Ataxia Telangiectasia Mutated Protein Research Articles

B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection.

Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection, as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in a host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses in vitro, suggesting that ATM is proviral in the context of infected cell cultures. The proviral role of ATM is also evident in vivo, as myeloid-specific ATM expression facilitates MHV68 reactivation during the establishment of viral latency. In order to better understand the complex relationship between host ATM and gammaherpesvirus infection, we depleted ATM specifically in B cells, a cell type critical for chronic gammaherpesvirus infection. B cell-specific ATM deficiency attenuated the establishment of viral latency due to compromised differentiation of ATM-deficient B cells. Further, we found that during long-term infection, peritoneal B-1b, but not related B-1a, B cells display the highest frequency of gammaherpesvirus infection. While ATM expression did not affect gammaherpesvirus tropism for B-1 B cells, B cell-specific ATM expression was necessary to support viral reactivation from peritoneal cells during long-term infection. Thus, our study reveals a role of ATM as a host factor that promotes chronic gammaherpesvirus infection of B cells.IMPORTANCE Gammaherpesviruses infect a majority of the human population and are associated with cancer, including B cell lymphomas. ATM is a unique host kinase that has both proviral and antiviral roles in the context of gammaherpesvirus infection. Further, there is insufficient understanding of the interplay of these roles in vivo during chronic infection. In this study, we show that ATM expression by splenic B cells is required for efficient establishment of gammaherpesvirus latency. We also show that ATM expression by peritoneal B cells is required to facilitate viral reactivation during long-term infection. Thus, our study defines a proviral role of B cell-specific ATM expression during chronic gammaherpesvirus infection.

The role of the ataxia telangiectasia mutated gene in lung cancer: recent advances in research.

Lung cancer is the leading cause of death due to cancer worldwide. It is estimated that approximately 1.2 million new cases of lung cancer are diagnosed each year. Early detection and treatment are crucial for improvements in both prognosis and quality of life of lung cancer patients. The ataxia telangiectasia mutated (ATM) gene is a cancer-susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. It has recently been shown to play an important role in the development of lung cancer. The main functions of the ATM gene and protein includes participation in cell cycle regulation, and identification and repair of DNA damage. ATM gene mutation can lead to multiple system dysfunctions as well as a concomitant increase in tumor tendency. In recent years, many studies have indicated that single nucleotide polymorphism of the ATM gene is associated with increased incidence of lung cancer. At the same time, the ATM gene and its encoding product ATM protein predicts the response to radiotherapy, chemotherapy, and prognosis of lung cancer, thus suggesting that the ATM gene may be a new potential target for the diagnosis and treatment of lung cancer.

In Vivo and In Vitro Effects of ATM/ATR Signaling Pathway on Proliferation, Apoptosis, and Radiosensitivity of Nasopharyngeal Carcinoma Cells.

The study investigated the ability of ataxia-telangiectasia mutated (ATM)/Rad3-related (ATR) signaling pathway to influence the proliferation, apoptosis, and radiosensitivity of nasopharyngeal carcinoma (NPC) cells. NPC tissues and corresponding adjacent normal tissues were collected from 143 NPC patients. The NPC CNE2 cells were assigned into a control group, X-ray group, CGK-733 group, and X-ray+CGK-733 group. The mRNA levels of ATM and ATR were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) and the protein levels of ATM and ATR using western blotting. The positive expression of ATM and ATR in tissues and nude mouse tumor tissues was determined by immunohistochemistry. Cell proliferation, migration, invasion, and apoptosis rates were analyzed by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, scratch test, transwell assay, and flow cytometry, respectively. A nude mouse model of NPC was established to observe tumor volume and growth. The mRNA levels of ATR and ATM and the expression of ATR and ATM protein in NPC tissues were significantly higher than those in adjacent normal tissues. The colony formation assay showed that the colony-forming rate decreased, showing radiation dose-dependent and CGK-733 concentration-dependent manners. Expression of ATM, ATR, Chk1, and Chk2 was evidently increased in the X-ray, CGK-733, and X-ray+CGK-733groups compared with the control group, and the aforementioned expression was highest in the X-ray+CGK-733 group among the four groups. The cell proliferation, invasion, and migration were decreased, tumor volume decreased and cell apoptosis increased in the X-ray, CGK-733, and X-ray+CGK-733 groups compared with the control group; the X-ray+CGK-733 group exhibited lowest cell proliferation, invasion and migration, smallest tumor volume, and highest cell apoptosis among the four groups. Inhibition of ATM/ATR signaling pathway reduces proliferation and enhances apoptosis and radiosensitivity of NPC cells.

