (ADS)

    Meng, H.; Kongoli, C.; Dong, J.; Wang, N. Y.; Ferraro, R. R.; Zavodsky, B.; Banghua Yan, B.

    2015-12-01

    A snowfall rate (SFR) algorithm has been developed for the Advanced Technology Microwave Sounder (ATMS) aboard S-NPP and future JPSS satellites. The product is based on the NOAA/NESDIS operational Microwave Humidity Sounder (MHS) SFR but with several key advancements. The algorithm has benefited from continuous development to improve accuracy and snowfall detection efficiency. The enhancements also expand the applicable temperature range for the algorithm and allow significantly more snowfall to be detected than the operational SFR. Another major improvement is the drastically reduced product latency by using Direct Broadcast (DB) data. The new developments have also been implemented in the MHS SFR to ensure product consistency across satellites. Currently, there are five satellites that carry either ATMS or MHS: S-NPP, NOAA-18/-19 and Metop-A/-B. The combined satellites deliver up to ten SFR estimates a day at any location over land in mid-latitudes. The product provides much needed winter precipitation estimates for applications such as weather forecasting and hydrology. Both ATMS and MHS SFR serve as input to a global precipitation analysis product, the NOAA/NCEP CMORPH-Snow. SFR is the sole satellite-based snowfall estimates in the blended product. In addition, ATMS and MHS SFR was assessed at several NWS Weather Forecast Offices (WFOs) and NESDIS/Satellite Analysis Branch (SAB) for its operational values in winter 2015. This is a joint effort among NASA/SPoRT, NOAA/NESDIS, University of Maryland/CICS, and the WFOs. The feedback from the assessment indicated that SFR provides useful information for snowfall forecast. It is especially valuable for areas with poor radar coverage and ground observations. The feedback also identified some limitations of the product such as inadequate detection of shallow snowfall. The algorithm developers will continue to improve product quality as well as developing SFR for new microwave sensors and over ocean in a project

  12. ATM protein is deficient in over 40% of lung adenocarcinomas

    PubMed Central

    Villaruz, Liza C.; Jones, Helen; Dacic, Sanja; Abberbock, Shira; Kurland, Brenda F.; Stabile, Laura P.; Siegfried, Jill M.; Conrads, Thomas P.; Smith, Neil R.; O'Connor, Mark J.; Pierce, Andrew J.; Bakkenist, Christopher J.

    2016-01-01

    Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide, and of the estimated 1.2 million new cases of lung cancer diagnosed every year, over 30% are lung adenocarcinomas. The backbone of 1st-line systemic therapy in the metastatic setting, in the absence of an actionable oncogenic driver, is platinum-based chemotherapy. ATM and ATR are DNA damage signaling kinases activated at DNA double-strand breaks (DSBs) and stalled and collapsed replication forks, respectively. ATM protein is lost in a number of cancer cell lines and ATR kinase inhibitors synergize with cisplatin to resolve xenograft models of ATM-deficient lung cancer. We therefore sought to determine the frequency of ATM loss in a tissue microarray (TMA) of lung adenocarcinoma. Here we report the validation of a commercial antibody (ab32420) for the identification of ATM by immunohistochemistry and estimate that 61 of 147 (41%, 95% CI 34%-50%) cases of lung adenocarcinoma are negative for ATM protein expression. As a positive control for ATM staining, nuclear ATM protein was identified in stroma and immune infiltrate in all evaluable cases. ATM loss in lung adenocarcinoma was not associated with overall survival. However, our preclinical findings in ATM-deficient cell lines suggest that ATM could be a predictive biomarker for synergy of an ATR kinase inhibitor with standard-of-care cisplatin. This could improve clinical outcome in 100,000's of patients with ATM-deficient lung adenocarcinoma every year. PMID:27259260

  13. A.t.m. observations on the X u.v. emission from solar flares

    NASA Technical Reports Server (NTRS)

    Brueckner, G. E.

    1976-01-01

    The paper summarizes observations of selected solar flares made with a far-UV spectroheliograph (190-465 A) and a UV spectrograph (900-1900 A) aboard Skylab. The emission lines used in the present analysis are identified, and three events are described in detail: the flare of June 15, 1973, a small subflare observed on August 9, 1973, and the flare of January 21, 1974. Ultraviolet images of two other events are also presented in an attempt to sketch a general picture of a flare as seen in this spectral region. It is found that a small kernel seems to be the source of the primary energy release of a flare. The size, electron density, and ion temperature of a typical kernel are estimated, and it is noted that hot clouds of coronal gas at 20 million K surrounded the observed kernels. It is speculated that flare kernels might be very thin channels through which high-energy particles, originating in deep layers, are ejected into the corona.

