Loss Of Ataxia Telangiectasia Mutated Research Articles

Mutations inString/CDC25inhibit cell cycle re-entry and neurodegeneration in aDrosophilamodel of Ataxia telangiectasia

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, we generated a Drosophila model of A-T. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, we hypothesize that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T.

Foxo3 Modulation of ATM and Oxidative Stress Mediates Distinct Functions in the Regulation of Hematopoietic Stem and Progenitor Cell Fate.

FOXO transcription factors are required for hematopoietic stem cell self renewal. In this study, we demonstrate that Foxo3 plays a specific and essential function in the regulation of both hematopoietic stem and progenitor cell fate. Foxo3 null mice display a myeloproliferative syndrome characterized by splenomegaly, a major expansion of the myeloid compartment in the blood, bone marrow and spleen, cytokine hypersensitivity of progenitors in hematopoietic organs and associated with the repression of the B lymphoid compartment. In addition, loss of Foxo3 leads to significant defects in hematopoietic stem cell numbers and activity. In particular, the numbers of long-term culture initiating cells (LTC-IC) was significantly reduced and the ability to repopulate lethally irradiated mice was severely compromised in Foxo3-defcient mice. This effect was mediated at least partially by enhanced accumulation of reactive oxygen species (ROS) in Foxo3-deficient hematopoietic stem cells as demonstrateda.by reduced QRT-PCR expression of several anti-oxidant enzymes leading tob.accumulation of ROS, (as measured by chloromethyl,dichlorodihydrofluorescein diacetate assay) in Foxo3 null hematopoietic stem cells, andc.in vitro and in vivo rescue of the phenotype using ROS scavengers.Furthermore, we demonstrate that while ROS accumulation results in suppression of Foxo3 null hematopoietic stem cell compartment, it enhances the activity of multipotential cells. By measuring RNA versus DNA content, and BrdU uptake, we determined that Foxo3-deficient hematopoietic stem cells exit quiescence (G0) and are impaired in their cycling at the G2/M phase. In particular, we identified ROS activation of p19ARF/p53 pathway and ROS-independent modulation of ataxia telangiectasia mutated (ATM) gene and p16INK4a, as major contributors to the interference with Foxo3-deficient hematopoietic stem cell self renewal and cycling. Loss of ATM has been shown to lead to hematopoietic stem cell deficiency. Importantly, we show that ATM gene expression is significantly suppressed in Foxo3-deficient hematopoietic stem cells suggesting that ATM lies downstream of Foxo3. Retroviral expression of a constitutively active form of Foxo3 in Foxo3-deficient bone marrow mononuclear cells enhances significantly the ATM expression suggesting that Foxo3 regulate expression of ATM gene. These combined findings suggest that Foxo3 functions in a tumor suppressor network to protect hematopoietic stem cells against deleterious effects of oxidative damage, to maintain hematopoietic lineage fate determination and to restrict the activity of long term repopulating hematopoietic stem cells. These findings provide insights into the mechanisms underlying hematopoietic stem cell fate.

The DNA damage signalling kinase ATM is aberrantly reduced or lost in BRCA1/BRCA2-deficient and ER/PR/ERBB2-triple-negative breast cancer

The ataxia-telangiectasia-mutated (ATM) kinase is a key transducer of DNA damage signals within the genome maintenance machinery and a tumour suppressor whose germline mutations predispose to familial breast cancer. ATM signalling is constitutively activated in early stages of diverse types of human malignancies and cell culture models in response to oncogene-induced DNA damage providing a barrier against tumour progression. As BRCA1 and BRCA2 are also components of the genome maintenance network and their mutations predispose to breast cancer, we have examined the ATM expression in human breast carcinomas of BRCA1/2 mutation carriers, sporadic cases and familial non-BRCA1/2 patients. Our results show that ATM protein expression is aberrantly reduced more frequently among BRCA1 (33%; P=0.0003) and BRCA2 (30%; P=0.0009) tumours than in non-BRCA1/2 tumours (10.7%). Furthermore, the non-BRCA1/2 tumours with reduced ATM expression were more often estrogen receptor (ER) negative (P=0.0002), progesterone receptor (PR) negative (P=0.004) and were of higher grade (P=0.0004). In our series of 1013 non-BRCA1/2 cases, ATM was more commonly deficient (20%; P=0.0006) and p53 was overabundant (47%; P<0.0000000001) among the difficult-to-treat ER/PR/ERBB2-triple-negative subset of tumours compared with cases that expressed at least one of these receptors (10 and 16% of aberrant ATM and p53, respectively). We propose a model of 'conditional haploinsufficiency' for BRCA1/2 under conditions of enhanced DNA damage in precancerous lesions resulting in more robust activation and hence increased selection for inactivation or loss of ATM in tumours of BRCA1/2 mutation carriers, with implications for genomic instability and curability of diverse subsets of human breast cancer.

