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Richard A. Gatti - One of the best experts on this subject based on the ideXlab platform.

  • arginine rich cell penetrating peptide dramatically enhances amo mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum
    Human Molecular Genetics, 2011
    Co-Authors: Liutao Du, Patrick L Iversen, Refik Kayali, Carmen Bertoni, Francesca Fike, Hailiang Hu, Richard A Gatti, Richard A. Gatti
    Abstract:

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

  • Stable Brain ATM Message and Residual Kinase-Active ATM Protein in Ataxia-Telangiectasia
    The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011
    Co-Authors: Jianmin Chen, Richard A. Gatti, Harry V. Vinters, Karl Herrup
    Abstract:

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

  • Immunoassay to Measure Ataxia-Telangiectasia Mutated Protein in Cellular Lysates
    Clinical Chemistry, 2004
    Co-Authors: Anthony W. Butch, Shareef A. Nahas, Helen H. Chun, Richard A. Gatti
    Abstract:

    Background: Ataxia-telangiectasia (A-T) is a neurologic disorder caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. A clinical diagnosis of A-T is confirmed by radiosensitivity testing and immunoblotting for ATM Protein. Because both of these tests have long turnaround times (≥3 months), we developed a rapid immunoassay to measure ATM Protein and determined its sensitivity and specificity for diagnosing A-T. Methods: Recombinant ATM Protein was used for standardization. Lysates of lymphoblastoid cell lines (LCLs) and peripheral blood mononuclear cells (PBMCs) from A-T patients, controls, and A-T heterozygotes were tested for ATM Protein by immunoassay. Results: Between-run imprecision (CV) was ≤13%. Nuclear lysates from control LCLs and PBMCs had ATM Protein concentrations of 49–610 μg/L and 48–943 μg/L, respectively. ATM Protein was not detectable in LCL nuclear lysates from 18 of 21 A-T patients. The three remaining A-T patients had trace amounts of ATM Protein, which was confirmed on immuoblots. ATM Protein was also detectable in whole-cell lysates from 4 × 106 cells at concentrations of 64–463 μg/L and 42–444 μg/L for control LCLs and PBMCs, respectively. A-T heterozygotes had ATM Protein concentrations of 52–98 μg/L. ATM Protein was stable in PBMCs stored for 1 month at −70 °C, but rapidly decreased after 1 day in unprocessed blood. Conclusions: This ATM Protein immunoassay can be used to confirm a diagnosis of A-T in 2 days on small numbers of PBMCs and can potentially identify A-T carriers and individuals at increased risk for cancer.

  • ATM Protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation.
    Biochemical and biophysical research communications, 2004
    Co-Authors: Helen H. Chun, Robert B. Cary, Fredrick Lansigan, Julian P. Whitelegge, David J. Rawlings, Richard A. Gatti
    Abstract:

    The ataxia-telangiectasia mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM Protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM Protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant Protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date.

  • improved diagnostic testing for ataxia telangiectasia by immunoblotting of nuclear lysates for ATM Protein expression
    Molecular Genetics and Metabolism, 2003
    Co-Authors: Helen H. Chun, Xia Sun, Shareef A. Nahas, Sharon N Teraoka, Chihhung Lai, Patrick Concannon, Richard A. Gatti
    Abstract:

    Abstract The laboratory diagnosis of ataxia–telangiectasia (A–T) currently relies upon measurement of serum alphafetoProtein (AFP) and cellular sensitivity to ionizing radiation. A previous report suggests that immunoblotting of whole cell lysates from lymphoblastoid cell lines (LCLs) might be informative for diagnosis. To further evaluate this possibility, and improve sensitivity, we performed immunoblotting for ATM Protein on nuclear lysates of 71 consecutive radiosensitive LCLs that were established from patients with clinical features suggestive of A–T. Fifty-two LCLs (73%) contained no detectable ATM Protein, with a representative sample ( N =25) testing negative for ATM kinase activity, having at least one ATM mutation, and having elevated AFP levels; these results confirmed the diagnosis. Seventeen LCLs (24%) expressed intermediate or normal levels of ATM Protein and exhibited normal ATM kinase activity; follow-up studies failed to detect ATM mutations and AFP levels were normal in all but three. Of the remaining two radiosensitive LCLs, one had 35% of normal Protein with normal kinase activity and no ATM mutations. The other LCL had 9% of normal Protein, with intermediate levels of kinase activity, a homozygous missense ATM mutation, and elevated AFP. Our data suggest that it is very uncommon to encounter bonafide A–T patients with more than trace amounts of ATM Protein. We conclude that immunoblotting for ATM Protein is of higher specificity for diagnosing A–T than radiosensitivity testing. In addition, we have documented in vitro radiosensitivity in other patients who share some clinical features with A–T.