  14. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage.

    PubMed

    Ortega-Atienza, Sara; Wong, Victor C; DeLoughery, Zachary; Luczak, Michal W; Zhitkovich, Anatoly

    2016-01-08

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Integrated network management framework for ATM-over-ADSL service

    NASA Astrophysics Data System (ADS)

    Hong, Won-Kyu; Yoon, Sung-Sook; Hong, Seong-Ik; Kim, Dong-Il; Jung, Mun-Jo; Song, Joong-Goo

    2001-11-01

    In this paper, we describe an integrated network management system for ATM over ADSL service provisioning. There are two distinct networks of ATM and Internet. Most of routers in Internet connected with WDM. The Network Access Server (NAS) in the Internet provides the Internet access service for the ATM over ADSL subscriber. The ATM network takes the roles of backbone network for the pure ATM PVC and SVC services and the access network for the ATM over ADSL service. In order to define the generic network model that can be commonly applicable for the backbone network for pure ATM service and the access network for ATM over ADSL service taking into account the scalability, we suggest two fragments of the topological fragment and connectivity fragment to maximize the scalability in accordance with the ITU-T G.805 layering and partitioning concepts and the RM-ODP information viewpoint. In addition, we propose the distributed computational model of the ATM over ADSL network management system using the RM-ODP computational viewpoint and TMN functional decomposition of FCAPS taking into account the functional distribution and the modularity. Lastly, we describe the scenario for providing the integrated ADSL service.

  16. ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

    PubMed Central

    Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

    2016-01-01

    Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

  17. Structural characterization of AtmS13, a putative sugar aminotransferase involved in indolocarbazole AT2433 aminopentose biosynthesis.

    PubMed

    Singh, Shanteri; Kim, Youngchang; Wang, Fengbin; Bigelow, Lance; Endres, Michael; Kharel, Madan K; Babnigg, Gyorgy; Bingman, Craig A; Joachimiak, Andrzej; Thorson, Jon S; Phillips, George N

    2015-08-01

    AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose-containing disaccharide moiety. The corresponding sugar nucleotide-based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X-ray structure at 1.50-Å resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT-I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose vs. pentose) and/or sugar C2 (deoxy vs. hydroxyl) substrate specificity.

  18. Radiobiological Characterization of Tuberous Sclerosis: a Delay in the Nucleo-Shuttling of ATM May Be Responsible for Radiosensitivity.

    PubMed

    Ferlazzo, Mélanie L; Bach-Tobdji, Mohamed Kheir Eddine; Djerad, Amar; Sonzogni, Laurène; Devic, Clément; Granzotto, Adeline; Bodgi, Larry; Bachelet, Jean-Thomas; Djefal-Kerrar, Assia; Hennequin, Christophe; Foray, Nicolas

    2017-08-07

    The tuberous sclerosis complex (TSC) syndrome is associated with numerous cutaneous pathologies (notably on the face), epilepsy, intellectual disability and developmental retardation and, overall, high occurrence of benign tumors in several organs, like angiofibromas, giant cell astrocytomas, renal angiomyolipomas, and pulmonary lymphangioleiomyomatosis. TSC is caused by mutations of either of the hamartin or tuberin proteins that are mainly cytoplasmic. Some studies published in the 1980s reported that TSC is associated with radiosensitivity. However, its molecular basis in TSC cells is not documented enough. Here, we examined the functionality of the repair and signaling of radiation-induced DNA double-strand breaks (DSB) in fibroblasts derived from TSC patients. Quiescent TSC fibroblast cells elicited abnormally low rate of recognized DSB reflected by a low yield of nuclear foci formed by phosphorylated H2AX histones. Irradiated TSC cells also presented a delay in the nucleo-shuttling of the ATM kinase, potentially due to a specific binding of ATM to mutated TSC protein in cytoplasm. Lastly, TSC fibroblasts showed abnormally high MRE11 nuclease activity suggesting genomic instability. A combination of biphosphonates and statins complemented these impairments by facilitating the nucleoshuttling of ATM and increasing the yield of recognized DSB. Our results showed that TSC belongs to the group of syndromes associated with low but significant defect of DSB signaling and delay in the ATM nucleo-shuttling associated with radiosensitivity.