Abstract 3807: ATM protein loss and clinical outcomes with platinum chemotherapy in patients with advanced solid tumors

Abstract Background Loss of ataxia telangiectasia mutated (ATM), a key protein regulating DNA damage repair signaling, appears to sensitize tumors to platinum chemotherapy in preclinical studies. In this study, the incidence of ATM loss in patients with advanced solid tumors and clinical outcomes with platinum chemotherapy was analyzed. Methods Archival tumor and biopsy samples of primary or metastatic sites of patients treated in a phase I trial unit were retrieved and ATM immunohistochemistry (IHC), as well as targeted next generation sequencing (NGS) was performed. Analytic validation and assay optimization of rabbit monoclonal antibody [Y170] to ATM (Abcam ab32420) was undertaken using known positive (VCaP xenograft material and GM14680 cell pellet) and negative (GM01526 cell pellet) controls. IHC slides were assessed by a trained pathologist for nuclear staining intensity of ATM and semi-quantitatively scored. ATM loss was defined as a nuclear H score of ≤10. Clinical data were collected retrospectively from case records. Duration on platinum therapy was defined as time from start of first platinum therapy to time to subsequent therapy (adjuvant platinum excluded). Results Overall, 679 samples of 587 patients were tested for ATM IHC, of which 517 samples had NGS performed; 5% of patients (n = 31) had ATM IHC loss, and 5% (n = 27) had somatic ATM mutations detected by NGS (Table 1). In 70 matched primary/metastatic or different metastatic sites, concordance of ATM IHC loss occurred in 99% (n = 69; 7 with ATM IHC loss) of samples. Of the colorectal cancer cohort (n = 223), duration on platinum therapy was significantly longer for the ATM loss group (11 vs 8 months, HR: 0.57, 95% CI: 0.35 to 0.93, p = 0.04). Discussion Loss of ATM expression was detected among 5% of this cohort of advanced solid cancers, primarily in breast and gastrointestinal tumors. Sensitivity to platinum therapy is significantly higher in ATM loss colorectal cancer. Further prospective studies with larger cohorts are required to validate these findings. ATM IHC and ATM mutation by NGS by tumor groupTumor typesTotalATM IHC loss (%)ATM mutation (%)Concomitant ATM loss and ATM mutationConcomitant TP53 mutation with ATM IHC loss or mutationTotalMisense nFrameshift nTruncating n(as % of those with ATM IHC loss)(as % of those with ATM IHC loss/mutation)Colorectal22316(7)9(4)544 (25)10 (48)Gynaecological1051(1)7(7)4125 (63)Thoracic801(1)3(4)211 (100)2 (67)Breast448(18)4(9)223 (38)4 (44)Upper GI372(5)1(3)12 (67)Hepatobiliary221(5)1(5)11 (50)Others762(3)2(3)21 (50)Total58731(5)27(5)14 (52)3 (11)10 (37)9 (29)24 (49) Citation Format: Raghav Sundar, Susana Miranda, Suzanne Carreira, Maxime Chénard-Poirier, Daniel Nava Rodrigues, Ines Figueiredo, Claudia Bertan, Wei Yuan, Desamparados Roda Perez, Ana Ferreira, Nina Tunariu, Joaquin Mateo, Johann de Bono. ATM protein loss and clinical outcomes with platinum chemotherapy in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3807. doi:10.1158/1538-7445.AM2017-3807

Unspecific white matter hyperintensity in T2-weighted imaging and dystonia as the first manifestations of ataxia telangiectasia