Luciana Chessa - One of the best experts on this subject based on the ideXlab platform.

  • ataxia telangiectasia phenotype genotype studies of ATM Protein expression mutations and radiosensitivity
    Molecular Genetics and Metabolism, 2000
    Co-Authors: Sara G Beckercatania, Gang Chen, Mee Jeong Hwang, Zhijun Wang, Xia Sun, Ozden Sanal, Luciana Chessa, Eva Y.-h. P. Lee, Eva Bernatowskamatuszkiewicz, Richard A. Gatti
    Abstract:

    Previous studies on a limited number of ataxia-telangiectasia (A-T) patients with detectable levels of intracellular ATM Protein have suggested a genotype/phenotype correlation. We sought to elucidate this possible correlation by comparing ATM Protein levels with mutation types, radiosensitivity, and clinical phenotype. In this study, Western blot analysis was used to measure ATM Protein in lysates of lymphoblastoid cell lines (LCLs) from 123 unrelated A-T patients, 10 A-T heterozygotes, and 10 patients with phenotypes similar to A-T. Our Western blot protocol can detect the presence of ATM Protein in as little as 1 microg of total Protein; at least 25 microg of Protein was tested for each individual. ATM Protein was absent in 105 of the 123 patients (85%); most of these patients had truncating mutations. The remaining subset of 18 patients (15%) had reduced levels of normal-sized ATM Protein; missense mutations were more common in this subset. We used a colony survival assay to characterize the phenotypic response of the LCLs to radiation exposure; patients with or without detectable ATM Protein were typically radiosensitive. Nine of 10 A-T heterozygotes also had reduced expression of ATM, indicating that both alleles contribute to ATM Protein production. These data suggest that although ATM-specific mRNA is abundant in A-T cells, the abnormal ATM Protein is unstable and is quickly targeted for degradation. We found little correlation between level of ATM Protein and the type of underlying mutation, the clinical phenotype, or the radiophenotype.

  • ATAXIA-TELANGIECTASIA: PHENOTYPE/GENOTYPE STUDIES OF ATM Protein EXPRESSION, MUTATIONS, AND RADIOSENSITIVITY
    Molecular genetics and metabolism, 2000
    Co-Authors: Sara G. Becker-catania, Gang Chen, Mee Jeong Hwang, Zhijun Wang, Xia Sun, Ozden Sanal, Eva Bernatowska-matuszkiewicz, Luciana Chessa, Eva Y.-h. P. Lee, Richard A. Gatti
    Abstract:

    Previous studies on a limited number of ataxia-telangiectasia (A-T) patients with detectable levels of intracellular ATM Protein have suggested a genotype/phenotype correlation. We sought to elucidate this possible correlation by comparing ATM Protein levels with mutation types, radiosensitivity, and clinical phenotype. In this study, Western blot analysis was used to measure ATM Protein in lysates of lymphoblastoid cell lines (LCLs) from 123 unrelated A-T patients, 10 A-T heterozygotes, and 10 patients with phenotypes similar to A-T. Our Western blot protocol can detect the presence of ATM Protein in as little as 1 microg of total Protein; at least 25 microg of Protein was tested for each individual. ATM Protein was absent in 105 of the 123 patients (85%); most of these patients had truncating mutations. The remaining subset of 18 patients (15%) had reduced levels of normal-sized ATM Protein; missense mutations were more common in this subset. We used a colony survival assay to characterize the phenotypic response of the LCLs to radiation exposure; patients with or without detectable ATM Protein were typically radiosensitive. Nine of 10 A-T heterozygotes also had reduced expression of ATM, indicating that both alleles contribute to ATM Protein production. These data suggest that although ATM-specific mRNA is abundant in A-T cells, the abnormal ATM Protein is unstable and is quickly targeted for degradation. We found little correlation between level of ATM Protein and the type of underlying mutation, the clinical phenotype, or the radiophenotype.