  19. Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

    PubMed Central

    Köcher, Sabrina; Rieckmann, Thorsten; Rohaly, Gabor; Mansour, Wael Y.; Dikomey, Ekkehard; Dornreiter, Irena; Dahm-Daphi, Jochen

    2012-01-01

    Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells. PMID:22730303

  20. ATM Expression Predicts Veliparib and Irinotecan Sensitivity in Gastric Cancer by Mediating P53-Independent Regulation of Cell Cycle and Apoptosis.

    PubMed

    Subhash, Vinod Vijay; Tan, Shi Hui; Yeo, Mei Shi; Yan, Fui Leng; Peethala, Praveen C; Liem, Natalia; Krishnan, Vaidehi; Yong, Wei Peng

    2016-12-01

    Identification of synthetically lethal cellular targets and synergistic drug combinations is important in cancer chemotherapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signaling and cell-cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its implications as a predictive biomarker for cancer chemotherapy remain unexplored. The present study evaluated ATM-induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, veliparib (ABT-888) and irinotecan (CPT-11), respectively. ATM expression was detected in a panel of gastric cell lines, and the IC50 against each inhibitors was determined. The combinatorial effect of ABT-888 and CPT-11 in gastric cancer cells was also determined both in vitro and in vivo ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy; however, they showed a higher therapeutic effect with ABT-888 and CPT-11 combinatorial therapy. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell-cycle progression and apoptosis in a P53-independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cells to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM-dependent regulation of cellular processes. Mol Cancer Ther; 15(12); 3087-96. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Alterations of ATM and CADM1 in chromosomal 11q22.3-23.2 region are associated with the development of invasive cervical carcinoma.

    PubMed

    Mazumder Indra, Dipanjana; Mitra, Sraboni; Roy, Anup; Mondal, Ranajit Kumar; Basu, Partha Sarathi; Roychoudhury, Susanta; Chakravarty, Runu; Panda, Chinmay Kumar

    2011-12-01

    To understand the importance of chr11q22.3-23.2 region in the development of cervical cancer, we have studied the genetic and epigenetic alterations of the candidate genes ATM, PPP2R1B, SDHD and CADM1 in cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CACX) samples. Our study revealed low expression and high alterations (methylation/deletion) (55-59%) of ATM and CADM1 genes along with poor patient outcome. The alterations of ATM and CADM1 are associated with the progression of tumor from CIN to Stage I/II, thus implying their role in early invasiveness. The two genes, PPP2R1B and SDHD, lying in between ATM and CADM1, have low frequency of alterations, and majority of the alterations are in CACX samples, indicating that their alterations might be associated with disease progression. Expressions (mRNA/protein) of the genes showed concordance with their molecular alterations. Significant co-alteration of ATM and CADM1 points to their synergic action for the development of CACX. Mutation is, however, a rare phenomenon for inactivation of ATM. Association between the alteration of ATM and CHEK1 and poor survival of the patients having co-alterations of ATM and CHEK1 points to the DNA damage response pathway disruption in development of CACX. Thus, our data suggest that inactivation of ATM-CHEK1-associated DNA damage response pathway and CADM1-associated signaling network might have an important role in the development of CACX.

  2. BNL-NYSERNet ATM project report

    SciTech Connect

    O`Connor, M.; Peskin, A.; Rabinowitz, G.

    1997-07-01

    In 1994, Brookhaven National Laboratory (BNL) and NYSERNet, Incorporated embarked on a joint project to develop a prototype Asynchronous Transfer Mode (ATM) Regional Network testbed. This project was funded as a three-year effort under a Cooperative Research and Development Activity (CRADA) agreement between the parties, with half the funds being provided directly by the U.S. Department of Energy and the remainder as an in-kind contribution by NYSERNet. This report documents that effort as it comes to a close, providing an account of the original goals, the accomplishments of the projects, and the results as they might apply to the future. It is useful to remember that, when the collaboration discussions first began in 1993, it was far from certain that ATM would be the technology of choice for the then-next generation of the Internet. That, of course, has turned out to be the case, which in retrospect makes this experience particularly valuable. The investigators were not totally prescient, however, and the project changed during its duration to account for changes in technology, available infrastructure, and other circumstances.