Objective: Ataxia telangiectasia (AT) is an autosomal recessive genomic instability syndrome characterized by cerebellar ataxia, immunodeficiency and cancer predisposition. Neurodegeneration in AT is closely associated with the absence or partial lack of the ataxia telangiectasia-mutated (ATM) kinase. ATM is a central player in maintaining cellular homeostasis. Magnetic resonance imaging (MRI) studies are usually without pathology in early infancy; in childhood, cerebellar atrophy and, in the second decade of life, hyperintensive lesions in the white matter occur. We present the first case in a toddler of genetically confirmed AT which mimics a leucencephalopathy. Methods: The diagnostic work-up was demonstrated and the clinical phenotype was described in 1 male patient with genetically confirmed AT. A salient neurological history including a video documentation of the treatment progress was obtained. Results: Patient presented firstly in our hospital at 18 months of age while walking, standing and sitting without support was not possible due to severe trunk dystonia and axillar muscular hypotonia. First symptom was recognized at 11 months of age with unstable sitting. MRI study showed white matter hyperintensity periventricular and parietal in T2-weighted imaging. Alpha-fetoprotein was 26.3 IU/L (<10), IgG and IgA were decreased. Two newly described heterozygote mutations were found in the ATM-gene. To ensure the diagnosis, ATM kinase activity and ATM protein levels were measured, showing the absence of detectable ATM protein and absence of ATM kinase activity. Therapy with carbidopa- levodopa was initiated twelve months ago with a maximum of 5 mg/kg per day; gait and up-right standing clearly improved under medication. Conclusion: In AT patients white matter lesions without cerebellar atrophy can occur early in childhood. Dystonia could be the first movement disturbance in AT. In atypical cases the diagnosis should be confirmed by measurements of ATM kinase activity and ATM protein levels.

Role of ataxia-telangiectasia mutated in hydrogen peroxide preconditioning against oxidative stress in Neuro-2a cells.

Ischemic preconditioning is an endogenous protective mechanism that may be triggered by exposure to hydrogen peroxide (H2O2). However, the exact mechanisms underlying preconditioning remain to be fully understood. Ataxia-telangiectasia mutated (ATM) is regarded as an essential endogenous protective protein against stress. The aim of the present study was therefore to investigate whether ATM mediates H2O2 preconditioning. Preconditioning of Neuro‑2a (N2a) cells with 100µM H2O2 for 90min resulted in protection from injury induced by a long period of exposure to 600µM H2O2. In addition, preconditioning with 100µM H2O2 activated ATM and increased ATM mRNA and protein expression levels in N2a cells. Furthermore, the protective effects induced by H2O2 preconditioning were attenuated by pretreatment with the ATM inhibitor, Ku55933, or ATM small interfering RNA. In conclusion, these findings suggested that ATM is involved in H2O2 preconditioning‑mediated protection against oxidative stress‑induced injury in N2a cells. To the best of our knowledge, the present study demonstrated, for the first time, that the ATM protein is a key mediator of H2O2 preconditioning.

Abstract B21: The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma

Abstract Glioblastoma (GBM) represents one of the most aggressive cancer types with the vast majority of patients succumbing to disease within the first five years. This dire prognosis reflects the limited efficacy of our frontline therapies which include radiation therapy and temozolomide (TMZ) chemotherapy. The cellular response to these therapies is critically mediated by DNA damage response signaling networks that are regulated by Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia And Rad3-Related Protein (ATR). Preliminary studies from our laboratory suggest the ATR inhibitor VX-970 has single agent efficacy in both established and primary GBM cell lines. Moreover, combined treatment with TMZ demonstrated therapeutic responses that, following regression analysis, were deemed synergistic (p&amp;lt;0.001). Validation studies using siRNA confirmed ATR as a central mediator of TMZ sensitivity as knockdown of ATM elicited no added benefit when tested in combination. VX-970 also enhanced radiation effects in neurosphere formation assays to suggest it may also be used as a radiosensitizer. While the specific mechanism responsible for these observations has yet to be determined, VX-970 treatment was found to exert anti-proliferative effects in a dose-dependent manner. This collection of work suggests that ATR inhibition may represent a worthwhile treatment strategy in GBM. Citation Format: Danielle Burgenske, Ann Mladek, Jann Sarkaria. The selective ATR inhibitor VX-970 enhances the therapeutic effects of standards of care in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B21.

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

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.