  • Defective Control of Apoptosis and Mitotic Spindle Checkpoint in Heterozygous Carriers of ATM Mutations
    Cancer research, 1999
    Co-Authors: Teruko Shigeta, Luciana Chessa, Domenico Delia, Masatoshi Takagi, Satoshi Iwata, Yusuke Kanke, Minoru Asada, Mariko Eguchi, Shuki Mizutani
    Abstract:

    Ataxia telangiectasia (AT) carrier-derived lymphoblastoid cell lines (AT-LCLs/hetero) with suboptimal ATM Protein expression were examined for the regulation of radiosensitivity, apoptosis, and mitotic spindle checkpoint in response to DNA-damaging agents. Although AT-LCLs/hetero showed intermediate radiation sensitivity, as determined by clonogenic assay, they were resistant to early-onset apoptosis, as much as AT patient-derived LCLs (AT-LCLs/homo). Furthermore, two of three AT-LCLs/hetero showed defective mitotic spindle checkpoint control in response to X-ray irradiation, which is a recently characterized biological feature in AT-LCLs/homo. Our findings indicate that carriers of ATM mutation have biological abnormalities due to haploinsufficiency of ATM Protein or dominant-negative effect of mutant ATM Protein. Thus, although it is still controversial whether ATM mutation carriers are at higher risk for cancer during adulthood, our findings based on in vitro biological indicators support the notion that at least some of such carriers are at a higher risk for cancer development than those without ATM mutation. Our findings may help to reevaluate epidemiological studies on cancer susceptibility in AT carriers.

  • The ataxia-telangiectasia gene product, a constitutively expressed nuclear Protein that is not up-regulated following genome damage
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Kevin D. Brown, Luciana Chessa, Yosef Shiloh, Yael Ziv, Sunanda N. Sadanandan, Francis S. Collins, Danilo A. Tagle
    Abstract:

    The product of the ataxia-telangiectasia gene (ATM) was identified by using an antiserum developed to a peptide corresponding to the deduced amino acid sequence. The ATM Protein is a single, high-molecular weight Protein predominantly confined to the nucleus of human fibroblasts, but is present in both nuclear and microsomal fractions from human lymphoblast cells and peripheral blood lymphocytes. ATM Protein levels and localization remain constant throughout all stages of the cell cycle. Truncated ATM Protein was not detected in lymphoblasts from ataxia-telangiectasia patients homozygous for mutations leading to premature Protein termination. Exposure of normal human cells to γ-irradiation and the radiomimetic drug neocarzinostatin had no effect on ATM Protein levels, in contrast to a noted rise in p53 levels over the same time interval. These findings are consistent with a role for the ATM Protein in ensuring the fidelity of DNA repair and cell cycle regulation following genome damage.

  • Predominance of Null Mutations in Ataxia-Telangiectasia
    Human molecular genetics, 1996
    Co-Authors: Shlomit Gilad, Luciana Chessa, Yael Ziv, Rami Khosravi, Dganit Shkedy, Tamar Uziel, Kinneret Savitsky, Galit Rotman, Sara Smith, Timothy J. Jorgensen
    Abstract:

    Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa Protein with a PI 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM Protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM Protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the PI 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM Protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.

Martin F. Lavin - One of the best experts on this subject based on the ideXlab platform.

  • Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Mark E. Graham, Martin F. Lavin, Sergei Kozlov
    Abstract:

    ATM (ataxia-telangiectasia mutated) Protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of Proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treATMent strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  • Identification of a Novel Protein Kinase Mediating Akt Survival Signaling to the ATM Protein
    The Journal of biological chemistry, 2002
    Co-Authors: Atsushi Suzuki, Martin F. Lavin, Gen-ichi Kusakai, Atsuhiro Kishimoto, Tsutomu Ogura, Hiroyasu Esumi
    Abstract:

    We identified a novel human AMP-activated Protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK, and SNARK, respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595-600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM Protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation.

  • Rapid radiation-induction of ATM Protein levels in situ.
    Pathology, 2001
    Co-Authors: Zhiming Fang, Martin F. Lavin, John H. Kearsley, C S Lee, Maria Sarris, Dedee F. Murrell, Katherine E Keating, Raymond A. Clarke
    Abstract:

    Ataxia-telangiectasia (A-T) is characterised by hypersensitivity to ionising radiation (IR), immunodeficiency, neurodegeneration and predisposition to malignancy. Mutations in the A-T gene (ATM) often result in reduced levels of ATM Protein and/or compromise ATM function. IR induced DNA damage is known to rapidly upregulate ATM kinase activity/phosphorylation events in the control of cell cycle progression and other processes. Variable expression of ATM levels in different tissues and its upregulation during cellular proliferation indicate that the level of ATM is also regulated by mechanisms other than gene mutation. Here, we report on the IR induction of ATM Protein levels within a number of different cell types and tissues. Induction had begun within 5 min and peaked within 2 h of exposure to 2 Gy of IR, suggesting a rapid post-translational mechanism. Low basal levels of ATM Protein were more responsive to IR induction compared to high ATM levels in the same cell type. Irradiation of fresh skin biopsies led to an average three-fold increase in ATM levels while immunohistochemical analyses indicated low expressing cells within the basal layer with ten-fold increases in ATM levels following IR. ATM high expressing lymphoblastoid cell lines (LCLs) which were initially resistant to the radiation-induction of ATM levels also became responsive to IR after ATM antisense expression was used to reduce the basal levels of the Protein. These results demonstrate that ATM is present in variable amounts in different tissue/cell types and where basal levels are low ATM levels can be rapidly induced by IR to saturable levels specific for different cell types. ATM radiation-induction is a sensitive and rapid radioprotective response that complements the IR mediated activation of ATM.

  • Epidermal Growth Factor Sensitizes Cells to Ionizing Radiation by Down-regulating Protein Mutated in Ataxia-Telangiectasia
    The Journal of biological chemistry, 2000
    Co-Authors: Nuri Gueven, Kum Kum Khanna, Dianne Watters, Katherine E Keating, Toshiyuki Fukao, P. Chen, Peter H. Rodemann, Martin F. Lavin
    Abstract:

    Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent Protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreATMent with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM Protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM Protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM Protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

  • Immunoblot analysis for laboratory diagnosis of ataxia-telangiectasia: use of Epstein-Barr virus-transformed or phytohemagglutinin-stimulated lymphoblasts for detection of ATM Protein.
    Journal of investigational allergology & clinical immunology, 2000
    Co-Authors: Toshiyuki Fukao, Dianne Watters, Martin F. Lavin, T Yoshida, Hideo Kaneko, X Q Song, H Tashita, Takahide Teramoto, Ryosuke Inoue, Naomi Kondo
    Abstract:

    Ataxia-telangiectasia (A-T) is a genetic disorder characterized by a progressive ataxia, immunodeficiency, neurological abnormalities, hypersensitivity to ionizing radiation, and predisposition to cancer. The gene responsible for A-T (ATM) has been cloned and shown to code for a 350 kDa polypeptide containing 3,056 amino acid residues. Detection of ATM mutations for laboratory diagnosis of A-T is laborious and not practical, unless there are common mutations in a population. We describe here immunoblot analysis for the detection of ATM in seven Japanese A-T patients from five families and in controls using ATM3BA antibody. ATM Protein was routinely and clearly detected in Epstein-Barr virus (EBV)-transformed or phytohemagglutinin (PHA)-stimulated lymphoblasts from controls. However, it could not be detected consistently in unstimulated peripheral blood mononuclear cells (PBMCs) from controls. We also detected ATM Protein in control fibroblasts, but the background was relatively higher than in control lymphoblasts. ATM Protein was not detected or dramatically decreased in EBV-transformed lymphoblasts from all seven patients tested and in fibroblasts from one patient. Immunoblot analysis using EBV-transformed or PHA-stimulated lymphoblasts represents a useful approach for laboratory diagnosis for A-T. The latter is especially preferable since it takes only 3 days to obtain sufficient cells for analysis.

Seema Kashyap - One of the best experts on this subject based on the ideXlab platform.

  • Prognostic relevance of ATM Protein in uveal melanoma and its association with clinicopathological factors.
    International journal of clinical oncology, 2019
    Co-Authors: J. Jha, M.k. Singh, Lata Singh, Neelam Pushker, Mandeep S Bajaj, Seema Sen, Seema Kashyap
    Abstract:

    Uveal melanoma (UM) is an intraocular malignancy commonly arising from choroid which can cause visual loss or metastasis. Ataxia-telangiectasia mutated (ATM) Protein is an activator of DNA damage response and its role in uveal melanoma (UM) is still unexplored. Therefore, the study aims to detect the expression and localization of ATM Protein and its association with clinicopathological parameters Expression of nuclear ATM (nATM) was investigated on 69 formalin fixed paraffin embedded choroidal melanoma samples by immunohistochemistry and validated by western blotting. Results were then correlated with clinical and histopathological parameters. Prognostic significance was determined by the Kaplan–Meier analysis and the multivariate analysis by Cox’s hazard proportional method. Loss of nATM was observed in 65% of cases, which was statistically significant with the reduced disease-free survival (p = 0.042). This loss was more frequently found in cases with high-risk histopathological factors like epithelioid cell type, tumor infiltrating lymphocytes and high pigmentation which might help in the progression of melanoma. On multivariate analysis, extraocular spread and loss of nATM were found to be independent prognostic factors (p 