  3. Modest increased sensitivity to radiation oncogenesis in ATM heterozygous versus wild-type mammalian cells

    NASA Technical Reports Server (NTRS)

    Smilenov, L. B.; Brenner, D. J.; Hall, E. J.

    2001-01-01

    Subpopulations that are genetically predisposed to radiation-induced cancer could have significant public health consequences. Individuals homozygous for null mutations at the ataxia telangiectasia gene are indeed highly radiosensitive, but their numbers are very small. Ataxia Telangiectasia heterozygotes (1-2% of the population) have been associated with somewhat increased radiosensitivity for some end points, but none directly related to carcinogenesis. Here, intralitter comparisons between wild-type mouse embryo fibroblasts and mouse embryo fibroblasts carrying ataxia telangiectasia mutated (ATM) null mutation indicate that the heterozygous cells are more sensitive to radiation oncogenesis than their normal, litter-matched, counterparts. From these data we suggest that Ataxia Telangiectasia heterozygotes could indeed represent a societally-significant radiosensitive human subpopulation.

  4. Modest increased sensitivity to radiation oncogenesis in ATM heterozygous versus wild-type mammalian cells

    NASA Technical Reports Server (NTRS)

    Smilenov, L. B.; Brenner, D. J.; Hall, E. J.

    2001-01-01

    Subpopulations that are genetically predisposed to radiation-induced cancer could have significant public health consequences. Individuals homozygous for null mutations at the ataxia telangiectasia gene are indeed highly radiosensitive, but their numbers are very small. Ataxia Telangiectasia heterozygotes (1-2% of the population) have been associated with somewhat increased radiosensitivity for some end points, but none directly related to carcinogenesis. Here, intralitter comparisons between wild-type mouse embryo fibroblasts and mouse embryo fibroblasts carrying ataxia telangiectasia mutated (ATM) null mutation indicate that the heterozygous cells are more sensitive to radiation oncogenesis than their normal, litter-matched, counterparts. From these data we suggest that Ataxia Telangiectasia heterozygotes could indeed represent a societally-significant radiosensitive human subpopulation.

  5. Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation.

    PubMed

    Beaumel, Sylvain; Picciocchi, Antoine; Debeurme, Franck; Vivès, Corinne; Hesse, Anne-Marie; Ferro, Myriam; Grunwald, Didier; Stieglitz, Heather; Thepchatri, Pahk; Smith, Susan M E; Fieschi, Franck; José Stasia, Marie

    2017-09-13

    NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47(phox) and p67(phox) to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Mutant IDH1 downregulates ATM and alters DNA repair and sensitivity to DNA damage independent of TET2

    PubMed Central

    Inoue, Satoshi; Li, Wanda Y.; Tseng, Alan; Beerman, Isabel; Elia, Andrew J.; Bendall, Sean C.; Lemonnier, François; Kron, Ken J.; Cescon, David W.; Hao, Zhenyue; Lind, Evan F.; Takayama, Naoya; Planello, Aline C.; Shen, Shu Yi; Shih, Alan H.; Larsen, Dana M.; Li, Qinxi; Snow, Bryan E.; Wakeham, Andrew; Haight, Jillian; Gorrini, Chiara; Bassi, Christian; Thu, Kelsie L.; Murakami, Kiichi; Elford, Alisha R.; Ueda, Takeshi; Straley, Kimberly; Yen, Katharine E.; Melino, Gerry; Cimmino, Luisa; Aifantis, Iannis; Levine, Ross L.; De Carvalho, Daniel D.; Lupien, Mathieu; Rossi, Derrick J.; Nolan, Garry P.; Cairns, Rob A.; Mak, Tak W.

    2016-01-01

    SUMMARY Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSC), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia. PMID:27424808

  7. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    PubMed Central

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  8. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    PubMed

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  9. MiR-203 inhibits tumor invasion and metastasis in gastric cancer by ATM.