Endosulfan induces cell dysfunction through cycle arrest resulting from DNA damage and DNA damage response signaling pathways

Our previous study showed that endosulfan increases the risk of cardiovascular disease. To identify toxic mechanism of endosulfan, we conducted an animal study for which 32 male Wistar rats were randomly and equally divided into four groups: Control group (corn oil only) and three treatment groups (1, 5 and 10mgkg−1·d−1). The results showed that exposure to endosulfan resulted in injury of cardiac tissue with impaired mitochondria integrity and elevated 8-OHdG expression in myocardial cells. Moreover, endosulfan increased the expressions of Fas, FasL, Caspase-8, Cleaved Caspase-8, Caspase-3 and Cleaved Caspase-3 in cardiac tissue. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of endosulfan (1, 6 and 12μgmL−1) for 24h. An inhibitor for Ataxia Telangiectasia Mutated Protein (ATM) (Ku-55933, 10μM) was added in 12μgmL−1 group for 2h before exposure to endosulfan. Results showed that endosulfan induced DNA damage and activated DNA damage response signaling pathway (ATM/Chk2 and ATR/Chk1) and consequent cell cycle checkpoint. Furthermore, endosulfan promoted the cell apoptosis through death receptor pathway resulting from oxidative stress. The results provide a new insight for mechanism of endosulfan-induced cardiovascular toxicity which will be helpful in future prevention of cardiovascular diseases induced by endosulfan.

Abstract P2-05-33: Refining the prognostic value of Ki67 biomarker by ataxia telangiectasia mutated (ATM) status in a retrospective study of early stage hormone receptor positive breast cancer

Abstract Purpose: The current study was designed to investigate the combined influence of ataxia telangiectasia mutated (ATM) and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (&amp;lt; 4 cm) and fewer than four positive lymph nodes. Methods: Formalin-fixed paraffin-embedded specimens of resected primary breast tumors from 532 patients diagnosed with early stage breast cancer were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using immunohistochemistry and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Results: Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p=0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). Low Ki67/high ATM expression independently predicts favorable disease survival in a multivariate model in low Ki67 subgroup of ES-HPBC (Stage I-III)Variablesp valueHR (95%CI)Low Ki67/high ATM vs Low Ki67/low ATM0.020.36 (0.15-0.88)ize (T1/2 vs T3/4)0.010.16 (0.04-0.69)LN status (- vs +)0.140.49 (0.20-1.25)LVI (_- vs +)0.160.51 (0.20-1.31)Grade (1/2 vs 3)&amp;lt;0.00010.28 (0.11-0.67)Age (&amp;lt;65 vs &amp;gt;65)0.010.26 (0.10-0.74) Conclusions: These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC. Citation Format: Feng X, Li H, Kornarga E, Dean M, Lees-Miller S, Riabowol K, Magliocco A, Morris D, Watson P, Enwere E, Bebb G, Paterson A. Refining the prognostic value of Ki67 biomarker by ataxia telangiectasia mutated (ATM) status in a retrospective study of early stage hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-33.

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

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.

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

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.

Silencing of ATM expression by siRNA technique contributes to glioma stem cell radiosensitivity in vitro and in vivo.

Evidence has shown that both high expression of the ataxia-telangiectasia mutated (ATM) gene and glioma stem cells (GSCs) are responsible for radioresistance in glioma. Thus, we hypothesized that brain tumor radiosensitivity may be enhanced via silencing of the ATM gene in GSCs. In the present study we successfully induced GSCs from two cell lines and used CD133 and nestin to identify GSCs. A lentivirus was used to deliver siRNA-ATMPuro (Agroup) to GSCs prior to radiation, while siRNA-HKPuro (Ngroup) and GSCs (Cgroup) were used as negative and blank controls, respectively. RT-qPCR and western blotting were performed to verify the efficiency of the siRNA-ATM technique. The expression of the ATM gene and ATM protein were significantly downregulated post-transfection. Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that the Agroup demonstrated weak cell proliferation and lower survival fractions post-irradiation compared to the C/Ngroups. Flow cytometry was used to examine the percentage of cell apoptosis and G2phase arrest, which were both higher in the Agroup than in the C/Ngroups. We found that the comet tail percentage evaluated by comet assay was higher in the Agroup than in the C/Ngroups. After radiation treatment, three radiosensitive genes [p53, proliferating cell nuclear antigen (PCNA), survivin] exhibited a decreasing tendency as determined by RT-qPCR. Mice underwent subcutaneous implantation, followed by radiation, and the resulting necrosis and hemorrhage were more obvious in the Agroup than in the Ngroups. In conclusion, silencing of ATM via the siRNA technique improved radiosensitivity of GSCs both invitro and invivo.

Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.

Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells treated with and without pharmacological kinase inhibitors revealed ATM-dependent chromatin remodeling at the MLL break site giving access to DNA repair proteins but also nucleases triggering MLL rearrangements. This chapter summarizes these methods for functional characterization of ATM in patient LCLs and human cell lines.

Assaying Radiosensitivity of Ataxia-Telangiectasia.

Ataxia-Telangiectasia (A-T) is a prototypical genomic instability disorder with multi-organ deficiency and it is caused by the defective function of a single gene, ATM (Ataxia-Telangiectasia Mutated). Radiosensitivity, among the pleiotropic symptoms of A-T, reflects the basic physiological functions of ATM protein in the double strand break (DSB)-induced DNA damage response (DDR) and also restrains A-T patients from the conventional radiation therapy for their lymphoid malignancy. In this chapter, we describe two methods that have been developed in our lab to assess the radiosensitivity of A-T patients: (1) Colony Survival Assay (CSA) and (2) Flow Cytometry of phospho-SMC1 (FC-pSMC1). The establishment of these more rapid and reliable functional assays to measure the radiosensitivity, exemplified by A-T, would facilitate the diagnosis of other genomic instability genetic disorders as well as help the treatment options for most radiosensitive patients.

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

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 invitro. 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.

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

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 (AtmL2262P/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 AtmL2262P/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.

Ataxia telangiectasia: a review.

Definition of the diseaseAtaxia telangiectasia (A-T) is an autosomal recessive disorder primarily characterized by cerebellar degeneration, telangiectasia, immunodeficiency, cancer susceptibility and radiation sensitivity. A-T is often referred to as a genome instability or DNA damage response syndrome.EpidemiologyThe world-wide prevalence of A-T is estimated to be between 1 in 40,000 and 1 in 100,000 live births.Clinical descriptionA-T is a complex disorder with substantial variability in the severity of features between affected individuals, and at different ages. Neurological symptoms most often first appear in early childhood when children begin to sit or walk. They have immunological abnormalities including immunoglobulin and antibody deficiencies and lymphopenia. People with A-T have an increased predisposition for cancers, particularly of lymphoid origin. Pulmonary disease and problems with feeding, swallowing and nutrition are common, and there also may be dermatological and endocrine manifestations.EtiologyA-T is caused by mutations in the ATM (Ataxia Telangiectasia, Mutated) gene which encodes a protein of the same name. The primary role of the ATM protein is coordination of cellular signaling pathways in response to DNA double strand breaks, oxidative stress and other genotoxic stress.DiagnosisThe diagnosis of A-T is usually suspected by the combination of neurologic clinical features (ataxia, abnormal control of eye movement, and postural instability) with one or more of the following which may vary in their appearance: telangiectasia, frequent sinopulmonary infections and specific laboratory abnormalities (e.g. IgA deficiency, lymphopenia especially affecting T lymphocytes and increased alpha-fetoprotein levels). Because certain neurological features may arise later, a diagnosis of A-T should be carefully considered for any ataxic child with an otherwise elusive diagnosis. A diagnosis of A-T can be confirmed by the finding of an absence or deficiency of the ATM protein or its kinase activity in cultured cell lines, and/or identification of the pathological mutations in the ATM gene.Differential diagnosisThere are several other neurologic and rare disorders that physicians must consider when diagnosing A-T and that can be confused with A-T. Differentiation of these various disorders is often possible with clinical features and selected laboratory tests, including gene sequencing.Antenatal diagnosisAntenatal diagnosis can be performed if the pathological ATM mutations in that family have been identified in an affected child. In the absence of identifying mutations, antenatal diagnosis can be made by haplotype analysis if an unambiguous diagnosis of the affected child has been made through clinical and laboratory findings and/or ATM protein analysis.Genetic counselingGenetic counseling can help family members of a patient with A-T understand when genetic testing for A-T is feasible, and how the test results should be interpreted.Management and prognosisTreatment of the neurologic problems associated with A-T is symptomatic and supportive, as there are no treatments known to slow or stop the neurodegeneration. However, other manifestations of A-T, e.g. immunodeficiency, pulmonary disease, failure to thrive and diabetes can be treated effectively.

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

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.