  • prognostic relevance of ATM Protein in uveal melanoma and its association with clinicopathological factors
    International Journal of Clinical Oncology, 2019
    Co-Authors: J. Jha, M.k. Singh, Lata Singh, Neelam Pushker, Mandeep S Bajaj, Seema Sen, Seema Kashyap
    Abstract:

    Uveal melanoma (UM) is an intraocular malignancy commonly arising from choroid which can cause visual loss or metastasis. Ataxia-telangiectasia mutated (ATM) Protein is an activator of DNA damage response and its role in uveal melanoma (UM) is still unexplored. Therefore, the study aims to detect the expression and localization of ATM Protein and its association with clinicopathological parameters Expression of nuclear ATM (nATM) was investigated on 69 formalin fixed paraffin embedded choroidal melanoma samples by immunohistochemistry and validated by western blotting. Results were then correlated with clinical and histopathological parameters. Prognostic significance was determined by the Kaplan–Meier analysis and the multivariate analysis by Cox’s hazard proportional method. Loss of nATM was observed in 65% of cases, which was statistically significant with the reduced disease-free survival (p = 0.042). This loss was more frequently found in cases with high-risk histopathological factors like epithelioid cell type, tumor infiltrating lymphocytes and high pigmentation which might help in the progression of melanoma. On multivariate analysis, extraocular spread and loss of nATM were found to be independent prognostic factors (p < 0.05). Our data suggest that loss of nATM Protein might serve as a poor prognostic marker in the pathogenesis of uveal melanoma which may lead to increased risk of metastasis.

Sergei Kozlov - One of the best experts on this subject based on the ideXlab platform.

  • Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Mark E. Graham, Martin F. Lavin, Sergei Kozlov
    Abstract:

    ATM (ataxia-telangiectasia mutated) Protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of Proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treATMent strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  • ATM Protein kinase: the linchpin of cellular defenses to stress
    Cellular and Molecular Life Sciences, 2011
    Co-Authors: Shahzad Bhatti, Sergei Kozlov, Ammad Ahmad Farooqi, Ali Naqi, Martin Lavin, Kum Kum Khanna
    Abstract:

    ATM is the most significant molecule involved in monitoring the genomic integrity of the cell. Any damage done to DNA relentlessly challenges the cellular machinery involved in recognition, processing and repair of these insults. ATM kinase is activated early to detect and signal lesions in DNA, arrest the cell cycle, establish DNA repair signaling and faithfully restore the damaged chromatin. ATM activation plays an important role as a barrier to tumorigenesis, metabolic syndrome and neurodegeneration. Therefore, studies of ATM-dependent DNA damage signaling pathways hold promise for treATMent of a variety of debilitating diseases through the development of new therapeutics capable of modulating cellular responses to stress. In this review, we have tried to untangle the complex web of ATM signaling pathways with the purpose of pinpointing multiple roles of ATM underlying the complex phenotypes observed in AT patients.

  • Low levels of ATM in breast cancer patients with clinical radiosensitivity
    Genome Integrity, 2010
    Co-Authors: Zhiming Fang, Dedee F. Murrell, Sergei Kozlov, Michael J Mckay, Rick Woods, Geoff Birrell, Carl N Sprung, Kiran Wangoo, Linda Teng, John H. Kearsley
    Abstract:

    Background and Purpose Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) Protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM Protein. Results Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM Protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM Protein levels < 55% compared to the mean for non-reactor controls. Conclusions ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM Protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

  • Low levels of ATM in breast cancer patients with clinical radiosensitivity
    Genome integrity, 2010
    Co-Authors: Zhiming Fang, Dedee F. Murrell, Geoff W. Birrell, Sergei Kozlov, Michael J Mckay, Carl N Sprung, Kiran Wangoo, Linda Teng, Rick G. Woods, John H. Kearsley
    Abstract:

    Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) Protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM Protein. Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM Protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM Protein levels < 55% compared to the mean for non-reactor controls. ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM Protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

  • cellular localisation of the ataxia telangiectasia ATM gene product and discrimination between mutated and normal forms
    Oncogene, 1997
    Co-Authors: Dianne Watters, Kum Kum Khanna, Magtouf Gatei, Padmini Kedar, Heather Beamish, Geoffrey Birrell, Kevin Spring, Deborah J Stenzel, Karen Hobson, Sergei Kozlov
    Abstract:

    The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted Protein sequence detected a similar to 350 kDa Protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM Protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation, We also provide evidence that ATM Protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM Protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.