    PubMed

    Zhou, Ping; Jiang, Nan; Zhang, Guo-Xia; Sun, Qing

    2016-08-01

    Gastric cancer is one of the most common malignancies in the world. A number of miRNAs are aberrantly expressed during the progression of gastric cancer. In this study, we aimed to investigate the role of miR-203 in the invasion and metastasis of gastric cancer and the potential mechanism of the effect of miR-203 on the tumor progression of gastric cancer. Our results showed that miR-203 was significantly downregulated in gastric cancer tissues and cells, while ataxia telangiectasia mutated kinase (ATM) was upregulated in gastric cancer tissues and cells and was directly regulated by miR-203. Ectopic overexpression of miR-203 inhibited the colony formation, migration, and invasion of gastric cancer cells. In addition, miR-203 overexpression significantly suppressed the protein level of Snail and obviously promoted the protein level of E-cadherin in gastric cancer cells. ATM knockdown phenocopied the effect of miR-203 overexpression. These results suggested that miR-203 suppressed the migration and invasion of gastric cancer through regulating the level of ATM-mediated-Snail and E-cadherin. MiR-203 might be a novel therapeutic strategy for the treatment of gastric cancer. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification

    PubMed Central

    Bassing, Craig H.; Sanda, Takaomi; Brush, James W.; Patel, Harin; Goff, Peter H.; Murphy, Michael M.; Tepsuporn, Suprawee; Gatti, Richard A.; Look, A. Thomas

    2010-01-01

    Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells. PMID:20566716

  11. Initiation of the ATM-Chk2 DNA damage response through the base excision repair pathway.

    PubMed

    Chou, Wen-Cheng; Hu, Ling-Yueh; Hsiung, Chia-Ni; Shen, Chen-Yang

    2015-08-01

    The DNA damage response (DDR) is activated by various genotoxic stresses. Base lesions, which are structurally simple and predominantly fixed by base excision repair (BER), can trigger the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway, a DDR component. How these lesions trigger DDR remains unclear. Here we show that, for alkylation damage, methylpurine-DNA glycosylase (MPG) and apurinic/apyrimidinic endonuclease 1, both of which function early in BER, are required for ATM-Chk2-dependent DDR. In addition, other DNA glycosylases, including uracil-DNA glycosylase and 8-oxoguanine glycosylase, which are involved in repairing deaminated bases and oxidative damage, also induced DDR. The early steps of BER therefore play a vital role in modulating the ATM-Chk2 DDR in response to base lesions, facilitating downstream BER processing for repair, in which the formation of a single-strand break was shown to play a critical role. Moreover, MPG knockdown rescued cell lethality, its overexpression led to cell death triggered by DNA damage and, more interestingly, higher MPG expression in breast and ovarian cancers corresponded with a greater probability of relapse-free survival after chemotherapy, underscoring the importance of glycosylase-dependent DDR. This study highlights the crosstalk between BER and DDR that contributes to maintaining genomic integrity and may have clinical applications in cancer therapy.

  12. ATM Technology Adoption in U.S. Campus Networking.

    ERIC Educational Resources Information Center

    Yao, Engui; Perry, John F.; Anderson, Larry S.; Brook, R. Dan; Hare, R. Dwight; Moore, Arnold J.; Xu, Xiaohe

    This study examined the relationships between ATM (asynchronous transfer mode) adoption in universities and four organizational variables: university size, type, finances, and information processing maturity. Another purpose of the study was to identify the current status of ATM adoption in campus networking. Subjects were university domain LAN…

  13. ATM LAN Emulation: Getting from Here to There.

    ERIC Educational Resources Information Center

    Learn, Larry L., Ed.

    1995-01-01

    Discusses current LAN (local area network) configuration and explains ATM (asynchronous transfer mode) as the future telecommunications transport. Highlights include LAN emulation, which enables the interconnection of legacy LANs and the new ATM environment; virtual LANs; broadcast servers; and standards. (LRW)

  14. Multimedia Applications in Heterogeneous Internet/ATM Environments.

    ERIC Educational Resources Information Center

    Wolf, Lars C.