Down‐regulation of ATM protein in HRS cells of nodular sclerosis Hodgkin's lymphoma in children occurs in the absence of ATM gene inactivation

The tumour component of classical Hodgkin's lymphoma (cHL), Hodgkin Reed-Sternberg (HRS) cells, are believed to be derived from germinal centre (GC) B cells but intriguingly display a characteristic loss of B cell receptor (BCR) expression. The precise mechanisms by which BCR-negative HRS cell progenitors survive negative selection during the GC reaction remain obscure. Individuals with ataxia telangiectasia, caused by biallelic inactivation of the DNA damage response gene, ataxia telangiectasia mutated (ATM), have a higher risk of cHL development. Here we show that, in contrast to normal GC B cells that expressed low but detectable ATM protein, ATM protein was not detected in HRS cells of 17/18 cases of paediatric cHL, all but one with nodular sclerosis (NS) subtype. A comprehensive analysis of the ATM gene in microdissected HRS cells of nine representative tumours showed no evidence of either loss of heterozygosity or consistent pathogenic mutations. Furthermore, bisulphite sequencing of the ATM promoter from HRS cells of five tumours also revealed the absence of hypermethylation. Since our microarray data suggested significantly reduced ATM transcription in HRS cells compared to GC B cells, we conclude that loss of ATM expression could be the result of alterations in upstream regulators of ATM transcription. Importantly, ATM loss in paediatric cHLs has clinical implications and could be potentially exploited to guide future, less toxic, tumour-specific treatments.

ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes

AbstractMutations in ATM (ataxia-telangiectasia mutated) cause ataxia-telangiectasia (AT), a disease characterized by neurodegeneration, sterility, immunodeficiency, and T-cell leukemia. Defective ATM-mediated DNA damage responses underlie many aspects of the AT syndrome, but the basis for the immune deficiency has not been defined. ATM associates with DNA double-strand breaks (DSBs), and some evidence suggests that ATM may regulate V(D)J recombination. However, it remains unclear how ATM loss compromises lymphocyte development in vivo. Here, we show that T-cell receptor β (TCRβ)–dependent proliferation and production of TCRβlow CD4+CD8+ (DP) thymocytes occurred normally in Atm−/− mice. In striking contrast, the postmitotic maturation of TCRβlow DP precursors into TCRβint DP cells and TCRβhi mature thymocytes was profoundly impaired. Furthermore, Atm−/− thymocytes expressed abnormally low amounts of TCRα mRNA and protein. These defects were not attributable to the induction of a BCL-2–sensitive apoptotic pathway. Rather, they were associated with frequent biallelic loss of distal Va gene segments in DP thymocytes, revealing that ATM maintains Tcra locus integrity as it undergoes V(D)J recombination. Collectively, our data demonstrate that ATM loss increases the frequency of aberrant Tcra deletion events, which compromise DP thymocyte maturation and likely promote the generation of oncogenic TCR translocations.

Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo

The ATM (ataxia telangiectasia mutated) protein kinase is activated under physiological and pathological conditions that induce DNA double-strand breaks (DSBs). Loss of ATM or failure of its activation in humans and mice lead to defective cellular responses to DSBs, such as cell cycle checkpoints, radiation sensitivity, immune dysfunction, infertility and cancer predisposition. A widely used biological marker to identify the active form of ATM is the autophosphorylation of ATM at a single, conserved serine residue (Ser 1981 in humans; Ser 1987 in mouse). Here we show that Atm-dependent responses are functional at the organismal and cellular level in mice that express a mutant form of Atm (mutation of Ser to Ala at position 1987) as their sole Atm species. Moreover, the mutant protein does not exhibit dominant-negative interfering activity when expressed physiologically or overexpressed in the context of Atm heterozygous mice. These results suggest an alternative mode for stimulation of Atm by DSBs in which Atm autophosphorylation at Ser 1987, like trans-phosphorylation of downstream substrates, is a consequence rather than a cause of Atm activation.

Analysis of the Ataxia Telangiectasia Mutated-Mediated DNA Damage Response in Murine Cerebellar Neurons

The DNA damage response is a network of signaling pathways that affects many aspects of cellular metabolism after the induction of DNA damage. The primary transducer of the cellular response to the double-strand break, a highly cytotoxic DNA lesion, is the nuclear protein kinase ataxia telangiectasia (A-T) mutated (ATM), which phosphorylates numerous effectors that play key roles in the damage response pathways. Loss or inactivation of ATM leads to A-T, an autosomal recessive disorder characterized by neuronal degeneration, particularly the loss of cerebellar granule and Purkinje cells, immunodeficiency, genomic instability, radiosensitivity, and cancer predisposition. The reason for the cerebellar degeneration in A-T is not clear. It has been ascribed by several investigators to cytoplasmic functions of ATM that may not be relevant to the DNA damage response. We set out to examine the subcellular localization of ATM and characterize the ATM-mediated damage response in mouse cerebellar neurons. We found that ATM is essentially nuclear in these cells and that various readouts of the ATM-mediated damage response are similar to those seen in commonly used cell lines. These include the autophosphorylation of ATM, which marks its activation, and phosphorylation of several of its downstream substrates. Importantly, all of these responses are detected in the nuclei of granule and Purkinje cells, suggesting that nuclear ATM functions in these cells similar to other cell types. These results support the notion that the cerebellar degeneration in A-T patients results from defective DNA damage response.

Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance.

In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. We discover in Drosophila that the two proteins control partially redundant pathways for telomere protection: the loss of ATM leads to the fusion of some telomeres, whereas the loss of both ATM and ATR renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. We provide evidence suggesting that both the loading of telomere capping proteins and normal telomeric silencing requires ATM and ATR in Drosophila and propose that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins.

Aberrant recombination involving the granzyme locus occurs in Atm−/− T-cell lymphomas

Ataxia telangiectasia (A-T) is an autosomal recessive disease caused by loss of function of the serine/threonine protein kinase ATM (ataxia telangiectasia mutated). A-T patients have a 250-700-fold increased risk of developing lymphomas and leukemias which are typically highly invasive and proliferative. In addition, a subset of adult acute lymphoblastic leukemias and aggressive B-cell chronic lymphocytic leukemias that occur in the general population show loss of heterozygosity for ATM. To define the specific role of ATM in lymphomagenesis, we studied T-cell lymphomas isolated from mice with mutations in ATM and/or p53 using cytogenetic analysis and mRNA transcriptional profiling. The analyses identified genes misregulated as a consequence of the amplifications, deletions and translocation events arising as a result of ATM loss. A specific recurrent disruption of the granzyme gene family locus was identified resulting in an aberrant granzyme B/C fusion product. The combined application of cytogenetic and gene expression approaches identified specific loci and genes that define the pathway of initiation and progression of lymphoreticular malignancies in the absence of ATM.

Single‐cell analysis of loss of heterozygosity at the ATM gene locus in Hodgkin and Reed‐Sternberg cells of Hodgkin's lymphoma: ATM loss of heterozygosity is a rare event

Hodgkin's lymphoma (HL) is a lymphoid malignancy characterized by the presence of rare neoplastic cells, Hodgkin and Reed-Sternberg (HRS) cells, scattered among a predominant population of inflammatory cells. On the basis of previously reported cytogenetic analyses, the ATM (ataxia-telangiectasia mutated) gene at 11q22-23 has been implicated in the etiology of HL. We therefore developed a single-cell PCR approach to detect ATM loss of heterozygosity (LOH) in HRS cells. Three microsatellites were investigated; 1 localized inside the ATM gene and the remaining 2 in close proximity. In 2 of the 15 lymph node samples, an allelic loss of the ATM gene locus was detected. ATM protein expression was examined in 8 cases (including 1 of the 2 cases with LOH) by immunohistochemistry. In the case associated with an allelic loss, the ATM protein was absent in the HRS cells, whereas in the 7 remaining cases, without detectable LOH at the ATM locus, nuclear ATM expression was observed. In the 2 HL cases with LOH, the ATM gene was sequenced following whole genome amplification of DNA isolated from microdissected HRS cells. In 1 of these 2 cases, a splice site mutation in the second ATM allele was found. This mutation could generate a premature termination codon leading to a marked instability and a rapid degradation of the resulting ATM mRNA transcripts. This latter event could explain the loss of the expression of the ATM protein in HRS cells as detected by immunohistochemistry in this particular case. As previously reported in some B-cell lymphomas, our results suggest that ATM genetic anomalies could play a role in the pathogenesis of a subset of HL cases.

Role of p53 and ATM in photodynamic therapy-induced apoptosis.

Photodynamic therapy (PDT) induces cell death through a laser light-activated photosensitizer and is a treatment option for tumors resistant to radio- and chemo-therapy. We investigated whether m-THPC-PDT induces cell death by necrosis and/or apoptosis, and whether these responses are modulated by p53 and/or ATM, two cancer-associated genes. Sensitivity of atm(+/+)p53(+/+), atm(+/+)p53(-/-), and atm(-/-)p53(-/-) mouse embryonic fibroblasts to m-THPC-PDT performed at a wavelength of 652 nm was determined by the MTT assay, trypan blue-exclusion, and the TUNEL and caspase3-cleavage apoptosis assays. c-Abl protein level was determined by immunoblotting. m-THPC-PDT rapidly induced cell death in a substantial fraction of cells by p53- and Ataxia telangiectasia mutated (ATM)-independent non-apoptotic processes. However, in the subset of apoptotic cells, apoptosis was reduced by loss of p53 and was even more reduced by the additional loss of ATM. Apoptosis correlated inversely with c-Abl level. p53 and ATM are not required for necrosis, but may be required for PDT-mediated apoptosis.