    1999-01-01

    Discussion of multimedia systems focuses on interaction approaches for the quality of service (QoS) architectures developed for the Internet and for asynchronous transfer mode (ATM). Highlights include interactions, videoconferencing, video on demand, a comparison of the ATM and IntServ QoS architectures, interaction models, and subordination…

  15. ATM: The Key To Harnessing the Power of Networked Multimedia.

    ERIC Educational Resources Information Center

    Gross, Rod

    1996-01-01

    ATM (Asynchronous Transfer Mode) network technology handles the real-time continuous traffic flow necessary to support desktop multimedia applications. Describes network applications already used: desktop video collaboration, distance learning, and broadcasting video delivery. Examines the architecture of ATM technology, video delivery and sound…

  16. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  17. Amplification of a novel c-Kit activating mutation Asn(822)-Lys in the Kasumi-1 cell line: a t(8;21)-Kit mutant model for acute myeloid leukemia.

    PubMed

    Beghini, Alessandro; Magnani, Ivana; Ripamonti, Carla B; Larizza, Lidia

    2002-01-01

    A subset of AML-M2/M4Eo patients has been shown to carry c-kit mutations suggesting that myelomonoblastic leukemia cells, disrupting core binding factor through t(8;21) or inv(16) chromosomal rearrangements, have a common differentiation stage suitable to c-kit mutation. In rare core binding factor leukemia patients an increased dosage of a mutated Asp816(Tyr/Val) kit allele is achieved through nonrandom duplication of chromosome 4 where the c-kit gene is located. The c-kit gene was studied in the core binding factor leukemia cell line Kasumi-1 with t(8;21) by fluorescence in situ hybridization and mutation analysis. The dosage of Asn822(Lys) mutated allele was evaluated by fluorescence semiquantitative PCR. The correct membrane homing of KIT receptor and its activating status was analysed by immunofluorescence and Western blotting respectively. We identified in the Kasumi-1 cell line a novel Asn822(Lys) ligand-independent c-kit activating mutation and demonstrated by semiquantitative PCR that the mutated allele is about fivefold amplified compared to the normal allele. Fluorescence In Situ Hybridization analysis revealed that c-kit amplification maps to minute 4cen-q11 derived marker chromosome, often carrying duplicated signals, which are unequally distributed in the cell population. The Asn822(Lys) mutation affects a highly conserved codon within the tyrosine kinase activation loop leading, likewise the Asp(816) mutants, to constitutive ligand-independent activation of the KIT receptor. Results obtained point to the Kasumi-1 cell line as powerful in-vitro model for further investigation of altered KIT signal transduction pathways in acute myeloid leukemia with core binding factor rearrangements and a useful tool for pharmacological therapeutic targeting.

  18. ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells.

    PubMed

    Nadkarni, Aditi; Shrivastav, Meena; Mladek, Ann C; Schwingler, Paul M; Grogan, Patrick T; Chen, Junjie; Sarkaria, Jann N

    2012-12-01

    Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

  19. FPGA Based Reconfigurable ATM Switch Test Bed

    NASA Technical Reports Server (NTRS)

    Chu, Pong P.; Jones, Robert E.

    1998-01-01

    Various issues associated with "FPGA Based Reconfigurable ATM Switch Test Bed" are presented in viewgraph form. Specific topics include: 1) Network performance evaluation; 2) traditional approaches; 3) software simulation; 4) hardware emulation; 5) test bed highlights; 6) design environment; 7) test bed architecture; 8) abstract sheared-memory switch; 9) detailed switch diagram; 10) traffic generator; 11) data collection circuit and user interface; 12) initial results; and 13) the following conclusions: Advances in FPGA make hardware emulation feasible for performance evaluation, hardware emulation can provide several orders of magnitude speed-up over software simulation; due to the complexity of hardware synthesis process, development in emulation is much more difficult than simulation and requires knowledge in both networks and digital design.

  20. PTEN enhances G2/M arrest in etoposide-treated MCF‑7 cells through activation of the ATM pathway.

    PubMed

    Zhang, Ruopeng; Zhu, Li; Zhang, Lirong; Xu, Anli; Li, Zhengwei; Xu, Yijuan; He, Pei; Wu, Maoqing; Wei, Fengxiang; Wang, Chenhong

    2016-05-01

    As an effective tumor suppressor, phosphatase and tensin homolog (PTEN) has attracted the increased attention of scientists. Recent studies have shown that PTEN plays unique roles in the DNA damage response (DDR) and can interact with the Chk1 pathway. However, little is known about how PTEN contributes to DDR through the ATM-Chk2 pathway. It is well-known that etoposide induces G2/M arrest in a variety of cell lines, including MCF-7 cells. The DNA damage-induced G2/M arrest resul